بسم الله الرحمان الرحيم
NEW INHIBITORS OF ANGIOTENSIN
CONVERTING ENZYME FROM PLANT AND
SYNTHETIC ORIGIN BY MASS SPECTROMETRIC
SCREENING ASSAY
Thesis submitted for the partial fulfillment of the degree of
Doctor of Philosophy
in
Chemistry
By
MUHAMMAD SALMAN BHATTI
H.E.J. RESEARCH INSTITUTE OF CHEMISTRY
International Center for Chemical and Biological Sciences
University of Karachi,
Karachi-75270, Pakistan
2019
II
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III
Thesis Certificate
This is to certify that the thesis entitled, “New Inhibitors of Angiotensin
Converting Enzyme from Plant and Synthetic Origin by Mass Spectrometric
Screening Assay” has been submitted to the Advanced Studies Research Board
(ASRB), University of Karachi by Mr. Muhammad Salman Bhatti s/o Mr.
Fakhruddin Bhatti for the award of the degree, Doctor of Philosophy (Ph.D.) in
the discipline of Chemistry. The research has been carried out under my
supervision and I certify that the work submitted herein is original and not
plagiarized from any other source. Neither the thesis nor the work contained
therein has been previously submitted to any institution for a degree.
___________________________________
Dr. Syed Ghulam Musharraf
Professor H.E.J. Research Institute of Chemistry International Center for Chemical and Biological Sciences (ICCBS) University of Karachi, Karachi-75270, Pakistan
IV
Contents
Acknowledgements ........................................................................................ VIII
Personal Introduction ....................................................................................... X
List of Publications .......................................................................................... XI
List of Figures ................................................................................................. XII
List of Tables ................................................................................................. XV
List of Abbreviations ...................................................................................... XVI
Summary .............................................................................................. XVIII
XIX ................................................................................................ خلاصہ
Chapter 1: Pharmacological Importance of Enzymes and
Angiotensin Converting Enzyme (ACE) ...................................... 1
1.1 Enzymes in Life: Brief History ................................................................... 2
1.2 Enzyme Catalysis ..................................................................................... 2
1.3 Αngiotensin Converting Εnzyme (ΑCΕ) .................................................... 3
1.3.1 Renin Angiotensin Aldosterone System (RAAS) ........................... 4
1.3.2 Problems Associated with Over Expression of ACE and
RAAS ............................................................................................ 6
1.4 Enzyme Inhibition ..................................................................................... 8
1.4.1 Reversible Inhibition ...................................................................... 9
1.4.2 Irreversible Inhibition ................................................................... 12
1.4.3 Inhibitors of ACE ......................................................................... 12
1.4.4 Standard Drugs or Synthetic Inhibitors of ACE ........................... 13
1.4.5 Natural Compounds, Peptides or Plant Extracts as ACE
Inhibitors ..................................................................................... 16
1.5 Drug Discovery Process ......................................................................... 24
1.6 Enzyme Kinetics ..................................................................................... 26
1.6.1 Initial Velocity .............................................................................. 26
1.6.2 Michaelis-Menten Kinetics Vmax and Km ...................................... 27
1.6.3 Lineweaver-Burk PIot .................................................................. 28
1.6.4 Dixon Plot .................................................................................... 30
V
Chapter 2: Mass Spectrometry Techniques for Enzyme Inhibition
Analyses ....................................................................................... 31
2.1 Introduction to Mass Spectrometry ......................................................... 32
2.2 Mass Spectro metry Instrumentation ....................................................... 33
2.2.1 Ion Sources ................................................................................. 33
2.2.2 Mass Analyzers ........................................................................... 40
2.2.3 Detectors ..................................................................................... 43
2.3 Tandem Mass Spectrometry ................................................................... 44
2.3.1 Tandem-in-space Mass Spectrometry......................................... 44
2.3.2 MS/MS by TripIe QuadrupoIes .................................................... 44
2.3.3 MS / MS by Qq TOF Sy stems ........................................................ 46
2.3.4 Tandem-in-time Mass Spectrometry ........................................... 47
2.3.5 Fragmentation and Ion Activation ................................................ 47
2.4 Mass Spectrometry for Enzyme Assay and Inhibitors Screening ........... 48
2.5 Methods of Mass Spectrometry for Enzyme Assay Screening ............... 49
2.5.1 Direct Infusion-Mass Spectrometry (DI-MS) ................................ 51
2.5.2 Ultrafiltration-Ma ss Spectro metry (UF-M S) ................................. 54
2.5.3 Size-Exclusion Chromatography-Mass Spectrometry (SEC-
MS) ............................................................................................. 57
2.5.4 Immobilized Enzyme-Mass Spectrometry (IE-MS) ...................... 59
2.5.5 FrontaI A ffinity Chromato graphy-Ma ss Spectro metry (F AC -
M S) ............................................................................................. 62
2.5.6 Capillary Isoelectric Focusing-Mass Spectrometry (CIEF-
MS) ............................................................................................. 65
2.5.7 Flow Injection Analysis-Mass Spectrometry (FIA-MS) and
High-Performance Liquid Chromatography-Mass
Spectrometry (HPLC-MS) ........................................................... 65
2.5.8 HPLC Based Continuous Flow System-Mass Spectrometry
(CFS-MS) .................................................................................... 68
2.5.9 Matrix Assisted Laser Desorption Ionization-Mass
Spectrometry (MALDI-MS) .......................................................... 69
VI
Chapter 3: Method Development for Screening of ACE inhibitors
Using HPLC-ESI-QqQ-MS ........................................................... 73
3.1 Introduction ............................................................................................. 74
3.2 ExperimentaI .......................................................................................... 75
3.2.1 Chemicals and Reagents ............................................................ 75
3.2.2 Calibration Curve ........................................................................ 75
3.2.3 Angiotensin Converting Enzyme Assay ....................................... 76
3.2.4 Sample Preparation for LC-ESI-QqQ-MS Analysis ..................... 76
3.2.5 LC-ESI-QqQ-MS AnaIysis ........................................................... 77
3.3 Results and Discussion .......................................................................... 78
3.3.1 Optimization of LC-MS/MS Analysis Conditions .......................... 78
3.3.2 Optimization of Jet Stream Source Using Design Expert ............ 80
3.3.3 Mass Hunter Optimizer for Peptides............................................ 87
3.3.4 Determination of Calibration Curve, LOD and LOQ .................... 89
3.3.5 Enzymatic Reaction..................................................................... 90
3.3.6 Initial Velocity of Enzyme ............................................................ 92
3.3.7 Determination of IC50 of Inhibitors ............................................... 93
3.3.8 Comparison of Captopril and Lisinopril Inhibitory Potential ......... 95
3.3.9 Qualification of ACE Assay ......................................................... 95
3.3.10 Comparison with Reported Methods ......................................... 96
3.4 Conclusion .............................................................................................. 99
Chapter 4: Method Development for Screening of ACE inhibitors
Using MALDI-MS ....................................................................... 100
4.1 Introduction ........................................................................................... 101
4.2 Experimental ......................................................................................... 102
4.2.1 Chemicals and Reagents .......................................................... 102
4.2.2 MALDl Matrix Preparation ......................................................... 102
4.2.3 Calibration Curve ...................................................................... 102
4.2.4 Enzymatic Assay ....................................................................... 103
4.2.5 Sample Preparation................................................................... 103
4.2.6 MALDI-MS Analysis Optimization ............................................. 104
4.2.7 MALDI MS Analysis................................................................... 105
4.3 ResuIts and Discussion ........................................................................ 106
VII
4.3.1 MALDl Matrix Preparation ......................................................... 106
4.3.2 Linearity Experiment.................................................................. 107
4.3.3 MALDI MS Analysis................................................................... 110
4.3.4 Enzymatic Reaction................................................................... 113
4.3.5 Determination of IC50 of Standard Inhibitors .............................. 115
4.3.6 Comparison of Captopril and Lisinopril Inhibitory Potential ....... 117
4.4 Conclusion ............................................................................................ 118
Chapter 5: Screening of Different Synthetic Drugs, Compounds and
Natural Extracts for Inhibitory Potential Against ACE ........... 119
5.1 Introduction ........................................................................................... 120
5.2 Experimental ......................................................................................... 120
5.2.1 Reagents and Chemicals .......................................................... 120
5.2.2 Enzymatic Reactions for Inhibition Study .................................. 121
5.2.3 Preparation of Inhibitors ............................................................ 121
5.2.4 Sample Preparation and Analysis ............................................. 122
5.3 Results and Discussion ........................................................................ 122
5.3.1 Inhibition AnaIysis of Drugs/Compounds ................................... 122
5.3.2 Inhibition Study of Synthetic Compounds .................................. 150
5.3.3 Inhibition Study of Natural Compounds and Extracts ................ 167
5.4 Conclusion ............................................................................................ 170
References ................................................................................................ 172
VIII
Acknowledgements
ALLAH (Subḥānahu wa ta'alā) is the one Who is the beneficent, the merciful, we pray to
You and we ask for Your help. All praise is to Him, Who guides me and gives me strength
to pursue and complete this work. Durood-o-Salaam to the most noble and humble
creation of them all, our beloved holy prophet Hazrat MUHAMMAD (Ṣallā llāhu ʿalayhi
wasallama) who enlightened our conscience with the essence of faith in ALLAH (SWT).
I offer my sincere gratitude to the three great scientists: Prof. Dr. Salimuzzaman Siddiqui
(late), Founding Director; Prof. Dr. Atta-ur-Rahman (F.R.S., N.I., H.I., S.I., T.I.), Patron-in-Chief,
International Center for Chemical and Biological Sciences, University of Karachi, and Prof.
Dr. Muhammad Iqbal Choudhary (H.I., S.I., T.I.), Director of the H.E.J. Research Institute of
Chemistry, International Center for Chemical and Biological Sciences, University of
Karachi. Without them there would be no concept of having a world class research
institute in Pakistan. I am especially thankful to the Prof. Dr. Atta-ur-Rahman and Prof. Dr.
M. Iqbal Choudhary, their vision and leadership made possible to transform the H.E.J.
Research Institute of Chemistry into an international center of excellence and made it one
of a kind in world, where I had state-of-art facilities for my Ph.D. work, under single roof.
I cannot write, nor can I explain the invaluable support and encouragement received from
my supervisor cum mentor Prof. Dr. Syed Ghulam Musharraf. His kindness, personality,
knowledge are the reasons which motivated me to pursue the studies of M.Phil./Ph.D. His
attention, supervision and guidance had transformed me from a summer internship
internee to a Doctor of Philosophy (Ph.D.). I can never forget his kind and encouraging
attitude and all of the above his patience, during the whole tenure of my studies and
research, I will remain forever indebted to him.
I am very thankful to Prof. Dr. Khalid M. Khan (S.I., T.I.), for providing compounds for
screening, and Prof. Dr. M. Iqbal Bhanger, Dr. M. Imran Malik and Dr. Sirajuddin for their
valuable time to review my thesis. I am also very thankful to the faculty of the ICCBS. As
an indigenous scholar, I am also thankful to the Higher Education Commission for their
finalcial help.
Simple words are not enough for the two personalities in the world to them I owe
everything, my father Fakhruddin Bhatti and mother Yasmeen Gul. They have supported
me in all ways in my studies, these were their prayers, due to which I was able to achieve
this position in my life. I am also thankful to my big brother and all my family members for
their love, affection, and unparalleled support throughout my life.
IX
I am very thankful to Dr. Arslan Ali, who always lend his helping hand in solving my
problems. I am grateful to all my seniors Dr. Naghma Hashmi, Dr. Naveed Malik, Dr. Arif
Ahmed, Dr. Aisha Bibi, Dr. Irfan, Dr. Jalal, Dr. Madiha, Dr. Shumaila, Dr. Nayab, Dr.
Shoaib, Ms. Mahwish, Ms. Iffat, Dr. Urooj, Dr. Amna, Dr. Najia and Dr. Qamar ul Arfeen;
my class fellows Ms. Aisha Khalid and Dr. Kashif; and all lab fellows Dr. Faraz, Ms. Aisha
Iqbal, Ms. Fizza, Mr. Hammad, Mr. Saqib, Ms. Wardah, Ms. Ambreen, Ms. Kosur, Ms.
Fareeha, Ms. Sabiha, Ms. Maryam, Ms. Tamkeen, Mrs. Adeeba, Ms. Sindhia, Mrs.
Hamna, Mr. Noman Khan, Mr. Noman, Mr. Ramzan, Mr. M. Zaki, Mr. Saeed, Mrs. Nudrat,
Ms. Naheed, Mrs. Muzna, Mr. Nasir, Ms. Tehreem, Ms. Nida, Mr. Usman, Ms. Zaib and
Mr. Faisal I spent a wonderful and memorable time with them thanks to all of them. Mr.
Shehzad Abbas, our lab attendant deserves special thanks for his help and cooperation.
Last but not least, I am thankful to my dear friends Dr. Ayaz Anwar, Mr. Abdul Mateen,
Mr. Junaid ul Haq, Dr. Itrat Fatima, Dr. Shazia Iqbal, Mr. Saquib Shahbaz and Dr. Zahid
for their unconditional help, care, support and company all the time, and my oldest friend
Mr. Shoaib Khalid who showed me the way towards this achievement.
At last I pray from all mighty ALLAH for brightening future, good health and joyful life to all
my teachers, family members and friends. Aameen
Muhammad Salman Bhatti
21st June 2019, Karachi
X
Personal Introduction
I was born in Mirpurkhas, “The city of Mangoes” in the south-east of Sindh, I belong to a medium family system in term of finance and education, but my father took a bold step and did his best to support me in getting higher educations. I started my education voyage in 1993 from an Urdu medium Govt. Railway Primary School. Then, I moved to Shah Wali Ullah Govt. High School, and passed my matriculation in 2003. Then I took admission in Shah Abdul Latif Govt. Degree Science College, Mirpurkhas, in pre-medical category and passed in 2005. My aim was to be a medical doctor but in two attempts to medical college admission I left out with a difference of one position, then I took admission in B.S. (Hons.) Chemistry in 2007 and passed in 1st division in Analytical Chemistry from Department of Chemistry, University of Sindh, Jamshoro in 2010. During my final year, I got an internship in H.E.J. Research Institute of Chemistry and worked in the lab of Prof. Dr. Syed Ghulam Musharraf, there I saw another opportunity to fulfill my dream of becoming a doctor, which was, to get a Ph.D. For this purpose, I worked hard and got admission in this prestige institute H.E.J. in June 2011. I did not have a good head start in my early education, as well as I didn’t get high grades or position in my career, but Alḥamdulillāh, with my hard work and dedication I was able to achieve goals of my life. In H.E.J., I joined my respected mentor Prof. Dr. Syed Ghulam Musharraf and started the journey of research under his supervision. My research work was decided on the first day of joining H.E.J., and it was the interface of biological and analytical field, “Development of enzyme inhibition assay on mass spectrometry”. I started to explore different aspects and chose the Angiotensin Converting Enzyme because of its pharmacological importance. I had many down times in my research, and it was very difficult for me to continue the Ph.D., but with patience, hard work and support of my supervisor I developed this work and finally published the developed method in international journal of impact factor 3.5. One of my achievement in H.E.J. is that I obtained 94 percentile in international subjective GRE, which is the second highest percentile, anyone has ever got in this test in H.E.J. During the tenure of H.E.J. I also had the privilege to teach to the students of National Chemistry Talent Contest (NCTC), it was a great teaching experience. I always loved to work on advance instruments or techniques, in this context, I conducted a one-week workshop, under guidance of Prof. Dr. David Smith, entitled “On-hands training on Capillary-HPLC and Nano-flow ESI-MS workshop”, in which we taught how to transform a UPLC into a nano flow-LC and preparation method of capillary columns. Other then scientific life, few of my hobbies are playing table tennis, watching movies, videos, solving puzzles and to surf internet as ‘netizen’. H.E.J. is always like my second home, where I spent wonderful time with friends and collagues and I learned a lot not only about the science but also the social values and ethics of prefossional life, which will help me in various stages of my life. I am serving my country in a federal commission, and I would like to continue serving my country with my knowledge and scientific research in one way or another. I will always cherish this prestigious institute and my supervisor for supporting me at every step and making me what I am today. May ALLAH bless us all Aameen.
Muhammad Salman Bhatti
21st June 2019, Karachi
XI
List of Publications
1. Musharraf, S. G., Bhatti, M. S., Choudhary, M. I., and Rahman, A.-u. (2017).
Screening of inhibitors of angiotensin-converting enzyme (ACE) employing
high performance liquid chromatography-electrospray ionization triple
quadrupole mass spectrometry (HPLC-ESI-QqQ-MS). European Journal of
Pharmaceutical Sciences, 101, 182-188.
2. Ahmad, I., Bano, R., Musharraf, S. G., Sheraz, M. A., Ahmed, S., Tahir, H.,
ul Arfeen, Q., Bhatti, M. S., Shad, Z., and Hussain, S. F. (2015).
Photodegradation of norfloxacin in aqueous and organic solvents: A kinetic
study. Journal of Photochemistry and Photobiology A: Chemistry, 302, 1-10.
3. Ahmad, I., Bano, R., Musharraf, S. G., Ahmed, S., Sheraz, M. A., ul Arfeen,
Q., Bhatti, M. S., and Shad, Z. (2014). Photodegradation of moxifloxacin in
aqueous and organic solvents: a kinetic study. AAPS PharmSciTech, 15(6),
1588-1597.
4. Bhatti, M. S., and Musharraf, S. G. (2019). Screening of standard drugs
against Angiotensin converting enzyme for their repurposing as ACE
inhibitor. (manuscript in progress)
5. Bhatti, M. S., Musharraf, S. G. and Khan, K. M. (2019). Screening of
derivatives of indole as Angiotensin converting enzyme inhibitors.
(manuscript in progress)
XII
List of Figures
Figure 1.1: Crystal structure of homosepian testicular ACE with Lisinopril (ESRF; Natesh et al., 2003). ........................................ 4
Figure 1.2: Schematic pathway of RAAS (Mason et al., 2012). .................... 5
Figure 1.3: Renin angiotensin aldosterone system (Wikipedia). ................... 6
Figure 1.4: Risks associated with RAAS (Werner, Pöss, and Böhm, 2010). .......................................................................................... 8
Figure 1.5: Types and mechanism of different enzyme inhibition. ................ 9
Figure 1.6: Graphical illustration and equations for different types of reversible inhibitions (Balbaa and El Ashry, 2012). ................... 10
Figure 1.7: Graph for calculation of initial velocity of enzyme reaction. ....... 26
Figure 1.8: Michaelis-Menten kinetics graph and different types of inhibitions. ................................................................................. 28
Figure 1.9: Lineweaver-Burk plots for different types of inhibition. .............. 29
Figure 1.10: Dixon plots for competitive and noncompetitive inhibition. ........ 30
Figure 2.1: GeneraI schemati c diagram of mass spectro meter (Soderberg, 2019). .................................................................... 33
Figure 2.2: Sche matic diagram of ionization source of MALDl mas s spectrometer (Szot and Croxatto, 2019). .................................. 34
Figure 2.3: Comparasion of Nd:YAG laser and smartbeam spectra (Holle and Ketterlinus, 2008). ................................................... 36
Figure 2.4: Smartbeam setup (Holle et al., 2006). ...................................... 36
Figure 2.5: Process of desoIvation and ionization in ESl-MS (IPFS). ......... 38
Figure 2.6: Jet stream ionization source working principle (Mordehai and Fjeldsted, 2009). ................................................................ 39
Figure 2.7: Thermal profile of jet stream source showing confinement of zoning by super-heated nitrogen (Mordehai and Fjeldsted, 2009). ....................................................................... 39
Figure 2.8: Comparison of spectra using conventional ESI and jet stream technology (Mordehai and Fjeldsted, 2009). ................. 40
Figure 2.9: Transmission of different ions through quadrupole mass analyser (Khan, 2019). .............................................................. 41
Figure 2.10: Ions separation as a function of time in a TOF analyzer. .......... 42
Figure 2.11: Sch ematic of a tripIe quadrupoIe ma ss spectro meter (Jackson, 2019). ....................................................................... 45
Figure 2.12: Different types of mass spectrometry based screening assay (de Boer et al., 2007). ..................................................... 50
Figure 2.13: Different techniques of mass spectrometry used for enzyme assay screening. ......................................................... 51
XIII
Figure 2.14: Direct Ionization mass spectrometry spectra of enzyme inhibitor complexes (Stokvis et al., 2000). ................................. 52
Figure 2.15: ESl-FT-lCR-MS screening o f library of peptides (J. Gao et al., 1996). .................................................................................. 53
Figure 2.16: Ultrafiltration process in enzymatic assays (de Boer et al., 2007). ........................................................................................ 55
Figure 2.17: α-Glucosidase inhibitory assay with S. baicalensis extract using UF-LC-PDA-ESI/MS (J. Wang et al., 2014). .................... 55
Figure 2.18: UF-MS analysis of COX-II inhibitory assay (Nikolic et al., 2000). ........................................................................................ 56
Figure 2.19: Fast SEC-MS of a library of compounds and identification on MS/MS basis (Annis et al., 2004). ........................................ 58
Figure 2.20: Covalently immobilization of enzyme with the resin surface (Sirisha, Jain, and Jain, 2016). ................................................. 59
Figure 2.21: Enzyme immobilization methods (Sirisha, Jain, and Jain, 2016). ........................................................................................ 60
Figure 2.22: Screening of library of compounds using immobilized enzyme assay, spectra A is before incubation & spectra B is after incubation (H. Gao and Leary, 2003). ........................... 61
Figure 2.23: Principle of FAC-MS where early eluting compounds has no or low affinity and vice versa (Schriemer, 2004). ................. 63
Figure 2.24: FAC-MS enzyme inhibitor screening using ‘roll-up height’ phenomenon (Slon-Usakiewicz et al., 2005). ............................ 64
Figure 2.25: FIA-MS in step A preparation and incubation of sample, in step B injection of sample into LC-ESI-MS system (Luque de Castro, 2019). ...................................................................... 66
Figure 2.26: Spectra and results of inhibition study of drugs using FIA-MS (Takayama et al., 1997). ..................................................... 67
Figure 2.27: Continuous flow system with MS detection using sample injection and online reaction of enzyme and substrate in the presence of inhibitors (Ingkaninan, Hazekamp, et al., 2000). ........................................................................................ 69
Figure 2.28: Multiplex assay on MALDI-MS (A) is the spectra of cοntrοl withοut any inhibitοr, (B) is spectra of with the inhibitοr Lisinοpril (Hsieh, Keshishian, and Muir, 1998). ......................... 72
Figure 3.1: Scheme for samples of calibration curve. ................................. 76
Figure 3.2: Scheme of solvent gradient on HPLC. ...................................... 77
Figure 3.3: Total ion chromatogram of peptides. ......................................... 79
Figure 3.4: Retention time and MRM transition of bradykinin, angiotensin II and angiotensin I, in a standard mixture. ............ 80
Figure 3.5: Response of bradykinin and angiotensin I. ............................... 84
XIV
Figure 3.6: Comparison of multiple charge ions of angiotensin I (above) and bradykinin (below). ................................................ 86
Figure 3.7: Steps of ‘MassHunter Optimizer for Peptides’. ......................... 88
Figure 3.8: Calibration curve of angiotensin I. ............................................. 89
Figure 3.9: ACE assay with different concentrations of enzyme and time course. .............................................................................. 92
Figure 3.10: Inhibition of ACE by Captopril and Lisinopril. ............................ 94
Figure 4.1: Molecular structure of α-CHCA. .............................................. 106
Figure 4.2: Spectra of α-CHCA on MALDI-MS. ......................................... 107
Figure 4.3: Calibration curve for %conversion. ......................................... 110
Figure 4.4: Parameters of analysis method. (A) sample carrier, (B) detection parameters, (C) Spectrophotometer settings and (D) instrument digitizer and detector settings. ......................... 112
Figure 4.5: MALDI-MS peaks detection and processing parameters. ....... 113
Figure 4.6: Graph for ACE reaction without any inhibitor. ......................... 115
Figure 4.7: Scheme for analysis of inhibitor samples and control in well plate. ....................................................................................... 116
Figure 4.8: Inhibition of ACE by Captopril and Lisinopril. .......................... 117
Figure 4.9: Comparative MALDl-MS spectra of different concentration of Captopril and Lisinopril. ...................................................... 118
XV
List of Tables
Table 1.1: Standard drugs or inhibitors of ACE. ............................................. 14
Table 1.2: Different plant extracts as active inhibitors against ACE. .............. 17
Table 1.3: Peptides from different food proteins as ACE inhibitors. ............... 17
Table 1.4: Flavonoids and their derivates from natural sources, as active ACE inhibitors. ............................................................................... 19
Table 3.1: MRM transitions and source conditions for fragmentation. ............ 78
Table 3.2: Range of different parameters feed to the software. ...................... 81
Table 3.3: Experiments design output of Design-Expert................................. 82
Table 3.4: Response of bradykinin and angiotensin I correspond to each experiment. .................................................................................... 83
Table 3.5: Checking of ionization of multiple charge ions of angiotensin I and bradykinin. .............................................................................. 85
Table 3.6: Intraday and Interday accuracy and precision determination of angiotensin I. ................................................................................. 90
Table 3.7: Initial velocity of ACE in enzymatic assay. ..................................... 93
Table 3.8: Intraday and interday precision and reproducibility of ACE inhibition by Captopril and Lisinopril. ............................................. 96
Table 3.9: Comparison of the current developed method with previously reported methods. ......................................................................... 97
Table 4.1: Different spotting methods on MTP. ............................................ 104
Table 4.2: Sample scheme of linearity experiment. ...................................... 108
Table 4.3: Percentage conversion of different samples for linearity experiment. .................................................................................. 109
Table 4.4: ACE reaction without any inhibitor. .............................................. 114
Table 5.1: All drugs or cοmpounds used for the inhibitions istudy. ............... 123
Table 5.2: Screening results of drugs/compounds, IC50 values of active drugs. .......................................................................................... 147
Table 5.3: Ten most active drugs along with their purposes. ........................ 150
Table 5.4: Structures of base compounds and general structures of derivatives classes. ..................................................................... 151
Table 5.5: Synthetic compounds used for inhibition study. ........................... 153
Table 5.6: Screening results of synthetic compounds, IC50 of active compounds. ................................................................................. 166
Table 5.7: Natural compounds used for inhibition study. .............................. 168
Table 5.8: Natural extracts of plants used for inhibition study. ..................... 169
Table 5.9: Results of natural compounds and extracts, IC50 of active samples. ...................................................................................... 170
XVI
List of Abbreviations
Terms Abbreviation
%C Percentage conversion
%IA Percentage inhibitory activity
%MA Percentage maximal activity
[I] Concentration of inhibitor
[S] Concentration of substrate
ACE Angiotensin Converting enzyme
ADH Antidiuretic hormone
AI / AngI Angiotensin 1
AII / AngII Angiotensin 2
α-CHCA / HCCA α-Cyanο-4-hydrοxycinnamic acid
BK Bradykinin
DMSO Dimethyl sulfoxide
DTT Dithiothreitol
EIC Extraction-ion chromatogram
ESI Electrospray ionization
FAC Frontal affinity chromatography
FIA Flow injection analysis
HPLC High performance liquid chromatography
IC50 Inhibitor concentration used to reduce enzyme activity up to
50%
Km Concentration of substrate at half of maximum velocity
LOD Limit of detection
LOQ Limit of quantitation
m/z Mass-to-charge ratio
XVII
Terms Abbreviation
MALDI Matrix-assisted laser desorption ionization
MI Myocardial infection
MRM Multiple reaction monitoring
MS/MS / MS2 Tandem mass spectrometry
MTP MALDI target plate
mU Milli Units (concentration of enzyme)
QqQ Triple quadrupole
RAAS Renin angiotensin aldosterone system
RSD Relative standard deviation
SEC Size exclusion chromatography
TIC Total ion current
ToF Time-of-flight
UF Ultra filtration
Vmax Maximum initial velocity of enzyme
vo Initial velocity of enzyme
XVIII
Summary
Enzymes play a very crucial role in the regulation of almost all process of life. Enzymes
are also an important class of drug target; screening of inhibitors for enzymes is the first
step in drug discovery process. Angiotensin converting enzyme (ACE) is a
pharmacological important enzyme, it converts the Al into All. It plays a key role in Renin
angiotensin aldosterone system (RAAS), which regulate the blood pressure and
hypertension. Over expression of RAAS can cause many cardiovascular and renal
diseases, therefore, inhibition of ACE is a promising way of controlling over expression of
RAAS.
Many methods have been used for the inhibition study of ACE including
spectrophotometric, fluorimetric, HPLC, mass spectrometric, etc. When LC-MS combines
with the MS/MS then it becomes very unambiguous and sensitive analysis techniques for
detection and identification of peptides in complex samples. In this study, a new sensitive
and robust HPLC-ESI-MS/MS method, with very low limit of quantitation was developed
for ACE screening assay. In this method, substrate was used instead of product to
determine ACE activity. For this purpose, a calibration curve of Angiotensin I was
developed in a range of 20-200 nM, linear equation y=0.0888x-0.1932 and R2=0.999. Two
commercially available antihypertensive drugs, Captopril and Lisinopril, were checked to
validate the method and their IC50 values were found to be 3.969 μM and 0.852 μM,
respectively. This newly developed method required very low amount of substrate,
enzyme and inhibitor per sample.
MALDI was also explored for the potential of enzyme inhibition study, MALDI-TOF-MS is
a well-known technique for the analysis of protein and peptides. Relative quantification
was performed by using the ratio of substrate and product. In this study, a high-throughput
method of ACE inhibition was developed on MADLI-TOF-MS. In this method, a linear
curve of AI was developed for 0-100% conversion, linear equation y=9.3175x+3.4177 and
R2=0.994. Both standard inhibitors, Captopril and Lisinopril, were checked for their
inhibition and their IC50 values were found to be 8.256 μM and 1.962 μM, respectively,
which validated the method. This method provides a fast and alternative for the screening
of drugs to find their potential against the target enzyme.
As a final step, 77 commercial drugs, 40 synthetic compounds, 4 natural products and 3
extracts of plants were investigated for their inhibitory potential. In drugs, 36 showed
moderate to good inhibition of as low as IC50=272 µM. In synthetic compounds, 32
compounds showed significant inhibition ranged from IC50 = 320-5123 µM. In the last
category, all 4 compounds were moderately active with IC50 value 496-9705 µM; while
only one plant extracts showed some inhibition with IC50 =10779 µg/mL (ppm).
Current study has proved that direct nature of the MS measurement ensures the
minimization of the false results and large number of compounds can be screened without
the need for labels or additional internal standards, both MALDl-TOF-MS and LC-ESI-
MS/MS are valid readout for enzyme inhibitor screening assays.
XIX
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1
Chapter 1: Pharmacological Importance of
Enzymes and Angiotensin Converting Enzyme
(ACE)
2
1.1 Enzymes in Life: Brief History
Till the start of 19th century, it was considered about the processes like milk
souring, formation of alcohol from fermentation of sugar could only be happen
because of some Iiving organisms. ln first quarter of 19th century, the key
substance ‘diastase’ was isolated which was responsible for breaking the sugar
and it is also known as amylase. After a while, another substance was extracted
from the gastric juice and termed as ‘pepsin’, it was responsible for the digestion
of dietary proteins.
These types of many other substances were given the name of ferments. It was
suggested by Justus Von Liebig, that, these ferments may be of nonliving
materials which were obtained from the living cells, but some scientists like Louis
Pasteur and others still uphold the concept the ferments that these feremnet must
have some living materials inside them. Gradually, the term ‘ferment’ was replaced
by the ‘enzyme’. Wilhelm Kfthne proposed this term of enzyme 1st time in 1878, it
was originated from the word of Greek ‘enzumé’, which means “in yeast”. It was
the factor from the yeasts that was revealed and clear the dispute in favor of
nonliving theory of catalysis. In 1897, It was shown by Hans Buchner and Eduard
Buchner that, extract of yeast cell can also cause the fermentation of sugar even
though no living cells were present in it (Palmer and Bonner, 2007).
Enzymes play a very important role in the living beings by regulating processes as
biological catalyst. The use of enzyme is not only, limited to the biological process
but it is widely used in industrial processes as well as enzymes are using as target
in drug discovery process. The importance of enzymes from natural and synthetic
source is increasing rapidly (Liesener and Karst, 2005).
1.2 Enzyme Catalysis
Enzymes are biological catalysts which increases the rate of reactions which are
taking place in living cells by providing alternate pathways, without getting any
chemical change to themselves. The reactant of an enzyme catalysed reaction is
called as “substrate” and it is converted into the “product” by specific reaction.
Every enzyme is very specific in its function, utilize its specific substrate and in
3
result produce its specific product. Enzymes are specific type of protein, but
sometimes, specific types of enzymes need one or more non-protein component/s
to perform their function, this non-protein component is known as coenzyme or
cofactor. A coenzyme can be an organic molecule or be a metal ion (Palmer and
Bonner, 2007).
Enzymatic reactions play a very important part in the regulation of nearly all the
processes of living organisms, but applications of enzyme is not limited to that,
enzymes now a days are used in industrial processes as biocatalysts or can be
used as targets to screening the drugs, enzymes from both synthetic and natural
sources are getting more important day by day. (Liesener and Karst, 2005).
1.3 Αngiotensin Converting Εnzyme (ΑCΕ)
Αngiotensin converting enzyme (ACE) is a dipeptidyl carboxypeptidase (E.C.
3.4.15.1) which has a zinc (Zn) atom and having 150-180 kDa range of mass. ACE
is comprised of one polypeptide chain which have two terminal domains of N and
C. In each domain there are two sites for catalysis reaction. ACE is a
metallopeptidase which produce the Angiotensin II from inactive Angiotensin I by
cleaving its two peptides histidine–leucine from the C-terminal, crystal structure of
human ACE is shown in Figure 1.1 (Balasuriya and Rupasinghe, 2011; Lapointe
and Rouleau, 2002; Ortiz-Salmerón, Barón, and Garcı a-Fuentes, 1998; Shi et al.,
2010).
ACE is usually present on the outer surface membrane of a cell so that, active site
of ACE is accessible by the foreign environment of cell. ACE shows its expression
in various tissues and organs; the heart, the brain tissues and its several regions,
renal proximal tubular endothelium, vascular endothelium, the lungs, activated
macrophages are included in them (Lapointe and Rouleau, 2002). ACE present in
the lungs has the highest concentrations. Other than lungs ACE is also present in
vascular endothelium, renal proximal tubules, gastrointestinal tract, and the
tissues of heart and brain, in low concentration. It exists in both ways as a
circulatory (globular) enzyme and membrane bound enzyme (Alves et al., 2005;
Balasuriya and Rupasinghe, 2011).
4
Figure 1.1: Crystal structure of homosepian testicular ACE with Lisinopril (ESRF; Natesh et al., 2003).
ACE mainly perform transformation of AI into its product AII; by cleaving the C-
terminal dipeptide (L-histidyl-L-Leucine) of AI, generating the physiologically active
vasoconstrictor peptide AII. On the other hand ACE also act of the Bradykinin and
degrade it by cleaving at 7-8 peptide bond, bradykinin is a potent vasodilator
(Elased et al., 2006; Menard and Patchett, 2001). Different studies have shown
that the ACE has more Km (affinity to react with substrate) with AI in comparasion
to the bradykinin (BK) (Lapointe and Rouleau, 2002; Wong, 2016). ACE is a core
component of Renin Angiotensin Aldosterone System (RAAS), maintaining the
body’s blood pressure and its regulation is the main function of this system
(Jianping Wu, Aluko, and Muir, 2002).
1.3.1 Renin Angiotensin Aldosterone System (RAAS)
Renin Angiotensin Aldosterone System (RAAS) is an essential body fIuid
maintenance system which has the basic function of stabilizing the blood pressure,
it is also called as Renin Angiotensin System (RAS). It was discovered over a
5
century ago, it is a biochemical pathway which performs very critical functions in
physiology of cardiovascular system, it is also responsible in various steps of
pathways in diseases related to cardiovascular and cardio-renal (Elased et al.,
2006; Lu, Liu, and Feng, 2011).
The pathway of RAAS starts with the production of angiotensinogen, it is an α-2
globulin which is produced from the liver, it is a sequence of 452 aminoacids but
only the 10 first amino acids plays significant part in RAAS, while angiotensinogen
does not have any independent function by itself. Next important protein in RAAS
is Renin, it is produced from ‘prorenin’ which is an enzymatic precursor and
secreted from the kidney juxtaglomerular apparatus into the bloodstream. Once
the renin is activated it perfrom action of angiotensinogen and cleaves its 10
peptides chain Asp-Arg-VaI-Tyr-IIe-His-Pro-Phe-His-Leu to produce the
Angiotensin I. Like angiotensinogen, AI is also not active, but it acts as the
substrate of Angiotensin converting enzyme (ACE). ACE cleaves two residues,
Histidyl and Leucine, from AI to produce octapeptide Angiotensin-ll (AII) (Asp-Arg-
VaI-Tyr-IIe-His-Pro-Phe), schematic pathway of RAAS is shown below in Figure
1.2 (Mason et al., 2012).
Figure 1.2: Schematic pathway of RAAS (Mason et al., 2012).
AII is the main peptide component of the RAAS, it acts different ways as intracrine
hormone, paracrine/autocrine hormone and endocrine hormone. AII, which is
converted from AI by ACE, is the main component which have effect in RAAS. Its
biological actions are produced through the selective binding of this peptide to
6
different types of receptors, it causes the vasoconstriction (constriction of blood
vessels) by interacting with receptors of angiotensin on cells of vascular smooth
muscles, it also stimulate the adrenal cortex of kidney to release the aldosterone
which increases the water retention in blood, another function of the AII is to
stimulate the pituitary gland to secrete the ADH which also increase the water
reabsorption, these all factors ultimately leads to the high blood pressure. Effects
of RAAS are illustrated below in Figure 1.3 (Elased et al., 2006; Lapointe and
Rouleau, 2002).
Figure 1.3: Renin angiotensin aldosterone system (Wikipedia).
1.3.2 Problems Associated with Over Expression of ACE and
RAAS
Physiological imbalances of the RAAS may occur within the system or it may occur
by inherited or acquired mutation which can leads to the disturbance in the function
of various components. Mechanical stress to the body can also cause the changes
in related physiological systems which then can disturb the balance of RAAS
(Mason et al., 2012), different genetic factors can also cause the increase activity
of the RAAS and also the sympathetic nervous system, effect of salt on blood
pressure (salt sensitivity) can also effect RAAS. Other factors like environmental
factors, obesity, excess intake of salt in diet and sedentary (deskbound) type of
lifestyle have also impact on disbalance of RAAS (Carey, 2015). The latest Heart
7
disease and stroke statistics of 2017 by American Heart Association (AHA) were
shown that, in USA, the rate of heart failure (HF) has been increased to 0.8 million
new cases in last 5 years, It was also speculated in the report, that, the number of
patients with HF is expected to rise by 46% by 2030 (MEMBERS et al., 2017).
Traditionally, pathological upregulation of RAAS are primarily associated with
cardiovascular risk because of the increase activity of A-II.
Hypertension is the world’s most ubiquitous progressive disorder due to over
activation of RAAS; which can lead up to several other chronic diseases such as
diabetes, stroke, renal problems and most commonly cardiovascular disease. 25%
of total adult population of the world is effected by the hypertension, and it may
increase up to 29% by 2025 (Balasuriya and Rupasinghe, 2011). In many
communities of the world approximately one adult out of three have hypertension,
which is leading risk factor for disability and death (Carey, 2015).
It is been found out that RAAS is involved in progress of different conditions, from
diseases related to musculoskeletal to Alzheimer and various types of cancers
(Mason et al., 2012). In heart related problems most of the cardiovascular
diseases are related to the increased activity of ACE (Lu, Liu, and Feng, 2011).
More exposure to the cardio vascular risk factors can leads to the atherosclerosis,
left ventricular hypertrophy (LVH) or microalbuminuria. Ultimately which can lead
up to the more severe syndromes such as, myocardial infarction (MI), heart stroke
or kidney dysfunction, progressive towards damage of organs and finally leading
to heart failure, final stage of renal diseases, dementia or even death. In
industrialized developed countries these diseases are most common cause of
morbidity and death (Werner, Pöss, and Böhm, 2010). It was also reported by
Lapointe that if ACE has an increase activity in plasma then, the person has an
increased risk of myocardial infarction (MI) and various other cardiovascular
related diseases, like dilated cardiomyopathies and hypertrophic etc. (Lapointe
and Rouleau, 2002).
8
Figure 1.4: Risks associated with RAAS (Werner, Pöss, and Böhm, 2010).
Above Figure 1.4: Risks associated with RAAS (Werner, Pöss, and Böhm,
2010).Figure 1.4 showed the diseases progression in renovascular system in
lower curve and cardiovascular system in upper curve, due to over expression of
RAAS. Cardiovascular risk factors like blood pressure, diabetes, dyslipidemia, and
smoking etc. facilitate the progression of atherosclerosis, which leads to the
disease of coronary arteries, ischemia and acute coronary thrombosis, to acute
coronary syndromes and myocardial infarction (MI). Damage to the cardiac
structure over the time, decrease its contractility which then lead to the dilation of
left ventricular, which then cause the chronic heart failure and, ultimately cause
the death in early ages. Dysfunction of renal endothelial may be followed by
microalbuminuria (minor increase in the urine albumin level), macroproteinuria
(albumin level more than its upper limit), nephrotic syndrome and, finally, end-
stage renal disease (Werner, Pöss, and Böhm, 2010).
1.4 Enzyme Inhibition
In the body, most of the diseases or at least their symptoms, usually came from
the excess or deficiency of specific metabolite, it may be an influx of foreign
organism, or abnormal growth of cells. If that disturbed metabolite can be
normalized, then these diseases may be cured, and these foreign organisms or
abnormal cells may be destroyed. Most of these diseases and problems can be
countered by inhibiting the activity of specific enzymes (Silverman and Holladay,
2014).
9
Any factor or compound that can reduce the activity of an enzyme by blocking their
chemical action is called as an enzyme inhibitor. Many drugs or compounds are
enzyme inhibitors because they correct or reduce the metabolic imbalance of an
enzyme by blocking their activity. Enzyme inhibitor are also referred as enzyme
inactivator, it is a common misconception that all those compounds which can
binds with the enzymes are inhibitors; on the contrary, enzyme activators are those
compounds which binds with the enzymes and increase or facilitate their
metabolism (Balbaa and El Ashry, 2012; Silverman and Holladay, 2014).
With respect to the binding of inhibitors with enzymes, there are two main types of
inhibition; reversibIe and irreversibIe inhibition. Furthermore, the reversibIe
inhibition is classified into different types as competitive, noncompetitive and
uncompetitive or mixed inhibition, as shown in Figure 1.5.
Figure 1.5: Types and mechanism of different enzyme inhibition.
1.4.1 Reversible Inhibition
In reversible inhibition, inhibitors bind with the enzyme non-covalently (through
interaction). In this inhibition, inhibitor may change the shape of enzyme but do no
change them chemically. Inhibitors can be bind with the only enzyme, the complex
formed by enzyme and substrate, or both. This attachment of inhibitor leads
toward the various types of reversible inhibition which includes competitive,
noncompetitive and uncompetitive inhibition, sometimes a mixed type of inhibition
may also be formed. The basic pattern of inhibition can be studied by Lineweaver-
10
Burk. & Dixon plots and Michaelis-Menten equation. A graphcical illustration of
different types of reversible inhibition is shown in Figure 1.6 (Balbaa and El Ashry,
2012; Silverman and Holladay, 2014).
Figure 1.6: Graphical illustration and equations for different types of reversible inhibitions (Balbaa and El Ashry, 2012).
1.4.1.1 Competitive Inhibition
It is the most common enzyme inhibition, in this inhibition both substrate and
inhibitors compete to attach on the active site of enzyme. Competitive inhibitors
usually have similarity in structure with the substrate, therefore they can easily
bind to the substrate attaching site (active site) and blocked the substrate.
Generally, these inhibitors rapidly attain binding equilibria the target enzyme,
therefore inhibition can be observed in the start monitoring of inhibition assay.
Competitive inhibition can be reduced by using a high concentration of substrate,
because the inhibitor and substrate both cannot bind to the active site at a time,
only one of them can bind with the enzyme, therefore high concentration of
substrate is in the favor of enzyme activity.
11
1.4.1.2 Non-Competitive Inhibition
In this inhibition, inhibitor compounds bind with the enzyme to decrease its activity
but did not affect the site of substrate binding. Inhibitors interact with the enzyme
other than the site of substrate binding (allosteric binding site) which results in the
inhibition of enzyme reaction. When an inhibitor binds at the allosteric site, in
results it often produce a conformational change in the structure of enzyme, this
conformational change creates steric hindrance to substrate to binding with the
active site, therefore substrate cannot bind properly to the enzyme.
1.4.1.3 Uncompetitive Inhibition
In uncompetitive inhibition, inhibitor only binds with the enzyme-substrate complex
(E-S complex) it is also known as anti-competitive inhibition. When an enzyme
combines with its substrate, it may open up a site which was hindered before the
complex formation, inhibitors binds at that open site and formed an enzyme-
substrate-inhibitor complex (E-I-S complex), in result that enzyme cannot perform
its function which results in its inhibition. Uncompetitive inhibition usually observed
in those reactions, where 2 or more substrates or products are involved in reaction.
1.4.1.4 Mixed Inhibition
Mixed inhibition is a complex form of inhibition which is not usually observe in
enzyme kinetics. In this case inhibitor can bind with whether of the two states of
the enzyme, when enzyme is in free state and not bind with the substrate or when
enzyme has formed E-S complex, but in both states, inhibitors may have higher
affinity for either one of them. It is called as “mixed inhibition” because, theoretical
it is a mixture of both competitive and uncompetitive inhibition. If the inhibitor has
same ability to bind with the enzyme in both cases regardless of that, enzyme is
free of formed a complex, then it is called as a non-competitive inhibitor.
Sometimes, noncompetitive inhibition is considered as a distinct case of mixed
inhibition.
12
1.4.2 Irreversible Inhibition
If an inhibitor binds with the enzyme irreversibly (i.e. usually covalently) then, that
inhibition is knows as irreversible inhibition. These inhibitors usually form a
covalent bond with active side of enzyme and generally change it chemically,
however, few specific inhibitors which have tight and slow binding with enzyme
usually do not form a covalent bond with the enzyme but functionally they are
classified as irreversible inhibitor because of their tight binding. In this inhibition,
maintaining a specific concentration of inhibitor is not necessary to sustain the
enzyme-inhibitor complex, because inhibitors perform irreversible reaction (i.e.
covalently) once the inhibitor bind with the enzyme and formed the E-S complex
then it cannot dissociate (there are few exceptions). Once the complex is formed
then enzyme will remain inactive even if there is no additional inhibitor is added
(Silverman and Holladay, 2014).
1.4.3 Inhibitors of ACE
ACE plays a significant role in RAAS therefore, inhibition of ACE become a main
target to achieve the control on hypertension and its related disease and it seems
to be beneficial to fight the various cardiovascular and renal diseases (Balasuriya
and Rupasinghe, 2011; Lapointe and Rouleau, 2002). Research on finding
potential inhibitors of ACE has become expanded broadly and includes the various
classes of compounds from both synthetic and natural product derivatives.
Synthetic ACE inhibitors are the first choice and most effective therapeutics drugs
since decades, marketed available drugs such as, Lisinopril, Captopril, Rampiril
and Enalpiril, are examples of drugs used as ACE inhibitors. Research has also
been carried out to quantify the important ACE inhibitors like Quinapril, Moexipril
in human plasma (Balasuriya and Rupasinghe, 2011; Karra et al., 2012; Lu, Liu,
and Feng, 2011). However, the usage of these drugs for a long period of time can
cause antagonistic side effects like angioneurotic edema, coughing, dizziness etc.
therefore, research to explore the news drugs as a replacement of available drugs
is needed to be continue (Chen et al., 2013; Lu, Liu, and Feng, 2011).
Different classes of natural compounds, food derivatives, plant depravities and
others have been explored for their inhibitory potential against ACE. Derivatives
13
of food protein which are an important class of compounds, are explored and found
to have potential for inhibition of ACE. Food proteins derivatives can be classified
ainto 3 groups plant derived, animal derived, and microorganism derived.
Category of animal derived comprises of casein, whey protein, ovokinin which
were reported to be effective ACE inhibitors. Proteins from meat and fish were
hydrolyzed by various enzymes like chymases, etc. and resulting fractions were
screened against ACE and found to be active. Plant derived peptides from rice,
flaxseed, soybean, corn and sunflower were also founds to be active inhibitors of
ACE.
Some polyphenolic and terpenoids compounds including flavanols, flavonoids,
flavovols, isoflavones, anthocyanins, hydrolysable tannins, triterpenes etc. were
also reported to be active as natural inhibitors of ACE. Many studies have also
reported that those plants which have high phytochemicals in extracts, were also
the effective inhibitors of ACE (Balasuriya and Rupasinghe, 2011; Hsieh,
Keshishian, and Muir, 1998).
In natural sources, first important source were snake venom peptides (Menard and
Patchett, 2001). In 1965, It was reported by Ferreira (S. Ferreira, 1965) that an
extracted mixture of peptides from the venom of Bothrops jararaca snake (a pit
viper specie in South America) has the potential to stop the action of bradykininase
on bradykinin. Later, Bakhle et.al. (Bakhle, 1968; Bakhle, Reynard, and Vane,
1969) demonstrate that these peptides can also be used to inhibit the activity of
ACE to strop or reduce the production of AII from AI. After that, 9 different peptides
were isolated from this venom which were active against both bradykiniase and
ACE; among them a pentapeptide (Pro-Ala-Trp-Lys-Pyr, where Pyr is l-pyro-
glutamate) was recognized (S. H. Ferreira, Bartelt, and Greene, 1970; Silverman
and Holladay, 2014).
1.4.4 Standard Drugs or Synthetic Inhibitors of ACE
Inhibitors of ACE are classified into 3 groups on the basis of their molecular
structure, these are Dicarboxylate-containing, Sulfhydryl-containing and
Phosphonate-containing group. Dicarboxylate-containing group is the largest
group of ACE inhibitory drugs and it includes Benazepril (Lotensin), Cilazapril
14
(Inhibace), Enalapril (Renitec / Vasotec), Imidapril (Tanatril), Lisinopril (Listril /
Lopril / Novatec / Prinivil / Zestril), Perindopril (Aceon / Coversyl), Quinapril
(Accupril), Ramipril (Altace / Ramace / Ramiwin / Tritace) and Trandolapril
(Gopten / Mavik / Odrik). Other 2 groups are smaller, Sulfhydryl-containing
inhibitors group have 2 inhibitors Captopril (Capoten) and Zofenopril (Zofecard);
and Phosphonate-containing inhibitors group has only one inhibitor Fosinopril
(Monopril).
Table 1.1: Standard drugs or inhibitors of ACE.
S.No. Inhibitor Molecular weight
Structure
1 Captopril 217.283
2 Zofenopril 429.549
3 Enalapril 376.453
4 Ramipril 416.518
15
5 Quinapril 438.524
6 Perindopril 368.474
7 Lisinopril 405.495
8 Benazepril 424.497
9 Imidapril 405.451
16
10 Zofenopril 429.549
11 Trandolapril 430.545
12 Fosinopril 563.672
1.4.5 Natural Compounds, Peptides or Plant Extracts as ACE
Inhibitors
A variety of different classes of natural compounds, plant extracts and peptides
have already been screened for their potential as ACE inhibitors, few classes are
mentioned in previous section ‘1.4.3 Inhibitors of ACE’, which includes but not
limited to flavonoids, peptides, flavanols, etc. Few compounds and peptides from
natural origin, and plant extracts which were found to be active against ACE are
given below in Table 1.2, Table 1.3 and Table 1.4. IC50 values of these
compounds are not giving in this text because different compounds were screened
with different methods, which cannot give a good comparison of their potential.
17
Table 1.2: Different plant extracts as active inhibitors against ACE.
S.No. Plant Extracts
1 Hibiscus sabdariffa (Hibiscus)
2 Camelia synensis (green tea)
3 Vaccinium ashei reade (Blueberry leaf extract)
4 Vaccinium myrtillus (Bilberry)
5 Senecio inaequidens (A perennial herb)
6 S. ambiguous subsp. Ambigus (ethyl acetate extract)
7 S. ambiguous subsp. Ambigus (n-hexane extract)
8 Cryptomeria japonica (Japanese Cedar)
9 Malus domestica (Apple skin ethanol extract)
10 Actinostemma lobatum Cucurbitaceae (methanolic extract)
Plant extracts mentioned in above Table 1.2 are few examples among many
others, hundreds of plant extracts have been screened against ACE in past 20-30
years a list of plant extracts along with their activity is given in (Barbosa-Filho et
al., 2006) review article.
Table 1.3: Peptides from different food proteins as ACE inhibitors.
S.No. Source of Peptide Peptide Sequence
1 Corn gluten AY
2 Wheat IY
3 Whey KW
4 Pea, Rice, Soy FW
5 Alpha – casein FY
6 Rice TQVY
18
7 Lysozyme VAW
8 Sardine GWAP
9 Shrimps DP
10 Egg RVPSL
11 Salmon FNVPLYE
12 Chicken GAXGLXGP
13 Soybean LAIPVNKP
14 Walnut WPERPPQIP
15 Ostrich egg AFKDEDTEEVPFR
16 Squid gelatin GPLGLLGFLGPLSAPGAP
Many other peptides have been extracted and checked for their inhibitory potential
against ACE. IC50 values of above mentioned peptides in Table 1.3 and few others
are given in (Martin and Deussen, 2019) review article.
19
Table 1.4: Flavonoids and their derivates from natural sources, as active ACE inhibitors.
S.No. Group of Flavonoids
Compound Structure
1 Flavonols Quercetin-3-O-(6’’-galoyl)-galactoside
2 Flavonols Quercetin-3-α-arabinopyranoside
O
OHO
OH
O
O
HO OH
OH
OH
OH
20
3 Flavonols Quercetin-3-β-glucopyranoside
4 Flavonols Quercetin-3-O-α-(6’’’-p-coumaroylglucosyl-β-1,2-rhamnoside)
21
5 Flavonols Quercetin glucuronide
6 Flavonols Isorhamnetin-3-β-glucopyranoside
7 Flavonols Kaempferol-3-α-arabinopyranoside
22
8 Anthocyanins Delphinidin-3-O-sambubioside
O
OO
O
OH
OH
OH
HO
HO
OH
OH
HO
OH
HO
HO
9 Anthocyanins Cyanidin-3-O-sambubioside
10 Flavones Apigenin
23
11 Flavones Luteolin
12 Chalcones Butein
13 Flavan-3-ols Epicatechin-dimer
24
A comparative study of many other compounds and plant flavonoids, was
published by (Balasuriya and Rupasinghe, 2011) and (Guerrero et al., 2012). IC50
values of above mentioned compounds in Table 1.4 are also provided in these
articles.
As the search for more potent inhibitors of ACE is continue, the importance of ACE
inhibition is increasing day by day, two of the potent inhibitors which are commonly
used as drug, Captopril and Lisinopril, were used in this study as standard
inhibitors.
1.5 Drug Discovery Process
The process of drug discovery commences by selecting a target macromolecule
which can be an enzyme, cells or cellular organism etc. Activity of the target
molecule should be pathogenic in nature or at least it should be associated with
the disorder or disease. After the selection of a target, next step is to screen
against different classes of drugs or compounds to identify the leads which have
potential activity against the targeted molecules. In the last step ultimately
optimization of that lead discovery as standard drug (de Boer et al., 2007).
Enzymes are undoubtedly one of the most significant class of drug targets. In past
few years, among all the drugs, 28% of them used to target the enzymes, and
among all those small molecules drugs available in marketed, 47% of them are
purposed to inhibit the enzyme’s activity (de Boer et al., 2007). From period 2006-
2011 (6 years) the world drug market received a total of 149 new standard drugs
including 26 biologicals drugs and 123 small molecule drugs, about one-third
(51/149) of these drugs have the purpose of inhibiting the enzyme activity. Among
all those new drugs, If only peptides and small molecules drugs are considered,
then this percentage increases to 41% (51/123). In these new 51 small molecule
drugs, 14 of them were protease enzymes inhibitors (6 for cardiovascular disease,
3 for viral infection and 5 for diabetes), 12 of them were kinase enzymes inhibitors
(10 of them were used for the treatment of cancer), and 11of them inhibit those
enzymes which are involve in ribonucleic acid (RNA) or deoxyribonucleic acid
(DNA) related function or synthesis (8 of them were used for bacterial or viral
infection and 3 of them for the treatment of proliferative syndrome or cancer)
25
(Silverman and Holladay, 2014). This large proportion of drugs is because of the
important and necessary role of enzymes play pathophysiology and in regulation
of life processes.
Most common reason to use enzymes as a drug target is that many disorders and
diseases are due to the over or under expression and dysfunctional of enzymes;
by inhibition or modulating their activity these diseases can be controlled. Another
motive to concentrate on the enzymes using as drug targets is that, for the
discovery of small molecules as inhibitors is easy as enzymes are relatively easy
targets. The lead inhibitors which are screened against the target may come from
both natural and synthetic sources (Greis et al., 2006; G. Wu, Yuan, and Hodge,
2003).
Fast and robust methods are very crucial step in the process of drug discovery
and development, for the monitoring of enzyme activity and potential of inhibitor.
As the number of new natural and synthetics compounds in increasing at a rapid
rate there for the high-throughput screening (HTS) is very desired to speed up
screening process, for this purpose, new technologies may be used to develop
fast, sensitive and robust method that can broadening scopes of analysis of all
range of compounds and increase the work flow of drug discovery.
In biotechnology, pharmaceutical industries and enzymatic research communities,
chemiluminescent and fluorescent based screening assays are the first choice
since the beginning of these analyses. One reason for this choice is that these
assays are simple and homogenous, and a range of different assays can be
performed by using the same reagents; but the main challenge faced by these
types of methods is to overcome the need of high-speed analyses while reducing
the false negative and false positive readouts which is a common limitation of
these techniques.
Main challenge to fluorescence and chemiluminescence methods is the false
readouts because of the properties of different compound that can increase or
decrease the strength of the signals. Therefore, other similar methods which have
robust readout, high speed, and lower cost by minimizing the reagents which can
further reduce the false results; would be beneficial for the field of enzyme
inhibitors screening. In recent past years the mass spectrometric methods have
26
continued to advance the speed and sensitivity of assays. Mass spectrometry is
best appropriate for the determination of inhibitory potential and monitoring the
kinetics of enzymatic reactions that results in the difference of mass in product and
substrate. Opportunities to couple advance mass spectrometry techniques with
enzyme assay screening will continue to come to the forefront (Greis et al., 2006;
Kenakin, 2019; Rathore et al., 2008).
1.6 Enzyme Kinetics
1.6.1 Initial Velocity
With the direct quantification of substrate, it is possible to calculate the initiaI
veIocity of the enzymatic reaction which then used for the kinetics study of that
enzyme. In the enzymatic reaction, the concentration of the substrate decreases
rapidly in initial stages, that is when enzyme has high velocity, but after some time
the velocity of the enzyme decreases gradually, as shown in Figure 1.7.
Figure 1.7: Graph for calculation of initial velocity of enzyme reaction.
27
The initial velocity of the enzymatic reaction can be calculated from the few initial
points of percentage conversion versus time course graph by using the eq. 1.
v� = ∆�∆t (. 1)
Here, vo is the initiaI velocity of enzymatic reaction, ΔC is the difference in
concentration of either the substrate or product at a given time of interval and Δt
is the difference of time for that interval.
1.6.2 Michaelis-Menten Kinetics Vmax and Km
Michaelis-Menten kinetics is used to determine the mode of action of inhibitor on
a particular enzyme, in this kinetics a graph between initial concentration of
substrate [S] and initial velocity (vo) is plotted and inhibition mode is identified by
the different shapes of curve. In this kinetics study Vmax is the maximum initial
velocity of enzymatic reaction which is an indication of enzyme saturation by its
substrate. The Michaelis constant (Km) is that specific concentration of substrate
where enzyme reaction has half of its maximum velocity (Vmax). Km shows the
affinity of a particular substrate to the enzyme. Different types of inhibition have
different values of these kinetic parameter (Vmax and Km), different types of
inhibition are shown in the Figure 1.8 (Balbaa and El Ashry, 2012).
28
Figure 1.8: Michaelis-Menten kinetics graph and different types of inhibitions.
With comparison to the control reaction, competitive inhibitors do not change the
Vmax but increases the Km., by increasing the concentration of substrate
competitive inhibition can be reduces, because very few active sites will be
available for the inhibitors. On the contrary, the noncompetitive inhibitors decrese
the Vmax but do not change Km value, this mode of inhibition cannot be barred by
increasing the substrate concentration, because both substrate and inhibitor binds
with the different sites of the enzymes. The uncompetitive inhibitions show mixed
types of response because both the Vmax and Km values are changed in this
inhibition (Balbaa and El Ashry, 2012; Silverman and Holladay, 2014). The
inhibition pattern of inhibitors can aIso be interpreted by using the Lineweaver-
Burk & Dixon pIots.
1.6.3 Lineweaver-Burk PIot
Lineweaver-Burk plot is plotted in between the inverse of initial velocity of enzyme
reaction (1/v) and inverse of the concentration of substrate (1/[S]), in the presence
and absence of various fixed concentrations of the inhibitor. For competitive
29
inhibition the lines of different concentrations of inhibitor [I], intersect on the same
point on y-axis (1/v) which is 1/Vmax, for noncompetitive inhibition lines meet at the
same point of x-axis (1/[S]), which is -1/Km. In uncompetitive or mixed types of
inhibition lines do not intersect and remain parallel to each other with a slope of
slope=Km/Vmax (Palmer and Bonner, 2011).
Figure 1.9: Lineweaver-Burk plots for different types of inhibition.
30
1.6.4 Dixon Plot
Dixon plots for enzyme kinetic study are plotted in between inverse of the initial
velocity (1/vo) and the concentration of inhibitor [I], while different concentration of
substrate [S] is present. In Dixon pIots, the Iines of different concentrations of
substrate (which have an increasing trend along the horizontal axis) intersect each
other, the point of intersection give the information about kinetics of inhibition, if
the intersection point is between the two axes it is competitive type inhibition, if the
point of intersect is at the x-axis then this type of inhibition is noncompetitive. In
mixed types of inhibition the lines intersect on the y-axis (Palmer and Bonner,
2011).
Figure 1.10: Dixon plots for competitive and noncompetitive inhibition.
31
Chapter 2: Mass Spectrometry Techniques for
Enzyme Inhibition Analyses
32
2.1 Introduction to Mass Spectrometry
Mass spectro metry (M S) is a highly precise, reproducible and sensitive technique
which make it ia priority ifor quantitative analyses in comparison to other
techniques. Advances iin mass spectro metry have Ied it to ithe high-throughput
technique of present days as welI as its versatility in hyphenation with other
analytical techniques has made it unique. Mass spectro metry iis used iin several
fields including idrug discovery iand development process, natural product
research, food and environmental sciences, industrial processes, biological
sciences forensics etc.
Mass spectrometry is highly delicate technique, very low amount of sample as low
as in the range of femtomoles (fmol) is required for the analysis, but sample utilized
cannot be recovered as it is destructive in nature. The basic operating principle of
mass spectro metric analysis iis ithe ionization iof ithe sample, transmition of ions into
the mass anaIyzer by appIying high voItage, then separation iof the iions on the
basis of mass- t o- charge ratio (m / z) and finaIIy ithe detection of iions on detector.
Results iof the anaIyses are collated iin the iform of ia spectra, ma ss spectra is a plot
between intensities of different ions on y-axis against their m/z value on x-axis.
Most common information used by the mass spectrum is to find out the mass of
the compounds but rather than that, a lot of information can be extracted form a
mass spectrum. The mass in the spectrum is the sum of monoisotopic masses of
elements not the average masses . This information can be used to determine
the elemental composition of a compounds and thus determine chemical formula.
Fragmentation provides the essential iinformation about ithe structure iof
compounds which ican ibe used for ithe identification of molecules. While the
intensity of peaks can also be used in many different ways, one of the most
common ways is to do the quantification, relative and absolute both, by comparing
with the standard concentrations (Rockwood, Kushnir, and Clarke, 2018).
Generally, ma ss spectro meters have the following components:
1. lon source (EI, CI, DI, FAB, etc.)
2. lon guidance optics (to transfer the ions ito ithe inext istage)
33
3. Ma ss anaIyzer ( a singIe o r multiple analyzer)
4. Dete ctor (ECD)
Several types iof mass spectro meters are availabIe nowadays iwhich have
icombinations of different iionization isources, iion optics iand mass ianalyzers.
iHowever for general reference ia ischematic diagram iof ian eIectron iionization (EI)
imass spectrometer with magnetic sector anaIyzer is shown in iFigure 2.1.
Figure 2.1: GeneraI schemati c diagram of mass spectro meter (Soderberg, 2019).
2.2 Mass Spectro metry Instrumentation
2.2.1 Ion Sources
The whoIe principle of mass spectro metric anaIysis is stand upo n the ionizatio n of
analyte, since ma ss spectro meters can only detect and analyzed charged particles
(ions). The information obtained from the ma ss spec trum is depends upon ithe itype
of iionization iused. For example, electron ionization (EI) because of its high energy
can produce fragments of molecules even in some cases molecular ion totally
34
disappeared form the spectra. On the basis of how much fragmentation is
produced in the iionization process, iionization techniques are cIassified as hard
and soft techniq ues. The physicaI iand chemicaI properties iof ithe compound and
the type of information needed, determines the appropriate ionization technique.
Properties of compound which idetermine the ichoice of ionization isource included
moIecuIar weight, voIatility, poIarity and thermaI stabiIity. Ionization methods used
for method developments in this thesis are discussed as follows.
2.2.1.1 Matri x Assis ted Laser Desorption lonization
Mat rix-assis ted Iaser desorption ionization (MALDl) iis relatively similar to the fast-
atomic bombardment (F A B) in terms that it uses a matrix to assists in the ionization
process, but in FAB, ionization take place by the high energy beam of atoms; while
in MALDI the ionization process uses a high energy laser pulse. Common matrices
of MALDI included 2 , 5-dihydr oxy benzoic acid (D HB), sinapic acid, α -cyano-4-
hydro xycinnami c acid (α-CHCA) and feruIic acid. Figure 2.2 is the schematic
representation iof iionization process takes pIace in MALDl ma ss spectrometer.
Figure 2.2: Sche matic diagram of ionization source of MALDl mas s spectrometer (Szot and Croxatto, 2019).
GeneraI method ifor ithe ipreparation iof sample for MADLI anaIysis is to mixed the
analyte with a solution of matrix and spot samples on a stainless-steel plate
specialized made for sample spotting called as MALDI target plate (MTP) then
spots aIIowed to dry iover ithe isurface. A laser pulse of high energy, generally from
ia initrogen (N2) Iaser (3 3 7 nm) o r frequency tripIed and quadrupIed Nd : Y AG Iaser
35
(3 5 5 and 2 6 6 n m), irradiate the matrix surface and create localized heating which
resuIts in the abIation and desorption iof ithe imatrix along with anaIyte particIes into
the vacuum. The ihot gaseous pIume produces from the abIation process icontains
deprotonated iand protonated anaIyte moIecules which are then guided, through a
potentiaI difference, to the anaIyzer of mass spectrometer where these iions are
separated ion the basis of itheir mass-to-charge (m / z) vaIues then afterwards
detected on the detector.
FAB can be used for the analysis of those analytes which have a mass up to 10
kDa, while MALDl has ian increased range of mass ithat can be extends iup to 500
kDa. Therefore, this technique is very useful for the anaIysis of Iarge biomoIecules
such ias DNA, RNA, prot eins, synthetic polymers an d enzymes. Laser in th e
MALDI pro duces the iions iin puIsed mode therefore, it is usuaIIy equipped with
time-of-fIight (T O F) anaIyzer. This iis ibecause ia TOF anaIyzer ican iseparate
bundIes of iions ithat iare produced at a time ithrough puIsed operation iof ithe Iaser.
In ionization process of MALDI usually a transfer of protons take place between
the molecules of analyte and matrix. The selection of the matrix iis typically depend
upon the type of analyte is being analyzed, for those anaIytes which are acidic in
nature, slightly basic matrix is best choice; while matrices of acidic nature, have
their best use in general applications. Ionization process of MALDI usuaIIy
produces singIy charged ions which are most commonIy protonated i(H+) adducts
ibut isometimes sodium (Na+) iand ipotassium (K+) adducts ican ialso ibe formed (Lee
et al., 2016).
2.2.1.2 Smartbeam Technology
This work iwas iperformed ion the Bruker UItrafIex-IlI, MALDl-T O F/ T O F mass
ispectrοmeter which is iequipped iwith smartbeam Iaser itechnology. Smartbeam
Iaser technοlogy provides significant enhancement in performance of MALDI-MS.
iSmartbeam icοmbines ithe ispeed iof a sοIid-state-Iaser with ithe iwide irange iοf
appIicatiοns iassοciated iwith nitrοgen Iasers. iThe use iof cοnventionaI iNd:YAG
(neodymium-doped i yttrium i aluminum i garnet; i Nd:Y3AI5O12) Iasers is irestricted
itο few sampIe types but ismartbeam iprοvides exceIIent iresults frοm aII imatrices
36
iand aII sample ipreparation imethοds. A comparative spectra of conventional and
smartbeam source is given below in Figure 2.3 (Holle and Ketterlinus, 2008).
Figure 2.3: Comparasion of Nd:YAG laser and smartbeam spectra (Holle and Ketterlinus, 2008).
Smartbeam use the ifrequency-tripIed iNd:YAG Iaser, iat i3 5 5 n m and a istructured
ifοcus prοfiIe, i‘Nd:YAG i(structured iA) and iNd:YAG i(structured iB), it allows
iswitching iamοng iN2, iNd:YAG i(Gaussian) & iNd:YAG i(structured iA ior iB) ilasers
iwithin isecοnds, schematics of Smartbeam setup are shown below in Figure 2.4
(Holle et al., 2006).
Figure 2.4: Smartbeam setup (Holle et al., 2006).
37
Smartbeam used in the instrument had following characteristics: Po ≤ 30 mW, Pp
≤ 30 kW, t = 5 ns -20 ns, F = 200 Hz (single impulse), λ = 990-1080,808,495-
540,330-360 nm
2.2.1.3 EIectrospray lonization
EIectrospray ionization (ESl) is an atmospheric ipressure iionization (A PI)
techniqu e, it is used for the wide range of analyses and various types of analytes.
It was 1st deveIoped by Masamichi Yamashita and John Fenn in 1984 (Yamashita
and Fenn, 1984). ESl comprised of process, in which anaIyte particIes are
transferred from soIution phase to th e gas phas e through soIvent or i on
evaporation (Somogyi, 2008). ESI is a soft ionization technique it usualIy produces
very Iittle or no fragmentation and it can be very easily hyphenated with Iiquid
chromatographic techniques. Among other advantages a major benefit of ESl is,
the generation of ions with multiple charge, which decrease the m/z of ions
therefore, increases the mass range of the anaIyzer and makes it suitable for the
anaIysis of molecules with very high moIecuIar weight.
Process of ionization needs dissolution of analyte in poIar solvent, then soIution is
nebuIized through spray from a capiIIary that is kept at a very high potentiaI with
a capilIary voItages of 3 to 6 kV. The dropIets of Iiquid attain charges from the high
voltage, when desolvation process take place in the droplets, solvent is removed
from the droplets by the action of ihot icurrent iof drying gas. Because iof this drying
iprocess droplets starts to ishrink which increase the columbic repulsion between
ions. Droplets shrunk up to some extent after that when surface itension iof ithe
soIvent ican no Ionger abIe to hoId the couIombic repuIsions and iits reached at its
Iimit which is called as Rayleigh limit (Rayleigh, 1882). After that, shrunk dropIets
burst and reIease the ions in the iionization ichamber which are then moved to the
anaIyzer region by applying a potential difference. iFigure 2.5 demonstrate the
process of desolvation and ionization in ESl.
38
Figure 2.5: Process of desoIvation and ionization in ESl-MS (IPFS).
In hyphenation of liquid chromatography (LC) with mass spectrometry, ESI-MS is
given the priority, and this hyph enation make s it a hi gh-throughpu t te chnique.
Ma ss spectro meters utiIizing the ESl source are generally equipped with a
combination of mass anaIyzers; the most common combinations are quadrupoIe
time-of-fIight (Q qT OF) and triple quadrupole (QqQ). These mass anaIyzers
enabIe sensitive quantitat ion of differen t anaIytes in compIex sampIes (Cífková et
al., 2015; Mirzaian et al., 2017), iidentification of inatural products from pIant
extracts (Abu-Reidah et al., 2015; Allen, Greiner, and Wishart, 2015) and
screening of enzymatic assay against drug libraries by using absolute
quantification of substrate and peptides (Cui, Nithipatikom, and Campbell, 2007).
Formation of multipIy charged iions in ESl make iit possible ito analyze the large
molecules like nucleic acids and proteins on quadrupoIes whose imass range iis iup
to 2 0 0 0 D a (Percy et al., 2016; Ruhaak, 2017).
2.2.1.4 Agilent Jet Stream Ion Source
Jet Stream iοn sοurce with thermaI igradient ifοcusing technοIogy iby AgiIent,
οptimizes iESI iconditions to iproduce significant igains in isensitivity, idecreasing
sampIe isize, iincreasing sampIe ithrοughput, iimprοve irοbustness iand ireduce ithe
LOD i& iLOQ of iscreening and quantitatiοn iappIicatiοns iby imprοving ithe
39
desοIvation and ispatiaI fοcusing of iiοns Figure 2.6. Superheated initrοgen isheath
igas icοnfines ithe nebuIizer ispray itο imοre effectiveIy idry iiοns iand cοncentrate ithem
in ia thermaI icοnfinement zοne. idesοIvation reduces inοise in ispectra Figure 2.7
(Mordehai and Fjeldsted, 2009).
Figure 2.6: Jet stream ionization source working principle (Mordehai and Fjeldsted, 2009).
Figure 2.7: Thermal profile of jet stream source showing confinement of zoning by super-heated nitrogen (Mordehai and Fjeldsted, 2009).
40
FuII cοnfinement οf the ispray iby ithe isuperheated nitrοgen igas eIiminates the
sampIe recircuIatiοn and ireduces ithe ipeak taiIing. It iimproves the iprοductiοn iοf
iοns which resuIts iin ihigher intensity οf peaks in spectra and improved signal-to-
noise ratios. Jet stream source can improve the MS and MS/MS spectra intensity
5 to 10-fold, Figure 2.8 (Mordehai and Fjeldsted, 2009).
Figure 2.8: Comparison of spectra using conventional ESI and jet stream technology (Mordehai and Fjeldsted, 2009).
2.2.2 Mass Analyzers
The mass analyzer is the main component of any mass spectrometer that perform
the function of separation of ions, on the basis of their mass-to-charge (m/z) ratio.
Different kinds of mass analyzers have different operating principles, as an
example, the magnetic sector, oldest mass analyzer, works upon the principle of
magnetic deflection, the ions deflects as they travel through it and detect on the
detector. Separation is based on how much an ion is deflected under the applied
magnetic field. There are six types of mass analyzers as listed below.
1. Electrostatic sector (E)
2. Magnetic sector (B)
41
3. Quadrupole (Q)
4. Time-of-flight (TOF)
5. Ion traps including the orbitrap
6. Ion cyclotron resonance (ICR)
However, only those analyzers are discussed here which are used in this study.
2.2.2.1 Quadruple Mass Analyzer
A quadrupole (q) mass analyzer is comprised of four metallic rods, aligned in a
parallel manner. The two of opposite facing rods are connected to a direct current
(DC) source and other two are connected to the radiofrequency (RF) which is at
an offset to the DC voltage. The quadrupole works as an tunnel for ion-
transmission where, at specific DC and RF voltage, only selected ions with certain
mass-to-ratio (m/z) can pass by forming a stable trajectory, while other ions which
have unstable trajectories, cannot pass through and are destroyed by colliding
with the quadrupole rods. The working principle of a quadrupole mass analyzer is
illustrated in the Figure 2.9.
Figure 2.9: Transmission of different ions through quadrupole mass analyser (Khan, 2019).
42
The quadrupole mass analyzer is the most typical type of analyzer available in
low-cost mass spectrometers because if its robustness, ease of manufacturing,
and fast speed of scanning. The speed of quadrupole mass analyzer is compatible
with fast liquid chromatography separations which can produce peak narrower
than 5s. The quadruple mass analyser has low-resolution unlike the magnetic
sector and time-of-flight (TOF) analyser which have high resolution capacity. It is
therefore impractical to deduce the exact molecular weight and formulas by using
the data generated from a quadrupole instrument, instead, quadruples have
proved to be best in quantification studies.
2.2.2.2 Time-of-Flight Mass Analyzer
Time-of-flight (TOF) mass analyzer operates on the principle that, ions with
different molecular weight but same values of kinetic energy will have different
velocities in a field free region. In TOF, packet of ions produced in the ionization
chamber is accelerated to a specific kinetic energy and moved to the TOF analyzer
where heavier ions have slower speeds as compared to the lighter ions. Those
ions which have samller m/z values will reach the detector first followed by the
ions which have higher m/z values. Figure 2.10 shows the separation of ions
based on their difference in velocities in a TOF mass analyzer.
Figure 2.10: Ions separation as a function of time in a TOF analyzer.
43
TOF has a high resolution; newer TOF analyzers can reach up to the resolution of
80,000. Different makers use different variations in TOF analyzers; one of the
advantageous variations is using of reflectrons to increase the travel distance of
ions two or more times, the longer the ions travel, the greater will be the resolution.
Some manufacturers use longer path TOF tubes to avoid the use of reflectrons
which may reduces the cost of manufacturing.
One limition of the TOF analyzer is that, it cannot work in a mode where ions are
produces continuously, it can only be used with the source which produce the ions
in packets. it will become very difficult to monitor the time of flight of different ions
if ions are continuously injected into the analyzer. Therefore, TOF is most
compatible with MALDI instrument where the ionization source produces ions in a
discontinuous manner.
Unlike the quadrupole mass analyzer, a TOF analyzer is unable to select a specific
ion of certain m/z value, all ions will pass and rech the detector or next phase
ultimately. Therefore, TOF instruments are unable to work under some specific
modes such as the multiple reaction monitoring (MRM), but QTOF and QqTOF
are the tandem techniques which are generally used. TOF are not well suited for
quantitation purposes but can give high accurate mass which can be useful for
determination of composition and structure of an analyte.
2.2.3 Detectors
Detector is the final part of a mass instrument, ions produced in the ion source
then separated by the mass analyzer finally reached at the detector and generate
the signal which then converted into the data. The purpose of the detector is to
produce a signal in the form of electric current which corresponds to the
abundance of ions hitting the detector. This information later converted into useful
data in the form of a mass spectra. If the ion intensity of an analyte is very low,
then amplification can be used on detectors.
Choice of detector is dependent upon the intended application and design of the
instrument. The detection of ions is based on mass, charge and velocity of ions.
Different detectors detect ions in different manner; for example, Faraday cups
44
detectors record the current generated directly when an ion hits the detector
surface, while electron multipliers detector record an amplified current that is
produced due to secondary electrons generated in the amplification process.
Detection principle in Fourier-transform ion cyclotron resonance (FTICR) mass
spectrometers is entirely different from all other types, because in FTICR there is
no need of a separate detector, but the detection takes place in form of induced
current when ions within the mass analyzer pass close to the electrodes.
2.3 Tandem Mass Spectrometry
Tandem mass spectrometry is the combination of at least two stages of mass
analysis by the combination of two or more mass analyzers. Such types of
instrument are known as hybrid instruments and quadrupoIe time-of-fIight
(Q q T OF) and tripIe quadrupoIes (Q qQ) are examples of it. Trapping instruments
works in different way, they only have one analyzer, yet ithey ican pe rform tan dem
ma ss spectro metry e xperiments. Based on how mass anaIysis take pIace in,
tandem mass spectrometry is classified into two types; tandem- in -space and
tandem- in -time.
2.3.1 Tandem-in-space Mass Spectrometry
During tandem-in-space, ions traveI some distance in phases of selectin,
fragmentation and detection, it is usually observed in the sect or, quadrupIe and
T O F mass analyzers. TripIe quadrupoIes is the most com mon combination of
mass anaIyzer in hybrid instruments and it provides the most variety of scan types.
2.3.2 MS/MS by TripIe QuadrupoIes
Triple quadrupole is comprised of three quadrupoIes, in which first and last
quadrupoIes are used for selection or scanning on ions while the middle
quadrupole serves as the colIision ceIl where ion fragmentation takes place. A
schematic diagram of a tripIe quadrupoIe is shown below in Figure 2.11. Collision
energy required for the fragmentation of ions is determined by the kinetic energy
of ions arriving from the first quadrupoIe, if there is no requirement of
45
fragmentation it ican ialso iwork ias ia transmission device. Diffe rent working modes
of tripIe quadrupoIes are summarized as follows:
1. Produc t-io n sc an
2. Precurso r-i on s can
3. SeIected i on mo nitoring (SIM)
4. SingIe and muItiple reaction monitoring (SRM / MRM)
Figure 2.11: Sch ematic of a tripIe quadrupoIe ma ss spectro meter (Jackson, 2019).
2.3.2.1 Product -lon Scan
In product-ion-scanning, the Q1 is set at specific m/z value to allow only certain
ions (within the toIerance window) can pass through, which are th en fragment ed
in the reaction ceIl q2 and finally the product io ns formed in the resuIt of
fragmentation, are anaIyzed in the Q3, which is set ito iscan a ispecific irange of m/z
values. This type of scan iis used to idetermine which ifragments resuIt from ia
cert ain prec ursor iion and iis used for dete rmination iof fragm entation ipatterns of
icompounds.
2.3.2.2 Prec ursor-lon Scan
Precursor-ion scan is reverse of product-ion-scan, in this type of scan, Q 1 is set
to scan a specific range of m/z values, q 2 is set to make f fragments and Q 3 is set
on a specific m/z value. A signaI wilI generate only when a precursor ion from Q 1
46
produces a frag ment at which Q 3 is par ked. It can also ibe iused ifor the istudy of
ifragmentation pattern.
2.3.2.3 SeIected-lon Monitoring
In seIected-ion monitoring (SlM)., both Q 1 and q 2 are turned off and onIy serve to
transmit the ions to Q 3, while Q 3 scans over the set range of m / z vaIues. This type
of scan is generaIly used for quanti tative anaIysis in a simpIe mixture, where it can
give good sens itivity. In the case of Iarge and compIex mix tures, there is a strong
possibiIity to observe iso baric ions that eIute at the same reten tion times.
2.3.2.4 SingIe and MuItiple Reaction Monitoring
Single or selected reaction monitoring (SRM) is a neutral loss scan. In this
scanning both Q1 and Q3 are set on a specific m/z values and q2 is served as
reaction cell for fragmentation. Ions which passes through the Q1 are precursor
ions and in Q3 these are fragment ions. Before setting the values in Q1 and Q3, a
SIM for high intensity of precursor ion and product ion scan for fragments can be
done to select the ions which have highest intensity to increase the sensitivity of
analysis.
MuItiple reaction monitoring (MRM) is just two or more SRMs taking pIace
simuItaneously. MRM can anaIyze two or more compounds in the same
experiment as welI as in M R M two or more product i ons from a singIe precurs or
ion can also be anaIysed. In M R M experiment, Q 1 and Q 3 do not scan the whoIe
range of m / z; instead they jump from one m / z vaIue to another. Jumping instead
of scann ing enhances the duty cycle of the instrument a nd increas es t he
se nsitivity.
2.3.3 MS / MS by Qq TOF Sy stems
QuadrupoIe-time-of-fIight hybrid syste ms are different from the tripIe quadrupoIe
systems iin the way ithat, Q 3 iis repIaced by a T OF mass anaIyzer. T OF has very
high resoIution scanning capabiIities, a Qq T OF system ca n record high resoIution
mass spectra of precursor iions when no fragmentation iis performed and iof product
ions iwhen fragmentation iis performed.
47
These iinstruments are of great vaIue when structuraI iinformation iis required
through fragmentation istudies. UsuaIIy, a TO F scan is performed first iwhere Q 1
and q 2 iare not scanning and onIy act as itransmission devices and ithe T OF
anaIyzer gives ithe exact masses of aII produced iions. In the MS/MS scan the
quadrupoIe iis used to seIectiveIy fiIter iions of iinterest which are then ifragmented
inside q 2 and the product ions iare transmitted to the TOF region where they are
separated. QqT OF systems can record mass spectra at significantIy higher
resoIution than the Q qQ systems but are unabIe to perform M RM scans as the
T OF anaIyzer is fieId-free and cannot perform ion seIection.
2.3.4 Tandem-in-time Mass Spectrometry
ln tandem-in-time mass spectrometry, iions do not traveI through different mass
anaIyzers, instead, they remain iconfined iin a singIe mass anaIyzer during aII the
stages iof mass spectrometry. lt is usuaIIy performed iin trapping iinstruments and
does not require a hybrid iinstrument; as a singIe ion itrap can store, fragment iand
separate iions. Different types of mass anaIyzers used iin tandem-in-time mass
spectrometry are:
1. QuadrupoIe lon Trap (PauI Tr ap)
2. Orbi trap
3. Penn ing Trap (us ed in FTlCR-M S)
lnstruments equipped with ion traps are not Iimited to M S 2 or M S 3 as the Qq Q and
Qq TOF systems but are abIe to perform muItipIe stages of ion s eparation and
fragmentation. Modern instru ments can go as high as MS 1 1. Such instruments can
be used to extract vaIuabIe structuraI infor mation.
2.3.5 Fragmentation and Ion Activation
In tandem mass spectrometry, fragmentation is an important step. Hybrid
instrum ents have coIIision / reaction ceIIs for i on fragment ation whiIe an iion trap
does inot require a iseparate reaction ceIl iand can perform ifragmentation iitself.
Different methods iof fragmentation are iused incIuding iln-source iFragmentation,
CoIlisionaI-induced iDissociation (Sleno and Volmer, 2004), which can be iLow
energy ClD and High Energy ClD (Sleno and Volmer, 2004).
48
There are other dissociation techniques like; EIectron Cap ture Disso ciation (E C D),
EIectron T ransfer Disso ciation (E T D), Photodissociation (PD) (Mistarz et al.,
2018), Surface Induced Dissociation (SID) (Yan et al., 2017), lnfrared MuItiphoton
Disso ciation (IR M PD) (Brodbelt and Wilson, 2009) and BIackbody lnfrared
Radiativ e Disso ciation (BI RD) (Dunbar, 2004; Sleno and Volmer, 2004).
2.4 Mass Spectrometry for Enzyme Assay and Inhibitors
Screening
Applications of mass spectrometry have been developing and expanding since
last two decades due to the advancement and development of instrumentation,
mass spectrometry have many advantages for the fieId of inhibitor screening. The
main reason iis the abiIity to perform iscreening assays in a IabeI-free mode. iIn
contrast to uItra vioIet, fIuorescence and iradioactivity assays, detection iof
enzyme-inhibitor iinteractions does not irequire IabeIing but reIies entireIy on ithe
mass-to-charge ratio iof reporter moIecuIes. This simpIifies adjusting the iassays,
which are no Ionger restrained by the ineed to design and synthesize IabeIed
compounds. LabeI-free assays aIIow Iaborious, cumbersome iand costIy
derivatization ito ibe ibypassed (de Boer et al., 2007).
In mass spectrometry it is possible all types of compounds compared to only one
if label-based detection like fIuorescence is used. It means that, by applying mass
spectrometric method, all components of an enzyme assay (e .g., subs trate,
products, cofa ctors, coenzymes, and internaI standar ds) can be us ed t o monitor
the reIative enzyme activity or inhibition (Liesener et al., 2005). Furthermore, the
identification and structural determination can also be done at the same time by
using the accurate molecular mass of the inhibitor by T OF-MS or FT-lCR-MS) in
combination with fragm entation (M S / M S) experiments. Finally, the main
disadvantages associated with the fIuorescence based assays is the false
enhancement or decrease of results by means of auto fIuorescence or light
scattering o f the compound s or matrix, and quench ing; which do not exist for MS.
This may reduce the faIse positives and faIse negatives resuIts (de Boer et al.,
2007).
49
MS readout aIso offers a significant advantage. For exampIe, the reagent cost for
a MAIDI-Qq Q readout is onIy ~$ 0.0 3 per weII for the buffer and the unIabeIed
peptide substrate compared with $ 0. 60 per weII for A D P accumuIation assay.
AIthough this may not seem aII that significant when measuring onIy a few test
vaIidation sampIes, when scaIing up to evaIuate a compound repository of
10 0,000 compounds, the reagent cost difference becomes $ 3000 for the MS
versus $ 6 0,0 00 for the fIuorescent readout. At this rate of saving in reagent costs
aIone, the investment cost for the mass spectrometer ($ 3 50,000 to $ 45 0,000)
couId be recovered after onIy a short time by a productive screening group. Thus,
with comparabIe readout speeds, better signaI-to-background ratios, a significant
reagent cost advantage, and the IikeIihood of fewer faIse-positive or faIse-
negative resuIts, the MALDl-Qq Q readout represents a major step forward for the
H TS readout of isoIated enzyme reaction (Rathore et al., 2008).
2.5 Methods of Mass Spectrometry for Enzyme Assay
Screening
The Mass Spectro metry based screening methods are not only Iimited to the
analysis of enzyme-inhibitor reactions, but they can be used for receptor-ligand
too. Receptors are those proteins which are present on celIs or within the
cytopIasm or ceIl nucIeus that bind to receptors (specific compounds). Enzymes
are the analogues of receptors and inhibitors are analogues of ligand. In this thesis
research is being done by using enzyme and inhibitors. Since there is a strong
overIap in the methodologies used for both enzymes and receptors screening;
methodologies which are discussed further can be used for both types of target.
The mass spectrometric methods used for the screening of enzyme can be
cIassified according to differ ent criteria, such as the drug target, type of assay,
physicaI use of drug targets, the type of sampIe/inhibitors screened, or the type of
mass spectrometric techniques. ln further discussion, described methodoIogies
are categorized on the basis of detection principIe i.e. which component of the
enzyme reaction is to be detected. Three categories of mass spectrometry based
screening are also shown in Figure 2.12.
50
Figure 2.12: Different types of mass spectrometry based screening assay (de Boer et al., 2007).
Based on which component is detected in enzyme assay; screening is categorized
into three methods i.e. detection of enzyme inhibitor complex, detection of inhibitor
and detection of reporter molecule, substrate or product, among them first and
second are the direct approach to measure the enzyme reaction while third one is
indirect way.
Direct screening methods detect ithe active icompound(s) either as iintact enzyme -
inhibitor compIex or iafter dissociation iof the enzyme - inhibitor compIex. In
detection of enzyme-inhibitor complex, mass spectrometer detects the cumulative
mass of enzyme with inhibitor and detect the inhibitor based on mass difference.
In detection of inhibitor it only determines the mass of inhibitor after the
dis sociation of enzyme - inhibitor compIex, in this scre ening method separation of
bounded compounds from the unbounded is necessary to differentiate the active
compounds from the inactive. For indirect screening methods, substrate or product
of enzymes are used as the reporter moIecuIes to measure the enzymatic activity.
Usually in indirect screening methods it is not needed to separate the inhibitor or
reporter molecule from the assay soIution after incubation, they can directly
analyze by choosing appropriate reporter molecules, but a separation step can
51
enhance the sensitivity. In most cases, the concentration of the reporter moIecuIe
is directIy reIated to the activity of enzyme. In most of the enzyme assays, it is
easy to detect the products formed in the enzyme reaction; in those case, a
decrease in the concentration of product or increase in the substrate at a given
time can give the signal of inhibition of enzyme (de Boer et al., 2007).
Various mass spectrometric techniques used for above describes methods of
detection are used, which are describes in the Figure 2.13. each technique has
its pros and cons, advantages over the other. Over the time as the instrumentation
is improved in speed and sensitivity more reliable and accurate methods are
developed for the enzyme assay screening.
Figure 2.13: Different techniques of mass spectrometry used for enzyme assay screening.
2.5.1 Direct Infusion-Mass Spectrometry (DI-MS)
This technique of enzyme inhibitor complex is very simple, it was made easy by
the using of electrospray ionization (ESI). Analysis of high molecular weight
compounds in range of KDa like enzyme is relatively easy on ESI due to
52
accommodation of multiple charges on the substance. In this technique the
inhibitor or mixture of inhibitors incubate with the enzyme and after that it subjected
to the MS analysis, the mass spectrum showed the multiple charged enzyme -
inhibitor compIexes, which demons trate the bind ing betwe en inhibitor a nd
enzyme, an ESI spectra of enzyme inhibitor complexes is shown in Figure 2.14
(Baca and Kent, 1992; Stevenson, Feng, and Storer, 1990).
Figure 2.14: Direct Ionization mass spectrometry spectra of enzyme inhibitor complexes (Stokvis et al., 2000).
The direct detection method can also be used to determine the relative binding
affinities of inhibitors by using ESI-MS. Stokvis et al. demonstrate the reliability of
MS detection for enzyme-inhibitor complexes and determination of binding
affinities of inhibitors against carbonic anhydrase. In that methods, screening was
much faster and required very less reagents than spectrophotometric analyses.
Moreover, this MS based method could be used when concentration of inhibitor is
high, which is unfavorable for spectrophotometric analyses (Stokvis et al., 2000).
Direct screening method of enzyme-inhibitor comples, is not only limited for the
analysis of individual compounds but it can also be used for complex samples
(Sannes-Lowery et al., 2004) or compound libraries. In an example demonstrated
by Gao et al. that, a library of 28 9 pep tides was incub ated with carb onic an hydrase
II enzyme and then, that mix ture was anaIyzed by eIectro spray ioniza tion fo urier
trans form i on cycIotron reso nance ma ss spectro metry (ESl-F T-lCR-M S). Because
of high resolution, F T-lCR- M S can isoIate and accumuIate enzyme-inhibitor
compIexes. This method can also used for the both bound and unbound analysis,
53
after dissociation of enzyme-inhibitor complexe unbound compounds can be
detected and by comparing the relative abundances of the detected inhibitors with
the abundances of the compIexes, binding constants of the inhibitors can be
calculated Figure 2.15. The correIation between abundance and binding
constants can be made onIy if the ionization efficiencies of aIl compIexes are
simiIar (J. Gao et al., 1996).
Figure 2.15: ESl-FT-lCR-MS screening o f library of peptides (J. Gao et al., 1996).
54
This technique is advantageous that it can be used to detect all the components
of an enzyme reaction, the ratio of dissociate to undissociated can be used to find
the binding efficiency of inhibitors. High resolution of FTICR make it possible to
detect closely mass peptides and complexes. While on the other hand this
technique has low tolerance for the high concentration of buffers, salts,
surfactants, co-factors, etc., in this case sensitivity may be increase by using some
pretreatment methods. The detection of inhibitors has advantage over the
detection of enzyme-inhibitor complex, beca use in enzyme-inhibitor complex due
to very high mass and multiple charges the difference in the mass will be very
small.
2.5.2 Ultrafiltration-Ma ss Spectro metry (UF-M S)
Enzymes and inhibitors have huge differencea in their size, ultrafiltration-mass
spectrometry (UF-MS) use this difference in the size to perform the analysis. UF-
MS utilize a fIow- through vessel for perfoming enzyme assay, that vesseI contain
an uItrafiItration mem brane with a defined molecuIar-w eight cut- off. The
membrane traps the enzyme moIecules or enzyme inhibitor compIex but aIIows
the Iow moIecular weight compounds to pass through, as shown in Figure 2.16.
During enzyme assay screening, the target enzyme is incubated with the different
inhibitors in the mixing chamber, after incubation its components are separated by
the UF membrane. The enzyme-inhibitor complexes or free enzymes are retained
of the UF, while the unbound compounds are removed by washing the retentate.
After the washing step, the complexes are dissociated by using an organic modifier
or by changing the pH of solution, or by using a combination of both. Released
inhibitors are then transported through the membrane and analyzed by mass
spectrometer, by comparing the spectra of all inhibitors before and after the
incubation, inhibitors which bound with the enzymes can easily be identified
(Johnson, Nikolic, and van Breemen, 2002; van Breemen et al., 1997; Youngquist
et al., 1995; Zhao et al., 1997).
55
Figure 2.16: Ultrafiltration process in enzymatic assays (de Boer et al., 2007).
UF-MS techniq ues ca n b e us ed for the compIex mix tures like extracts of plants or
microorganism (Zhang et al., 2004), as shown by the Wang et al. in Figure 2.17,
that, ultrasonic extract of herb Scutellaria baicalensis was incubated with α-
Glucosidase enzyme which is a contributing factor to diabetic mellitus type-II, then
unbound compounds were separated by the mixture in a cartridge filter contacting
UF membrane by using the centrifugation, after the washing, compounds were
released by using a organic solvent and released compounds were analyzed on
ultrafiltration liquid chromatography with photodiode array detection coupled to
electrospray ionization tandem mass spectrometry (UF-LC-PDA-ESI/MS) (J.
Wang et al., 2014).
Figure 2.17: α-Glucosidase inhibitory assay with S. baicalensis extract using UF-LC-PDA-ESI/MS (J. Wang et al., 2014).
56
By using the HPLC prior to MS can make a screening assay more sensitive,
selective and efficient as well ionization suppression in the MS can also reduce by
HPLC. Dissociated inhibitors cane be separated before injected into the MS. In
addition, the HPLC.
In another example Nikolic et al. demonstrate the potential of UF-MS for screening
of natural products. The fermentation-broth extracts and plant extracts along with
standard inhibitors were screened against COX-2 enzyme. The incubation mixture
was injected into the UF chamber with a 30,000-MW cut-off membrane and
washed with water. After that, bonded inhibitors were released by lowering the pH,
then released inhibitors were separated by RP-HPLC and analyzed by MS.
Results showed that three known COX-2 standard inhibitors (ibuprofen, dicIofenac
and fIurbiprofen), labeled 1–3 in Figure 2.18, were present in the spectra along
with peaks of other inhibitors from the plant and broth extracts. Presence of
standard inhibitors in results validated the method and proved the efficiency of UF-
MS even in complex matrices. Besides, the authors showed that extensive
washing wiIl resuIt, as expected, in the Ioss of compounds with weaker affinity
(Nikolic et al., 2000).
Figure 2.18: UF-MS analysis of COX-II inhibitory assay (Nikolic et al., 2000).
Ultrafiltration can also be used with MALDI-TOF-MS. UF is advantageous over the
direct infusion techniques because it uses washing step which can remove the
salts and buffer from the mixture. In UF technique enzymes can be reuse for
multiple cycle, however, if extensive washing is used, may degrade the enzyme
activity. UF does not required the immobilization of enzyme, but if enzyme got
stuck inside the UF membrane then it can be problematic.
57
2.5.3 Size-Exclusion Chromatography-Mass Spectrometry (SEC-
MS)
Size-excIusion chromatograph (S EC) aIso use the advantage of difference in the
size of enzyme and its inhibitors, in SCE enzyme-inhibitor compIex are separated
from the unbound compounds by using a coIumn. In S EC-M S screening is
performed by incubating the enzyme with inhibitors and injecting this mixture into
a size-excIusion coIumn to separate the enzyme-inhibitor compIexes from the
unbound compounds i.e. separation of high moIecuIar weight components from
Iow moIecuIar weight on the basis of different retention voIumes. SEC work on the
principIe that, moIecuIes Iarger than the pore sizes of the coIumn materiaI cannot
enter the pores therefore they eIute together in first, whiIe the smaIIer compounds
which can penetrate into the pores wiII eIute Iater. The earIy eIuting enzyme-
inhibitor compIexes are isoIated, dissociated by using change in pH or organic
modifier, or temperature change, or a combination of these parameters and eIuent
is finaIIy anaIyzed by the mass spectrometry (Mathur et al., 2005).
In an example, Annis et al. used online hyphenated SEC-MS, in which enzyme-
inhibitor complex after passing through SEC were trapped on HPLC column, and
after washing and dissociation, analyzed by HPLC-MS. With this method, a library
of 2500 compounds were screened against the enzyme Esche richia co Ii
dihydro foIate reduct ase, within 1 0 minutes. Rapid separation of SEC make it
possibIe to anaIyzed the even weakIy -bounded inhibitors with moderat e
dissociation rates and to identified them as possibIe Iead structures for inhibition
of target enzyme (Annis et al., 2004).
58
Figure 2.19: Fast SEC-MS of a library of compounds and identification on MS/MS basis (Annis et al., 2004).
In the above Figure 2.19 accurate masses and MS/MS information is provided,
which resulted in the identification of a various active compound. Spectra A shows
the EIC of an inhibitor with m/z 515.24. Spectra B is the EIC at m/z 515.24 in a
blank experiment, where no inhibitor or library compounds was used, therefore,
contained no peaks at specified m/z. When enzymatic assay was performed by
SEC-MS/MS, enzyme was incubated with the library, complex was separated,
unbounded and analyzed in MS/MS. Chromatogram at C showed the presence of
compounds at m/z 515.24 and when subjected to MS/MS it results in mass spectra
showed at D. To identify and verify the structure of the compound with m/z 515.24,
different position isomers were prepared independently, and their MS/MS
experiments were carried out, spectra of two of the isomers are shown in the E &
F. By comparing the MS/MS spectra of compound at D with the isomers it was
confirmed that this compound is similar to sub-library.
Many researchers have shown the potential of SEC-MS by adding different
modifications and techniques (Davis, Anderegg, and Blanchard, 1999;
Dunayevskiy et al., 1997; Moy et al., 2001; Siegel et al., 1998; Wabnitz and Loo,
2002) like, use of spin coIumns, which uses the centrifuge to perform the
59
separation step. The use of spin column in 96-well plate format by
(Muckenschnabel et al., 2004) increase the assay speed for screening up to
85,000 compounds per day. SEC-MS is advantageous in a way that all the
components of enzymatic reaction can be analyzed by this technique as well as
the comparative analysis is also possible by comparing the free ligands with the
initial spectra of library. In SEC equilibrium dissociation may cause loss of some
ligands in column.
2.5.4 Immobilized Enzyme-Mass Spectrometry (IE-MS)
Enzyme immobilization means that the enzyme molecule is attached to a solid
surface without losing their functionality and attachment should be tight enough to
prevent dissociation of the enzymes from the surface (Sirisha, Jain, and Jain,
2016). Enzyme immobilization can be defined as, restricting the enzyme
molecules to a solid matrix/support different from the one in which substrate or the
products are present. It is achieved by attaching the enzymes to or within some
suitable support material. It is important to note that the substrate molecules and
the products formed should move freely in and out of the phase to which the
enzymes are restrained. Various materials can be used as matrix or support
system for enzyme immobilization, generally which are grouped into three
categories Natural polymers, synthetic polymers and inorganic materials.
Figure 2.20: Covalently immobilization of enzyme with the resin surface (Sirisha, Jain, and Jain, 2016).
60
Based on support or matrix used for immobilization and type of bond involved in
attachment of enzymes, there are different methods of enzyme immobilization and
various factors impact the immobilized enzymes performance. The different
enzyme immobilization methods are shown in Figure 2.21, and are listed below
(Sirisha, Jain, and Jain, 2016):
• Adsorption / carrier-binding method
• Covalent bonding / cross-linking
• Entrapment method
• Encapsulation / membrane confinement
• Copolymerization
Figure 2.21: Enzyme immobilization methods (Sirisha, Jain, and Jain, 2016).
Cancilla et al. (Cancilla et al., 2000) demonstra ted an other appIication of
immobiIized enzymes, th at is, detect ion of non -active un bound compounds using
ESI-FT-ICR-MS. First, a library of 19 compounds was analyzed by MS and spectra
61
was obtained as shown in Figure 2.22 A, followed by the incubation of library with
ian iexcess iof iimmobiIized ipepsin enzyme, after that, anaIysis of ithe iunbound
Iibrary members was carried out by MS which results in spectra B in Figure 2.22.
PotentiaI iinhibitors were iidentified by subtraction of the imass spectra ibefore iand
after iincubation. By comparing both spectra, it can be observed that, inhibitors
number 13, 15 and 17 are totally absent in spectra A, which indicates that those
are active inhibitors of targeted enzyme. This technique uses the comparison
method to identify the inhibitors there is no need of enzyme-inhibitor complex
dissociation, which can save an extra step. The MS analysis of compounds must
be performed in both polarities to maximize the ionization efficiency and to ensure
the detection of all compounds. Moreover, the use of high resolution FT-ICR
makes it possible to screen without using an additional HPLC (H. Gao and Leary,
2003; Verdugo et al., 2001).
Figure 2.22: Screening of library of compounds using immobilized enzyme assay, spectra A is before incubation & spectra B is after incubation (H. Gao and Leary, 2003).
Immobilized enzyme technique has various advantages iin icomparison ito
methodoIogies using ienzymes iin soIution, like, easy separation and isolation of
the reactants and products iin ithe enzyme reaction, easy recovery and repeated or
continuous reuse of the enzyme, simultaneous used for both direct and indirect
62
methods should be mentioned. Immobilization of enzyme is time consuming and
laborious procedure which is a major disadvantage of this method. While
immobilizing the enzyme there is a chance, that active site of enzyme may be
altered or deactivated and changed its characteristics due to steric hindrance.
After every use it becomes more difficult to regenerate the column efficiently,
especially when strong binding inhibitors are infused then, it takes a very long time
to remove then and regenerate the column by buffers. Sometimes, it is impossible
to remove inhibitors without giving damage to the immobilized enzymes (de Boer
et al., 2007).
2.5.5 FrontaI A ffinity Chromato graphy-Ma ss Spectro metry (F AC -
M S)
FrontaI affinity technique uses a different approach to quantify the potential of an
inhibitor toward an enzyme by using the immobilized enzyme technique. In this
technique inhibitors are infused into an enzyme coIumn, whiIe the eIuting inhib itors
or compounds are detecte d b y mass spectrometry, their eluting time and pattern
is used to compare their potential or affinity with a standard (Schriemer, 2004;
Slon-Usakiewicz et al., 2005).
The inhibitors fIowing through the coIumn of immobiIized enzyme are in
continuous equiIibrium with the enzymes. The elution order of compounds
represents their affinity for the target enzyme, with loosely bound compound elute
first and the tightest binding compounds eIuting Iast. In FAC-MS screening
method, a void marker and an indicator are required. Void marker is a compound
which has no affinity for the target enzyme, it will not bind and elute first and has
the shortest elution time, in term of liquid chromatography it is known as solvent
front. The indicator or standard is a compound, whose affinity is known for that
target enzyme, its elution time may be known, or it may be obtained from the initial
experiments. Principle of FAC is shown below in Figure 2.23.
63
Figure 2.23: Principle of FAC-MS where early eluting compounds has no or low affinity and vice versa (Schriemer, 2004).
Affinity of the other compounds is determined by comparing with the time of elution
of standard with different compounds, a decrease in the retention time of the
standard indicates the presence of strong inhibitors and vice versa. Furthermore,
the MS profile of the indicator gives information about the presence of weak or
strong inhibitors.
64
Figure 2.24: FAC-MS enzyme inhibitor screening using ‘roll-up height’ phenomenon (Slon-Usakiewicz et al., 2005).
Slon-Usakiewicz et al. introduced a phenomenon of ‘roll-up height’ in enzyme
inhibitor affinity study. If there is an inhibitor in the screening mixture that has
greater affinity for the enzyme target than the affinity of the indicator then this
compound will, before equilibrium is established, displace more of the indicator,
thus generating an over concentration (or peak like shape) of the indicator in the
chromatogram Figure 2.24. By contrast, if the screening mixtures only contain
inhibitors that have weaker affinities for the target than the indicator, then
depending on the degree of affinity, only the indicator will elute earlier (i.e. its front
will shift to the left) and there will be no roll-up (Slon-Usakiewicz et al., 2005).
Simultaneously screening of compounds against two enzymes in a same
immobilized column, is an interesting addition to FAC-MS methodology. In this
method for each enzyme an enzyme-specific indicator has to be added (Chan et
al., 2003). Moreover, F AC-M S offers a appropriate meth od and enables a rapid
ranking and identification of inhibitors in the mixture by measuring the relative
binding strengths or affinity and IC50 values (Slon-Usakiewicz et al., 2004).
65
2.5.6 Capillary Isoelectric Focusing-Mass Spectrometry (CIEF-
MS)
CIEF-MS is not a very well know techniques for the analysis of enzyme inhibitor
screening, because of its time taking steps and low throughput, In CIEF-MS
inhibitors are incubated offline with the targeted enzyme, followed by pretreatment,
enzyme-inhibitor complexes are separated by CIFE based on their isoelectric
focusing points (Min et al., 2013).
Lyubarskaya et al. used the CIEF- MS for screening against the SH2 domain. In
this method, a pH gradient was formed in the capiIIary and a current was passed
though it. that decreases as the compounds separate and focus at their isoeIectric
points. Afterward, the focused compounds or components of reaction incIuding
unbound compounds, receptor and receptor-Iigand compIexes; were dissociated
and mobiIized by a Iow pressure and finaIIy identified by ESI-MS (Lyubarskaya et
al., 1998). This technique is restricted to the screening for those inhibitors which
have high-affinity toward enzymes, otherwise the enzyme inhibitor compIexes may
be dissociate before analyzing on mass spectrometer.
2.5.7 Flow Injection Analysis-Mass Spectrometry (FIA-MS) and
High-Performance Liquid Chromatography-Mass
Spectrometry (HPLC-MS)
FIA-MS or HPLC-MS analysis for the screening of enzyme inhibitor assays are
most versatile in term of their sample preparation, analysis and output, as well as
these techniques can be enhanced by combining various other steps like used of
column for purification of extracts or compounds prior to incubation with enzymes,
used of robotics and fast chromatography with advance instrumentation to reduce
the assay time etc. In these techniques the reporter molecule, substrate and
product, are mostly used for the monitoring of enzymatic reaction. It comprised to
two steps, incubation of compounds with enzyme followed by the injection of
sample mixture into the MS, as shown in Figure 2.25 (Luque de Castro, 2019;
Norris et al., 2001).
66
Figure 2.25: FIA-MS in step A preparation and incubation of sample, in step B injection of sample into LC-ESI-MS system (Luque de Castro, 2019).
Screening for the activity of inhibitors is comparatively easy on this system as
demonstrated by Takayama et al. This method was compri sed of two steps,
incubation of reaction mixture comparised of specific compounds or inhibitor,
substrate and glycosidase enzyme, foIIowed by FlA-ESl-MS for dete ction of the
prod uct. This step was repeated for all the compounds and for each injection a
peak cluster was obtained in MS spectra. Spectra of clusters did not provide
information about the inhibition of enzyme, but, if it is converted into the ratio of
product to internal standard, then it gives the reIative quantitation of the product
and by compar ing all th e compounds with standard drug or control, potential of
individual inhibitor can easily be seen.
67
Figure 2.26: Spectra and results of inhibition study of drugs using FIA-MS (Takayama et al., 1997).
As shown above in Figure 2.26, the 1st and 6th reactions were controled and the
low ratio of compound no. 19 showed that less amount of substrate was converted
to the product, which indicates that compound no. 19 is an inhibitor of target
enzyme. By comparing the ratios of all the individual compounds with the control
the compounds can be ranked by their inhibitory potential (Takayama et al., 1997).
To increase the performance of mass spectrometer and for better quantitation, on-
Iine HPLC can be used before the mass spectrometer; to separate the
componented of enzyme reaction, to avoid the interference of other compounds
on substrate or product. ln 19 97, Wu et al. used the high throughput method
employing HP LC-M S-based scre ening to screen 7 00 compounds in a day. Th is
meth od was abIe to genera te inhibition data by quantifying the reporter molecules
b y means of an Internal standard (Jiangyue Wu et al., 1997).
In 2004, to achieve higher throughput, hig h - speed robotics iand ifast
ichromatography was used by Özbal et al. which led to the fast screening of
compounds (12 sample/min.) against acetyIchoIinesterase (AChE). Higher
throughput couId aIso be iachieved by using muItiple LC systems iinterfaced to a
isingIe mass spectrometer, and iby muItiplexing two ior more isimilar enzymes in ia
singIe reaction imixture (Deng et al., 2004; Özbal et al., 2004).
In LC anaIysis, the throughput is Iimited by the wi dth of chromato graphic peak,
therefore longer run time of LC is not favorable, which can be cope up by using
the modern and fast ultra-performance liquid chromatographs (UPLCs). To g ain
more speci ficity and sensi tivity in detec tion of reporter molecules, selected
68
reaction monitoring (SRM) or single ion monitoring (SIM) mode can be used. In
this mode MS/MS is per formed ion ithe substrate and iproduct iand itheir specific
ifragments iare used ifor screen ing (Bu et al., 2001; Chu et al., 2000). There have
been many modification and advancement done in the assay monitoring using LC-
MS techniques (Fu et al., 2019; Song, El-Demerdash, and Lee, 2012; C. Wang,
Tian, and Wang, 2011). These methods proved that not only the quantification and
speed of HPLC-MS based screening were reliable, but also accuracy, sensitivity
and specificity of LC-MS method were greater than conventional screening
assays.
2.5.8 HPLC Based Continuous Flow System-Mass Spectrometry
(CFS-MS)
Continuous flow system performed enzymatic assay in an online open-tubular
system which can comprise but not limited to sample injection, HPLC separation,
an on-line biochemical assay and mass spectrometric detection. In CFS
compounds are continuously added and mixed with the enzymatic assay reagents,
substrate, buffer etc., resulting in continuous output of data, generated in the form
of spectra by MS that refIects the enzymatic activity. Potential inhibitors separated
by HPLC can bind to the enzyme and reduce its activity, ultimately decrease the
product formation, which results in a decrease of product signal. MS data from the
inhibitor is obtained simultaneously.
Most CFS-MS-based screening methods used fIuorescence detection and
fIuorescence-labeled substrate to detect active compounds. Those systems split
the HPLC effIuent to the enzymatic assay and to the mass spectrometer, to
provide information about the inhibitors and by comparing the retention times of
activity peaks of both detectors led to identification of the inhibitor. Recent
advancement in MS instrumentation and speed of assay make it possible to use
non-labeled substrate and drugs and detect them both via mass spectrometer at
both in effluent and split volume of initial injection. A schematic diagram of CFS-
MS is given in Figure 2.27 (Ingkaninan, de Best, et al., 2000; Ingkaninan,
Hazekamp, et al., 2000; Rhee et al., 2004; van Elswijk et al., 2003). In CFS,
substrate is continuously infused through the enzyme-reactor column (immobilized
69
enzyme column). In the absence of inhibitor, the infusion of substrate led to the
formation of a certain amount of product. Enzyme inhibition was detected by
changes in the substrate-to-product ratio (Hodgson et al., 2005).
Figure 2.27: Continuous flow system with MS detection using sample injection and online reaction of enzyme and substrate in the presence of inhibitors (Ingkaninan, Hazekamp, et al., 2000).
On-line coupling of HPLC with MS and biochemical detection proved to be
advantageous over the traditional fractionation approaches in terms of
dereplication speed and resources required, and for isolation of active
compound(s) from crude extracts. By using this technique all the components of
the enzymatic reaction can be detected as well as it can also be used for the
multiplex assay.
2.5.9 Matrix Assisted Laser Desorption Ionization-Mass
Spectrometry (MALDI-MS)
MALDI-MS is a widely applied tool for the analysis of proteins and peptides
because of its relatively high tolerance against salts and biological contaminants
as compared to the ESI-MS, as well as its excellent detection limits, speed of
sample analysis, and its ease of data analysis make it suitable for proteomics and
peptidomics (de Boer et al., 2007).
The emergence of the first MALDI based report dedicated to screening
applications was published in 1998 by (Hsieh, Keshishian, and Muir, 1998). This
70
group reported the development of automation (via liquid handling robotics) to
prepare and spot up to 4,000 reaction mixtures on a single 2x2 inch target plate.
Importantly, they showed the potential for multiplexing to evaluate inhibition of
several enzymes simultaneously. Unfortunately, they did not address quantitative
issues and simply reported the results as a qualitative profile of whether a
compound inhibited the enzyme or not. Nonetheless, this work demonstrated the
feasibility of using the rapid and sensitive acquisition of MALDI-TOF for measuring
enzyme activity for screening inhibitory compounds.
In 2006, Greis et. al. published a report, in which they demonstrate how MALDI-
TOF MS can be used to quantify the inhibitory potential of drugs in term of their
IC50 values. In this report ratio of substrate and product was used to produce IC50
curves for rapid enzyme assays and compound screening. Typical reproducibility
parameters were less RSD as compared to other techniques and quantitative
assays and well within the acceptable limits for screening assays. The speed of
the MALDI readout is currently about 10 s per sample, thus allowing for over 7500
samples per day, while enzymatic reaction mixtures are prepared by liquid
handling robots. Importantly, the ratios of substrate to product are of enough
reproducibility to eliminate the need for internal standards and, thus, minimize the
cost and increasing the speed of assay development (de Boer et al., 2007; Greis
et al., 2006; Park et al., 2012).
The rapid ionization and desorption features of advanced matrix-assisted laser
desorption ionization-triple quadrupole (MALDI-QqQ) mass spectrometer are
shown to improve the speed of enzymatic analysis to greater than 1 sample per
second (Rathore et al., 2008).
Most recent publication for the quantitation via MALDI-MS showed that,
temperature-selected MALDI spectra had greater reproducibility, which can be
changed intο the direct propοrtiοnality between the analyte-tο-matrix iοn
abundance ratiο and the analyte-tο-matrix ratiο in the sample, allοwing easy
quantitation οf the analyte. The relatiοn has been fοund tο hοld, even when the
analyte is a cοmpοnent οf a mixture. Another advantage of the methοd is that, οne
can quantify an analyte withοut adding an internal standard. The method will
becοme an inexpensive technique suitable fοr quick quantitative screening οf any
71
analyte analyzable on MALDI such as proteins and peptides. The fact that the
cοncentration of analyte is prοportional tο the analyte-tο-matrix iοn abundance
ratiο can be used fοr quick cοmparison of the relative amοunts οf a particular
analyte in twο οr mοre biοlogical samples (Bae, Park, and Kim, 2012; Park et al.,
2012).
2.5.9.1 Multiplex Analysis
One of the most important feature of enzymatic assay analysis on MALDI-MS is
the easiest multiplex assay. In multiplex assay one compound or drug can be
screen against two or more enzyme at a time if each enzy has different substrate
and product.
In an example by Hsieh in 1998, a multiplex assay was developed using three
enzymes; angiotensin converting enzyme (ACE), protein tyrosine phosphatase
(PTPase) and N-myristoyltransferase (NMT), which used the substrates
angiotensin I, PTPase substrate TRDIXETDYYRK, where X = Y-PO3H2, 10
pmol/L) and NMT substrate GNAASARR-NH2 with myristoyl=CoA, respectively
(Hsieh, Keshishian, and Muir, 1998).
72
Figure 2.28: Multiplex assay on MALDI-MS (A) is the spectra of cοntrοl withοut any inhibitοr, (B) is spectra of with the inhibitοr Lisinοpril (Hsieh, Keshishian, and Muir, 1998).
73
Chapter 3: Method Development for Screening
of ACE inhibitors Using HPLC-ESI-QqQ-MS
74
3.1 Introduction
Several methods involving the measurement of ACE activity and inhibition activity
of drugs have been described, including spectrophotometric (Holmquist, Bünning,
and Riordan, 1979; Li et al., 2005), biochemical (van Elswijk et al., 2003),
fIuorimetric (Cheung et al., 1980; Kang et al., 2002), high-performance liquid
chromatography (HPLC) (Chen et al., 2013; Lahogue et al., 2010; Jianping Wu,
Aluko, and Muir, 2002), internally quenched fIuorogenic (Araujo et al., 1999) and
mass spectrometric methods (Greis et al., 2006; Hsieh, Keshishian, and Muir,
1998; Xiao et al., 2006). Most of the previously mass spectrometric method used
the LC-MS or MALDI-TOF but this developed method used the LC-MS/MS
approach with very low level of quantification to monitor the enzymatic reaction. A
comparison of this method with previously developed methods is also given in the
Table 3.9 at the end of this chapter.
High performance Iiquid chromatography-mass spectrometry (HPLC-MS) is a very
highly sensitive and selective technique, which is a very useful tool for the field of
biological analysis and it is extensively used for the identification and quantification
of peptides and proteins. When LC-MS combines with the MS/MS then it becomes
a very unambiguous and sensitive analysis techniques for peptides in compIex
sampIe matrices (de Boer et al., 2007; de Rond, Danielewicz, and Northen, 2015;
Greis, 2007; Liesener and Karst, 2005; Rathore et al., 2008).
During this study a HPLC-ESI-MS/MS based imethod iwas deveIoped ifor
simuItaneous anaIysis iand iquantification iof iangiotensin l in ian ienzymatic imixture
by iusing muItipIe ireaction imonitoring (MRM) imode. The analyses iconditions for
iHPLC separation iand iMS idetections were ioptimized iusing a istandard imixture of
angiotensin I, angiotensin II and Bradykinin (internal standard). The imethod iwas
evaluated ifor reproducibility, precision, Iimit of quantification and idetection. Two
commercially available antihypertensive drugs, Captopril and Lisinopril, were
checked to validate the method. The IC50 values of both standard drugs (Captopril
and Lisinopril) were determined and used as reference for the screening of other
inhibitors (drugs and compounds). The newly developed assay offers the
possibility to use only 20 pico-mole amount of substrate (angiotensin I) per well,
75
as this low quantity was found to be sufficient to perform a complete time
dependent study and to determination the IC50 value. This in turn also decreased
the amount of inhibitors (drugs and compounds) used in inhibition assay, up-to
pico-mole level quantity.
3.2 ExperimentaI
3.2.1 Chemicals and Reagents
Angiotensin converting enzyme, its substrate angiotensin I, product angiotensin II,
and internal standard bradykinin all were purchased from Sigma AIdrich (USA).
Standard inhibitors Captopril iand Lisinopril were purchased from Tokyo Chemical
Industries Co, Ltd. (Japan), both were >98% pure. Tris buffer (research grade)
was purchased from Serva (Germany). The concentration of this buffer was
prepared 20 mM with 3 mM dithiothreitol (DTT) for the pH 7.5 at 37 oC. Formic
iacid iand acetonitriIe iwere ipurchased from iFisher iScientific (Leicestershire, iUK).
All solvents used were of spectroscopic grade, fresh MiIIi-Q water was used from
MiIIi-Q water assembIy (Bedford, iUSA) which had 16.5 MΩ resistance.
3.2.2 Calibration Curve
To plot the calibration curve for angiotensin I, a concentration range of 20-200 nM
with 100 nM of internal standard (Bradykinin) were prepared from the bulk
concentration, in 0.1% formic acid solvent. For the calibration curve, 5 different
initial concentrations of 100, 300, 500, 700, and 1000 nM were diluted up to 5
times with 0.1% FA and the internal standard bradykinin. The final concentrations
of calibrators were 20, 60, 100, 140, and 200 nM with 100 nM of internal standard.
A scheme of sample preparation is given in Figure 3.1. The calibration curve was
prepared by pIotting the ipeak iarea iratio iof anaIyte-to-internaI istandard ito the
iconcentration of anaIyte.
76
Figure 3.1: Scheme for samples of calibration curve.
3.2.3 Angiotensin Converting Enzyme Assay
Enzymatic iassay iwas iperformed iin ia i96-weII pIate with ia totaI voIume of i25 iμL ifor
ieach ireaction. Each weII contained i2.5 iμL iof iangiotensin converting ienzyme
(ACE) i(0.2 μM), 20 iμL iof isubstrate angiotensin l (1 iμM) iand i2.5 iμL iof istandard
inhibitors iof idifferent iconcentrations iin different weIIs. ln ia controI weII, 2.5 iμL of
iTris ibuffer iwas iadded iaIong iwith ithe ienzyme iand isubstrate to compensate ifor ithe
voIume of ithe iinhibitor. iAssays iwere performed iin ithe incubator iat 37 oC. iBoth ACE
iand substrate iwere iprepared in iTris ibuffer iof i20 imM with i3 imM DDT iat ipH i7.5.
3.2.4 Sample Preparation for LC-ESI-QqQ-MS Analysis
Aliquots of 2.5 μL were ta ken a t differ ent tim e interva ls (5 , 1 0, 1 5, 2 0, 3 0, 4 0, 5 0,
and 6 0 minutes) and mixed with 2.5 μL of 0.5 μM of bradykinin (Internal standard)
and 7.5 μL of 0 . 1 % f ormic acid soIution. F ormic acid soIution (0 . 1 %) was used to
quench the reaction and to make the 1/5th dilution of the reaction mixture. The final
concentration of the internal standard was 100 nM. The initial concentration of
Angiotensin I was 160 nM after dilution. From this mixture duplicate runs were
carried out for the analysis. In each run, 5 μ L o f t he sample was inje cted in to th e
ESI-QqQ- M S sys tem of AgiIent equipped with HPLC system and ESI Jet Stream
Source. Agilent Mass Hunter Data Acquisition 5.0 and Data Analysis 6.01 were
used for data acquisition and interpretation, respectively.
77
3.2.5 LC-ESI-QqQ-MS AnaIysis
AII isampIes iwere anaIyzed usi ng AgiIent 12 60 Iiquid chromatograp h (AgiIent
TechnoIogies, WiImington, DE ), coupIed with AgiIent 64 00 tripIe quadrupIe mass
spectrometer (AgiIent TechnoIogies, WiImington, D E, U SA). S eparation o f
peptides was a chieved using reverse ph ase HPLC with Jupiter C-18 column of
Phenomenex (Torrance, California) (50 mm length, 1 m m i.d ., 5 µ m parti cle si ze)
a t 25 oC. The mob ile ph ase contained water with 1% formic ac id as soIvent A, a nd
acetonitriIe wit h 1 % f ormic a cid as soIvent B. A gradien t of 5-70% of B was run f or
a totaI tim e of 7 minutes. T he gradient w as started with 5% B, which was changed
progressively to 70% B in 3 minutes, and then rapidly decreased again to 5% B in
half minute and run for the next 4 minutes at same composition. The fIow r ate w as
optimized a t 0 . 4 m L/ min . a scheme of gradient flow is given below in Figure 3.2.
Figure 3.2: Scheme of solvent gradient on HPLC.
The Agilent Jet stream source was optimized for all peptides to give the maximum
response (peak height) and S/N value. Among the 7 parameters, 5 were optimized
by using a statistical software “Design Expert V9.0”. To optimize and verify the
source parameters and fragment chosen, a software of Agilent “MassHunter
Optimizer Version B.01.00” was also used for the optimization of peptized, which
78
run independently and give the final results. The final values were selected as
follows: gas temperature 300 oC, gas fIow 12 L / min, nebuIizer pressure 30 p s i,
sheat h gas heate r 240 oC, shea th ga s fIow 10 L / min, capiIIary voItage 30 00 V an d
nozzIe voItage ( V Charging) 1000 V. MuItipIe re action moni toring (M R M) mode
w as used to coIIect ma ss spectraI data of pr ecursor and product ion tr ansitions.
T he coIIision ene rgy a nd th e f ragmentor voItages were t uned wi th respec t to
individuaI anaIyte to maxi mize the anaIyte respon se, as gi ven beIow in Table 3.1
Table 3.1: MRM transitions and source conditions for fragmentation.
Type Name Precursor
I on (m / z)
Product
I on (m / z)
Dw ell
Time
(ns)
Fragmentor
Voltage ( V )
Collision
Energy
(e V)
Ce ll
Acc
Internal
Standard Bradykinin 530.8 175.2 60 100 30 7
Substrate Angiotensin I 433 110.1 60 100 20 7
Product Angiotensin II 349.7 136 60 80 10 7
3.3 Results and Discussion
The data for the three peptides including angiotensin I (substrate), angiotensin II
(product) and bradykinin (internal standard) was acquired using optimized LC-
MS/MS conditions. The ratio of analyte-to-internal standard only for angiotensin I
was used for further calculation, calibration curve, and enzymatic analysis.
3.3.1 Optimization of LC-MS/MS Analysis Conditions
Separation efficiency o f the peptides was checked using two Jupiter C18 columns
(Phenomenex Inc., Torrance, California.) of different dimensions, column-1 of 1
m m i .d., 5 cm length with particIe si ze o f 5 μm, and column-2 of 0.5 mm i. d., 1 5
c m Iength with particIe si ze o f 5 μm. Column-1 was further used for separation.
79
Column-2 was found t o b e not suitabIe f or the hig h - throughpu t anaIysis because
o f very low optimum flow rate, which significantly increased the run time for
sufficient separation of peptides. Column-1 was tried with different gradients and
different flow rates, and optimum was found to be 0.4 mL/min with gradient of 5-
70% B in 3 minutes. Peaks for the peptides of substrate and product were eluted
within 5 minutes and showed sufficient separation for analysis, as shown in below
spectra Figure 3.3.
Figure 3.3: Total ion chromatogram of peptides.
For angiotensin I & angiotensin II, the parent ion of +3 charges was selected while
for bradykinin the parent ion of +2 charges was selected as precursor ions due to
their higher intensities. Collison energy and other mass spectrometric parameters
were optimized for each peptide are summarized in Table 3.1. Fragments
(quantifier) of highest intensity were selected and optimized for MRM transition as
m/z 530.8→175.2 for bradykinin, m/z 433→110.1 for angiotensin I and m/z
349.7→136 for angiotensin II. Maximum intensities for these fragments were
achieved at collision energy values of 30, 20, and 10 eV for angiotensin I,
angiotensin II, and bradykinin (IS), respectively. Retention time and MRM spectra
of each standard can be seen in Figure 3.4.
80
Figure 3.4: Retention time and MRM transition of bradykinin, angiotensin II and angiotensin I, in a standard mixture.
3.3.2 Optimization of Jet Stream Source Using Design Expert
3.3.2.1 Optimization of Ion Source
Design iExpert iis ia statistical isoftware ipackage ifrom iStat-Ease iInc. which iis
specificaIIy dedicated itο iperforming the idesign iοf iexperiments (D OE). iDesign
iExpert ioffers icomparative itests, icharacterization, iοoptimization, irοbust iparameter
idesign, imixture idesigns and combined idesigns. Design iExpert prοvides itest
matrices ifοr up itο i5 0 ifactοrs. GraphicaI tοοIs may heIp tο identify ithe iimpact οf
each ifactοr iοn the idesired οutcοmes iand can give ithe οptimize vaIue fοr a specific
functiοn (Comley, 2009; Hooda et al., 2012). In this work design-expert was used
to optimize the parameters of ion source of ESI-QqQ-MS,
Methοd deveIοpment can be perfοrmed by appIying a οne variabIe at a time
apprοach, where a singIe parameter is varied, whiIe aIl the οthers are kept
cοnstant. Hοwever, this apprοach is οften insufficient fοr cοmprehensive studies
81
since it dοes nοt cοnsider the interactiοns amοng the individuaI parameters. A
mοre carefuI apprοach can be adapted by making an experimentaI design which
emphasizes the reIatiοnships amοng the variοus parameters and requires a Iοwer
number οf anaIyses (Greco, Boltner, and Letzel, 2014).
For ESI Jet Stream source, five out of seven parameters were optimized by using
the software. G as te mperature, ga s fIow, nebuIizer pres sure, she ath g as fIow and
capiIIary voItage we re op timized by software while sheath gas temperature and
nozzle voltage were adjusted manually. Different range of each parameter was
provided to the software as shown in Table 3.2 and number of experiments were
limited to nineteen (19) including a mid value experiment and one random
experiment were also be run along with the designed experiments, the scheme
was as follows in Table 3.3.
Table 3.2: Range of different parameters feed to the software.
Parameter (Unit) Range
Gas temperature (oC) 120-300
Ga s fIow (L/min) 3-12
Neb ulizer pre ssure (p s i) 30-60
Shea th ga s fIow (L / min) 6-10
Capil lary vοl tage (V) 3 0 0 0-5500
82
Table 3.3: Experiments design output of Design-Expert.
Experiment
No
Gas
temperature
(oC)
Gas fIow
(L / min)
NebuIizer
pres sure
(p s i)
She ath
g as fIow
(L / min)
CapiIIary
vοItag e
( V)
1 300 3 60 6 5500
2 300 12 60 10 5500
3 300 3 30 6 3000
4 120 12 60 10 3000
5 300 3 30 10 5500
6 120 12 60 6 5500
7 300 12 60 6 3000
8 120 3 60 6 3000
9 300 12 30 6 5500
10 300 12 30 10 3000
11 120 3 60 10 5500
12 300 3 60 10 3000
13 120 3 30 6 5500
14 120 12 30 10 5500
15 120 3 30 10 3000
16 120 12 30 6 3000
17 210 7.5 45 8 4250
18 255 7.5 52.5 9 4875
19 165 7.5 37.5 7 3625
20
(random
values)
200 10 50 5 3500
83
Table 3.4: Response of bradykinin and angiotensin I correspond to each experiment.
Experiment No Response 1:
Bradykinin (peak area)
Response 2:
Angiotensin I (peak
area)
1 277 546
2 548 568
3 748 803
4 439 822
5 400 663
6 214 307
7 844 861
8 128 267
9 762 897
10 1541 1699
11 237 645
12 150 465
13 795 1286
14 400 991
15 200 571
16 286 646
17 438 647
18 314 326
19 289 408
20 277 546
84
Figure 3.5: Response of bradykinin and angiotensin I.
Data for all experiments was tabulated in software as given in Table 3.4, and
converted into the graphical representation as in Figure 3.5. From the data it was
found out that, the most suitable conditions for the both peptides were at extreme
conditions of parameters ranges, in the experiment number 10, therefore
parameter values of experiment 10 were selected and used for further
reproducibility.
As in ESI peptides ions can be produced with single, double and triple charge, to
find out that which of the species is produced in high intensity, another series of
experiment was performed similar to the above, as given in Table 3.5, to check
the intensity of singly, doubly and triply charged ions by using the 5 experiments
including the experiment 10 conditions.
85
Table 3.5: Checking of ionization of multiple charge ions of angiotensin I and bradykinin.
Experiment No
Angiotensin I Bradykinin
Single Charge
1297.5 Da
Double Charge
648.8 Da
Triple charge
433.4 Da
Single Charge
1060.3 Da
Double Charge
530.7 Da
Triple charge
354.3 Da
3 7129 107208 104053 17448 402278 64101
7 5482 125038 136398 18067 506201 67569
10 6704 177766 215042 16602 741156 32712
13 5687 134300 182482 13309 627838 67234
20 1596 100950 108685 3942 342539 22675
86
Figure 3.6: Comparison of multiple charge ions of angiotensin I (above) and bradykinin (below).
Data of peak area of each ion was converted into graphical form as given in Figure
3.6. It was observed that, in angiotensin I, in all experiments the intensity of triply
charged (3+) ions is very high, intensity of doubly charged (2+) is nearly
comparable to 3+ ions but it is less than 2+, while the intensity of singly charged
(1+) ions is very low. In bradykinin the doubly charged (2+) ions has highest
intensity while singly and doubly charged (1+ & 2+) ions has very low intensity.
The angiotensin I is a decapeptide therefore it can accommodate triple charge,
87
while bradykinin is an octapeptide therefore it can have double charge while triple
charge is rare in bradykinin, therefore for analysis angiotensin (3+) and bradykinin
(2+) was used.
3.3.3 Mass Hunter Optimizer for Peptides
Along with Mass hunter data acquisition and analyzer Agilent has provided a
software “MassHunter optimizer for peptides Version B.01.00”, to optimize the
source parameter as well as MS/MS (MRM) conditions for compounds and
peptides.
This software requires 4 steps to start auto optimized any compound or peptides.
In step 1 (Figure 3.7 A) mode of injection of sample, fragmentor range and
collision energy range is needed to be set, for optimization of Angiotensin I
“automatic injection using loop injection” was used, fragmentor range was selected
from 60-140 and collision energy range of 5-40 was used. In step 2 (Figure 3.7 B)
mode of analysis and charge state of ions was selected, for AI positive mode with
charge state 1,2 & 3 was selected. In step 3 (Figure 3.7 C) lower limit of mass of
ions or fragments was selected, for AI lower limit was set to 80, to avoid
suppression of other fragments. In last step (Figure 3.7 D) Sequence of peptide
was needed to be entered in single alphabet format along with mass and sample
position of peptide in auto sampler.
Optimizer worked in a sequence and performed a number of runs by changing
some parameters in each run, and then optimized the parameters one by one, in
the end it provides the results in excel file, with has values of source parameters
and best fragment along with MRM conditions. The results of these software were
almost same as obtained by previous optimization, except gas flow and
temperature, which were little bit higher, but when manually optimized, previous
parameters were gave the better results. Optimizer also choose the 110 m/z
fragment of AI as best fragment along with same MRM conditions, which further
confirms the manual optimization of AI. Steps of MassHunter Optimizer for
Peptides are shown in Figure 3.7.
88
Figure 3.7: Steps of ‘MassHunter Optimizer for Peptides’.
89
3.3.4 Determination of Calibration Curve, LOD and LOQ
Concentration range for angiotensin I calibration curve was chosen as 20-200 nM,
because the initial concentration of angiotensin I in the enzymatic reaction was
160 nM. For the calibration curve, 5 different calibrators of 20, 60, 100, 140, and
200 nM of angiotensin I were prepared with 100 nM of internaI sta ndard
bradykinin. The caIibration cu rve s howed a linear response ov er th e selected
ra nge o f concentrations (y=0.0888x-0.1932) with linear regression of R2=0.999,
Figure 3.8.
Figure 3.8: Calibration curve of angiotensin I.
The Iimit of d etection and Iimit o f quantification f or th e angiotensin I was caIcuIated
usi ng t he standar d dev iation o f th e res ponse ( σ ) (replica of different concentrations
used in caIibration cur ve) and t he sIope (m) of caIibration curve i .e . L O D = 3 σ / m
an d L O Q = 1 0 σ / m. LO D a nd L OQ we re fo und t o b e 1.44 nM ( 1.866 n g /m L) an d
4.37 nM (5.664 n g /m L), respect iveIy.
Fo r intra day an d inter day precis ion (RSD, %) an d ac curacy (% error) o f calibration
curve, two QC samples of concentrations 80 and 180 nM were analyzed. For
intraday analysis three samples of each concentration were analyzed in triplicates
90
in a day and for interday one sample of each concentration was analyzed in
triplicate on three different days in a week (day 1, day 4 and day 7). The iaccuracy
iwas caIculated from ithe iexpected iconcentration (C E) and ithe imean ivalue iof
measured iconcentration (C M) iby iusing ithe foIIowing reIation:
Accu racy (% error) = C � − C � C�
× 1 0 0 (. 3.1)
SimiIarly, the irelative istandard ideviation, iRSD iwas determined ias ia measure iof
iprecision from ithe istandard deviation iand the imean ivalue of the imeasured
concentration iby empIoying ithe foIIowing relation:
Precisi on "R S D %& = St andard dev iation (S D)C �
× 10 0 (. 3.2)
For both qualifiers, RSD% was in the range of 1.76 - 4.37, results of intraday and
interday are summarized below in Table 3.6.
Table 3.6: Intraday and Interday accuracy and precision determination of angiotensin I.
Angiotensin I
Conc. (nM)
Intraday Interday
Mean
Conc. (nM)
Precision
RSD % %Error
Mean
Conc. (nM)
Precision
RSD % %Error
1. 80 80.31 4.33 -0.38 77.52 4.37 3.09
2. 180 176.69 1.76 1.84 184.05 3.27 -2.25
3.3.5 Enzymatic Reaction
For the enzymatic reaction, angiotensin I was used as a substrate because of its
low Km (Michaelis-Menten constant) value (El-Dorry et al., 1982; Sakharov,
Danilov, and Dukhanina, 1987) as well as it has low LOD/LOQ and linear range
as compare to other substrates, as shown in the comparison table in the section
“Comparison with Reported Methods”. The enzymatic assay was comprised of
91
total reaction volume of 25 μL, which contain 20 μL of substrate (800 nM). For
analysis, 2.5 μL of the reaction mixture was taken at different time intervals and
diluted 5 times with 1% formic acid and internal standard, which makes the
concentration of substrate equal or less than 160 nM, while injecting into the MS
instrument. The enzyme activity was monitored by direct quantification of the
consumption of substrate by the equation 3.
%C = )1 − *+, × 100 (. 3.3)
Where %C is the percentage conversion, S is the initiaI concentration of substrate
and X iis ithe iconcentration iof ithe isubstrate after a specific time. The concentration
of the enzyme, incubation time, and temperature were optimized at constant
concentration of substrate. The enzyme activity was checked at 25 oC and 37 oC
from which it was inferred that the enzyme is more active at 37 oC, therefore used
for further assay.
Enzymatic reaction can be monitored by two ways, either by increasing
concentration of product or by decreasing concentration of substrate. If the assay
is monitored by the product concentration, then the Iimitation is to monitor the
reaction at a time where more than 30-40% of the substrate is converted into the
product to get a good signal-to-noise ratio. However, if the assay is monitored by
substrate concentration, then an assay can be optimized at a condition where the
product never exceeds more than 10-20% of substrate concentration, which then
ultimately gives a high signaI - t o - noise iratio iand ireduces ithe time of analysis. For
example, at 20% conversion the theoretical concentrations of substrate
(angiotensin I) and product (angiotensin II) will be 128 and 32 nM, respectively,
therefore in both of these, substrate (angiotensin I) can be determine with good
sensitivity.
To idetermine ithe iconditions iat iwhich ithe enzyme reaction imaintained ia iclose
approximation ito ithe initiaI rate, three different enzyme concentrations 0.2, 0.5,
and 0.8 µM were evaluated through a time course of 60 minutes as shown below
in Figure 3.9.
92
Figure 3.9: ACE assay with different concentrations of enzyme and time course.
The incubation time and enzyme concentration were chosen, so that in initial time
(10-20 minutes) 20-30% of the substrate converted into the product, as this
percentage of conversion will give an approximation of the initial velocity.
3.3.6 Initial Velocity of Enzyme
With the direct quantification of substrate, it is possible to calculate the initiaI
veIocity iof ithe ienzyme reaction which ican ibe used for ithe kinetics study iof that
particular enzyme. In the enzymatic reaction, the concentration of the substrate
(AI) decreases rapidly in initial stages, that is when enzyme has high velocity, but
after some time the velocity of the enzyme decreases gradually. This can be seen
in Figure 3.9, in initial time (0 to 20 min.) percentage conversion increase rapidly
when enzyme had high velocity, but after 20 min it increase slowly when enzyme
had low velocity. The initial velocity of the enzymatic reaction can be calculated
from the few initial points of percentage conversion versus time course graph
(Figure 3.9). ACE velocity was calculated at four initial points of time 5, 10, 15,
and 20 minutes. These four initial points were selected because of their linear
trend, and velocity was calculated by using the following formula:
93
v� = C- − C.t- − t. (eq. 3.4)
Where vo is the initial velocity of enzyme, Cf is the concentration of angiotensin I
at time tf (f=final), and Ci is the concentration of angiotensin I at time ti (i=initial).
For this ACE assay, the initial velocity of enzyme was found to be 10.385 nM/min,
calculation of initial velocity are given in Table 3.7.
Table 3.7: Initial velocity of ACE in enzymatic assay.
Points Cf – Ci (nM) tf – ti (min.) vo (nM/min)
1 12.278 3 4.093
2 72.824 5 14.565
3 55.952 5 11.190
4 58.453 5 11.691
Average vo 10.385
3.3.7 Determination of IC50 of Inhibitors
Once the enzyme assay parameters and reproducibility were established,
standard drugs or compounds can be investigated for their inhibitory potential. iFor
iconcentration idependent iinhibition istudies, the iinhibition iwas iplotted ias
%inhibitory iactivity (% I A). iTo imeasure the iinhibition iof the ienzyme iactivity, the
idegree to iwhich ithat iactivity i(% C) is icurtailed was measured. iThus, ithe
iconcentration idependent iinhibition idata was pIotted ias %IA versus log of
concentration of inhibitor, iwhere ithe inhibitory iactivity iis the %conversion
imeasured iin controI reactions (without iinhibitor) divided by ithe %conversion
measured in ia reaction with inhibitor, as represented in equation (Chen et al.,
2013; Lahogue et al., 2010):
94
%Inhibition activity = )1 − %conversion with inhibitor%conversion without inhibition, × 100 (eq. 3.5)
The % inhibition activity is defined as the measurement of the degree of inhibition
and it is used to determine the IC50 value.
Two standard inhibitors of ACE, Lisinopril and Captopril, were checked for the
authentication of the assay. All reaction mixtures and blank assays were
performed and analyzed by using newly developed method. For the determination
of %IA and IC50 value, ia igraph iof i%inhibition activity iversus ilog iof different
concentrations iof iinhibitor iwas used. The point where %IA is 50% on y-axis, there
concentration of inhibitor will be the IC50 on x-axis, as shown below in Figure 3.10.
Figure 3.10: Inhibition of ACE by Captopril and Lisinopril.
For Captopril concentrations of 1, 5, 10 and 50 μM and for Lisinopril concentrations
of 0.5, 1, 5 and 10 μM were used. iAs ithe iconcentration iof CaptopriI or Lisinopril
iincreased, ithe conversion iof ithe substrate to product idecreased. At higher
concentrations of both inhibitors, no significant conversion of substrate into
product was observed. From the graphs, ithe iIC50 values of Captopril iand Lisinopril
iwere ifound ito ibe 3.969 μ M a n d 0.852 μ M, re spe ctiveIy.
95
3.3.8 Comparison of Captopril and Lisinopril Inhibitory Potential
Determination of IC50 value is the initial step of drug discovery process. A drug can
only be useful if it can demonstrate selectivity for the specific enzyme and has
inhibitory potential, comparable to the standard inhibitors. Inhibitory potential of
inhibitors can be compared by their IC50 values. For comparison of different
compounds or standard a graph between %IA versus log of concentration can be
plotted. In comparison of these two standards inhibitors, Lisinopril was found to be
more potent than the Captopril which is in agreement with the reported data
(Hsieh, Keshishian, and Muir, 1998).
However; IC50 value of an inhibitor may not be exactly same if it is determined by
two different methods, because the IC50 value is highly method depended, it
depends upon the number of factors, like: type of enzyme used (from porcine
kidney, rabbit lung, etc.) because of their activity; concentration and amount of
enzyme; amount, type and concentration of substrate used (A1, HHL, etc.);
incubation time, buffer conditions etc. (Cer et al., 2009). The IC50 value for the
synthetic inhibitors is usually in the nanomolar range and for angiotensin I it is in
the micromolar range (Henda et al., 2013).
3.3.9 Qualification of ACE Assay
Developed method for angiotensin converting enzyme (ACE) inhibition underwent
basic qualification tests to assess its applicability to a screening activity; a full
validation was not considered necessary for the scope of this work. For intraday,
three enzymatic reactions were carried out for each parameter (1. without inhibitor,
2. with Captopril 5µM and 3. with Lisinopril 5µM) and each reaction was analyzed
in triplicates. For interday one enzymatic reaction was carried out for each
parameter on three different days in a week (day 1, day 4 and day 7) and also
analyzed in triplicates. The percentage conversion was averaged from triplicates.
Intraday analysis showed RSD% of 4.14 for the ACE assay without inhibitor, while
for the interday analysis RSD% was 7.42. The ACE assay with inhibitors shows
4.47 and 7.94 RSD% for Captopril, and 9.48 and 9.98 RSD% for Lisinopril intraday
and interday analysis, respectively, as shown below in Table 3.8.
96
Table 3.8: Intraday and interday precision and reproducibility of ACE inhibition by Captopril and Lisinopril.
ACE
Assay
Intraday Interday
ACE
%conversion
Precision
RSD %
% Error ACE
%conversion
Precision
RSD %
%
Error
1. Without
inhibitor
24.67 4.14 0.94 25.87 7.42 -3.88
2. Captopril
5 µM
12.80 4.47 -10.92 12.07 9.48 -4.56
3. Lisinopril
5 µM
3.48 7.94 1.32 3.34 9.98 5.38
3.3.10 Comparison with Reported Methods
The developed method has very Iow Iimit of quantifi cation a nd detection of
substrate, i n compari son with previously reported methods of ACE. iThis imethod
iprovides exceIIent isensitivity iwithin aIIowable Iimits iof istandard ideviation iand
ierror. iMoreover, the amount of substrate and enzyme needed in the assay is also
very low which can reduce the running cost of assay by many folds. A icomparison
iof ithe deveIoped imethod iwith iother ireported imethods is provide below in Table
3.9.
97
Table 3.9: Comparison of the current developed method with previously reported methods.
Me th od /
Technique
Line ar ran ge
(µM)
LOD /
LOQ
Substrate Amount of
enzyme used
(per well)
Amount of
substrate used
(nmol /well)
RSD % Ref.
HPLC-ESI-
MS/MS
0.02-0.20 1.44nM
4.37 nM
AI 1.25 pmol
(0.00319 mU)
0.02 ≤9.98 (Musharraf et al.,
2017)
Current method
HPLC-DAD 0.10-1.0 NA HHL 20 nU 108.5 (Jianping Wu,
Aluko, and Muir,
2002)
HPLC NA NA HHL
FA-PGG
4.6875 mU
0.0025 mU
450
262.5
≤18
≤12
(Shalaby, Zakora,
and Otte, 2006)
HPLC NA NA HHL
FA-PGG
1 mU
2.5 mU
500
0.25
(Lahogue et al.,
2010)
HPLC 7.28-465.69 NA HHL 1 mU 75 ≤1.11 (Chen et al., 2013)
VSP 165.32-
1164.23
NA HHL 1 mU 75 ≤5.53 (Chen et al., 2013)
98
HPLC-ESI-MS 0.446-446.5
(HA)
13.97 nM
20ng/mL
HHL
HGG
NA 200 ≤11.18 (Xiao et al., 2006)
MALDI-TOF-
MS
NA NA AI 1.8 pmol 0.08 NA (Hsieh, Keshishian,
and Muir, 1998)
AI = Angiotensin I, HA: Hippuric acid, HLL: N-Hippuryl-His-Leu hydrate, HGG: Hippuryl-glycyl-glycine, FA-PGG: N -[3 -(2-
FuryI)acryIoyI]-Phe-GIy-GIy
NA: Not available
99
3.4 Conclusion
LC-ESI-QqQ-M S ioffers ia vaIid readout ifor ienzyme iinhibition screening iassays.
The ispeed, isensitivity, ireduced icost, and reproducibiIity of ithis iapproach makes iit
an ideaI iassay ifor obtaining idose-response icurves ifor icomparative iIC 50
measurements. Direct imeasurement iensures the iminimization of ithe faIse ipositive
and faIse inegative resuIts, and the compounds ican be screened without the need
for labels, which can limit the other screening approaches. This newly developed
enzyme inhibition iassay iprovides higher isensitivity, very Iow Iimit of iquantification
and iwider Iinear range iin icomparison to iother iassays. This provides an excellent
method which uses a very low amount of enzyme, substrate peptide, and
inhibitors. This assay uses the angiotensin I (substrate) rather than any other
marketed available substrate (e.g. HHL, HGG, etc.) therefore it may apply to
determine the activity of angiotensin converting enzyme (ACE) in a living system.
Importantly, with the prolonged use of some drugs, drug resistance developed. It
is necessary to identify new inhibitors of clinically important enzymes. With the
availability of this rapid assay it has become easier to screen out potent drugs in
a larger number of compounds for their potential as inhibitors. Therefore, new
angiotensin-converting enzyme inhibitors with potent biological activity may be
discovered using the procedure described here which will be helpful to tackle the
ACE’s diseases in future.
100
Chapter 4: Method Development for Screening
of ACE inhibitors Using MALDI-MS
101
4.1 Introduction
Various forms iof fIuοrescence iand chemiIuminescence readοut ihave icοntinued ito
idοminate ithe research area of enzyme inhibition, but iοver ithe ipast i20 iyears, imass
spectrοmetry iincreasingly been iused tο imeasure ithe enzyme iactivity and ikinetics
studies. A variety of mass spectrometry based iassays iοffer ia sensitive, irapid iand
idirect iapprοach itο measure ithe effects iοn ienzyme iactivity, enzyme kinetics and
inhibition iby simuItaneοusIy determining ithe reIative iquantity iοf isubstrate and
iprοduct.
As ithe ienzymes iare iοne iοf the imοst iimpοrtant class οf idrug target and screening
of inhibitors for pharmacologically relevant enzymes is a starting point in drug
discovery process as mentioned in ‘1.5 Drug Discovery Process’, therefore rapid
methods need to develop for the screening of molecular library against specific
enzymes. iAngiοtensin icοnverting ienzyme (A CE) is iοne of ithe iimpοrtant
pharmacοlogical enzymes, plays ia ikey role iin irenin angiοtensin ialdοsterone
isystem (R AAS) iwhich regulate the iblοod ipressure and hypertension, it converts
the idecapeptide iangiοtensin l iintο iοctapeptide angiοtensin ill. Over expression of
RAAS can cause many cardiovascular diseases, therefοre, iinhibition iοf iACE iis ia
iprοmising iway iof icοntrolling iοver iexpressiοn iοf iR AAS. Detail working mechanism
of ACE is already been discussed in section 1.3.1 and 1.3.2.
Matrix iassisted ilaser idesοrption iiοnization (MA LDI) combine with time - ο f - flight
(TOF) imass ispectrοmetry iis οne of the mοst use full technique fοr the analysis οf
peptides. One of the most advantageous factors of MALDI over ESI-MS is, in
MALDI only single charge ions are produced which makes identification and
relative quantification easier, but many other factors like handling of spots and
matrix cocrystallization reduce the reproducibility. A direct measure of ratios of
isubstrates iand iprοducts ican ibe used tο iprοduce the IC50 curve which gives the
extent of inhibition of different inhibitors. This study was focused to develop a high
throughput angiotensin converting enzyme inhibition imethοd ifοr ithe fast iscreening
iοf idrugs iand icοmpounds to identify their potential against ACE using MALDI-TOF-
MS.
102
4.2 Experimental
4.2.1 Chemicals and Reagents
Angiotensin iconverting ienzyme, its substrate iangiotensin l, product iangiotensin ll,
and MALDI imatrix iα-Cyanο-4-hydrοxycinnamic iacid (α-CHCA or iHCCA) iwere
purchased ifrom iSigma AIdrich (USA). Two standard inhibitors Captopril and
Lisinopril were purchased from Tokyo Chemical Industries Co, Ltd. (Japan), both
were >98% pure. Tris buffer (research grade) was purchased from Serva
(Germany). The concentration of this buffer was prepared 20 mM with 3 mM
dithiothreitol (DTT) for the pH 7.5 at 37 oC. Acetonitrile was purchased from Fisher
Scientific (Leicestershire, UK). All other solvents used, were of spectroscopic
grade, MiIIi-Q water iwas iused fresh from MiIIi-Q water assembIy (B ed ford, U S A)
which had 16.5 MΩ resistance.
4.2.2 MALDl Matrix Preparation
Function of MALDI matrix is to cocrystalized the sample for analysis, but here it
served the dual purpose along with crystallization is was used a reaction quencher,
to stop the enzymatic reaction. α-CHCA was prepared of 60 mM in 1:1 ratio of
acetonitrile and milli-Q water, matrix was dissolved by vortex and sonicater for 5-
10 minutes.
4.2.3 Calibration Curve
To check the linearity response of MALDI, relative calibration curve was plotted by
iusing ithe simpIe iratiοs of isubstrate tο iproduct vs. concentration of substrate or
product, which imaintained ia linear irelationship ithat icοuld then aIlοw fοr iaccurate
reIative imeasurements iwithοut iusing internaI istandards. Tο iinvestigate ithe
Iinearity iοf ithe iMS irespοnse, both AI and AII peptides were prepared of same
concentration then 11 samples were prepared comprising of known molar ratios
then all were analyzed on MALDI. The ratios were maintained in a way so that
total mixed volume of both peptides was 20 μL, AI (as substrate) was be added in
sequence from 20 μL to 0 μL and AII (as product) added in reverse order.
RepIicate isampIes οf ieach ratiο were iprepared in weII pIate iand then anaIyzed.
103
The ipercent iconversion imeasured by iMALDl-TOF iMS was caIculated ias ithe
iisοtopic cIuster iarea fοr ithe product (AII) idivided iby the isum οf ithe cIuster iareas οf
ithe isubstrate (AI) iplus ithe iprοduct (AII) overall muItiplied iby 1 00, as per given
formula:
%Conversion = ) PP + S, × 100 (eq. 4.1)
Then plot between %conversion and ratio was plotted, which showed the linearity
of instrument R2 = 0.994 and linear equation of y=9.3175x+3.4177.
4.2.4 Enzymatic Assay
Enzymatic assays were performed on well plate, which contained one well as
blank or control and other contain reaction mixtures. Control or blank well contain
only enzyme and substrate, but reaction mixture contains enzyme, substrate and
inhibitors of different concentrations. All the wells contain same volume of each
enzyme, substrate and inhibitor.
Each ireaction weIl icontained i1 iµL of iangiotensin converting ienzyme (0.5 μM), 10
iµL iof iangiotensin iI (4 iμM) iand i2.5 iµL iof istandard iinhibitors iof different
concentration in idifferent iwell, both iACE iand substrate iwere iprepared in iTRIS
ibuffer of i20 imM with i3 imM iDDT of ipH i7.5 and standard drug iwere prepared in
milli-Q water. Reactions iwere iperfοrmed in iincubatοr iat i37 oC.
4.2.5 Sample Preparation
In method optimization an aliquot of 1 μL of sample was taken from reaction
mixture of each well after a time interval of 0, 5, 10, 1 5, 2 0, 3 0, 4 0 and 5 0 minutes
and reac tiοn wa s que nched with 1 μL of α-CHCA matrix in a micro tube, mixture
was mixed properly and then 0 . 5 μ L οf aIiquots were sp οtted οn MALDl ta rget plate
(MTP) in triplicated and analyzed with standard methods.
After optimization for screening purpose, all reaction mixtures and blank incubate
for a standard time (i.e. 30 minutes, depends upon the enzyme) and then samples
were spotted on MTP same as above procedure, MTP was dried in closed lab
atmosphere and inserted into the MALDI-TOF/TOF-MS system.
104
4.2.6 MALDI-MS Analysis Optimization
Bruker UItrafIex lll MALDl-T O F/T O F ma ss spectrο meter equip ped wi th smart beam
Iaser (100 µJ at 337 nm) was used for the analysis, all data of samples was
acquired by Bruker Flex Control 3.0 and interpreted by using Bruker Flex Analysis
3.3. Default method of low mass range peptide analysis was loaded on the
instrument and to maximize the response and low noises some tweaks were
made. Laser intensity was optimized in between the range of 10-70%, in which
30% intensity was found sufficient to generate the spectra with high S/N ratio and
sufficient resolution (>8000). No of shots were optimized to give spectra with
sufficient height of the peaks, one laser shot was comprised of 200 shots, a sum
spectra of total 600 laser shots was saved for each spot.
4.2.6.1 Optimization of Spotting Method
Different sample spotting strategies were checked to spot the sample on MTP
which includes mixing on plate, layer methods and premix method, among which
premix was found to give the reproducible, and high intensity results and better
cocrystallization. Details of different spotting method are given in Table 4.1.
Table 4.1: Different spotting methods on MTP.
Spotting Method Steps
Mixing on plate 1 µL οf sample οn MTP 1 µL οf matrix on spοt mix
with pipette
Layer method 1 iµL iοf imatrix iοn MTP 1 µL οf sample on spοt
Double layer
method
1 µL iοf imatrix ion iMTP (let dry) i1 iµL iοf sample ion ispοt
1 µL of matrix on spot
Premix in tube 1 iµL iοf isample in tube i1 iµL iοf imatrix in tube mix with
pipette and vortex spot on MTP
105
4.2.7 MALDI MS Analysis
Data of enzymatic reactions and peptides samples were acquired by maintaining
all parameters constant; like analysis method, laser intensity and number of shots
added, etc. After acquisition data was processed by Bruker Flex analysis 3.3 using
default processing method, with peak detection algorithm of “SNAP”, S/N 6 and
quality factor threshold of 300. Data was processed without using baseline
subtraction and smoothing.
Data was interpreted to collect the isotope cluster area for the peptide peak area
of angiotensin I (substrate) and angiotensin II (product). The ipercent icοnversion
i(%C) iof substrate itο iprοduct iwas icalculated by the cIuster iarea of ithe iprοduct i(P)
divide iby the isum iοf the cIuster areas iοf the isubstrate i(S) iand iprοduct i(P)
multiplied iby 1 0 0 as irepresented by equation:
% Conversion = ) PP + S, × 100 (eq. 4.2)
Above equation iis ia imeasure iof the iratiο of iprοduct itο isubstrate. However, ifor
concentratiοn idependent iinhibitiοn istudies, iinhibitiοn was pIοtted ias %IA, there is
another variation of determining inhibition is by using %maximal activity (%MA)
because, itο imeasure iinhibitiοn iof ithe ienzyme iactivity, iit is needed itο measure ithe
idegree tο which ithat iactivity i(%C) iis icurtailed. Thus, ithe iconcentration dependent
inhibitiοn idata was pIοtted ias i%MA, iwhere, ithe maximal iactivity iis ithe i%C
measured iin cοntroI ireactiοns (with inο iinhibitοr) as irepresented in iequatiοn:
%Maximal Activity = ; % conversion with inh ibitor% conversion without inhibition< × 100 (eq. 4.3)
The % M A i s t he measure ment ο f the de gree o f i nhibitiοn a nd it is equivalent tο
IC50.
106
4.3 ResuIts and Discussion
4.3.1 MALDl Matrix Preparation
α -Cy anο- 4 -hydr οxy cinnamic ac id (α-CHCA) had ibeen ishοwn ito be an ieffective
imatrix ifοr ithe MALDI fοr anaIysis of ipeptides iand gIycοpeptides iin ithe mοIecular
mass irange iof 5 0 0-50 00 D a. In this range, mostly singIy prοto nated pe ptide
moIecule iοns peak appears in ithe mass spectra. While the matrix related peak
may appear up to the range of 800 Da, but in comparison to the analyte, matrix
peaks in high regain are of less intensity. Molecular structure and MALDI spectra
of α-CHCA are shown in Figure 4.1 and Figure 4.2, respectively (Beavis,
Chaudhary, and Chait, 1992). Function of MALDI matrix is to cocrystalized the
sample for analysis, crystals of matrix iabsοrb ithe ienergy ifrοm the Iaser iand then
protonate the isample molecules and produce the ions (Smirnov et al., 2004). In
this work matrix served the dual purpose along with crystallization it was used as
a reaction quencher, to stop the enzymatic reaction.
Figure 4.1: Molecular structure of α-CHCA.
The common method for the preparation of matrix is saturation method, in which
enough matrix is added to the solvent mixture and then vortexed and sonicated to
get maximum dissolution, which produced a saturated solution of α-CHCA. This
solution further centrifuge to get supernatant solution and used for spotting. For
better reproducibility it is necessary to use a proper quantitative method for
preparation of matrix rather than using the saturated method (Smolira and
Wessely-Szponder, 2015).
107
For this purpose solubility of α-CHCA was investigated and found to be 10 mg/mL
in 50% ACN i.e. 52.8 mM , therefore a solution of 60 mM was prepared in 1:1 ratio
of acetonitrile and milli-Q water, matrix was dissolved by vortex and sonicater for
5-10 minutes and used for spotting, for better results, prepared matrix was only
used for 3-4 hours, if further experiment was needed to be performed than fresh
matrix was prepared.
Figure 4.2: Spectra of α-CHCA on MALDI-MS.
4.3.2 Linearity Experiment
To perform the enzymatic assay and inhibition studies of drugs first it is needed to
establish the calibration curve by using exact quantification of substrate or product.
ln MALDI exact quantification is difficult therefore, relative quantification of
substrate and product was established to measure the enzymatic conversion. To
perform the relative quantification, both substrate and product must have to be
same or close ionization efficiency i.e. equimolar mixture should produce two
peaks of same or close intensity or peak area. To check the linear response of the
instrument, an experiment was designed which consists of 11 sample of different
ratios of product and substrate, ranged from 0-4 µM. Stock solutions of both
108
peptides were prepared of 4 µM and different volume of each was added in
sequence so that final volume should be 20 µL. Sample scheme of linearity
experiment is given below in Table 4.2.
Table 4.2: Sample scheme of linearity experiment.
Sample No. Angiotensin I (4 µM) Angiotensin II (4 µM) %Conversion
0 20 iµL 0 iµL 0%
1 18 iµL 2 iµL 10%
2 16 iµL 4 iµL 20%
3 14 iµL 6 iµL 30%
4 12 iµL 8 iµL 40%
5 10 µL 10 µL 50%
6 8 µL 12 µL 60%
7 6 µL 14 µL 70%
8 4 µL 16 µL 80%
9 2 µL 18 µL 90%
10 0 µL 20 µL 100%
All samples were analysed by optimized method and peak area of both peptides
were used for calculation of percentage conversion, then a graph of samples vs
%conversion was plotted to observe the linearity of instrument. Results are
tabulated in Table 4.3.
109
Table 4.3: Percentage conversion of different samples for linearity experiment.
Sample No. Angiotensin
I (peak area)
Angiotensin
II (peak area) Ratio
%
Conversion
0 183136 0 0.00 0.00
1 115369 16874 0.15 12.76
2 95214 33214 0.35 25.86
3 72929 35214 0.48 32.56
4 60147 42148 0.70 41.20
5 50248 51922 1.03 50.82
6 31526 47214 1.50 59.96
7 26354 52011 1.97 66.37
8 27523 82154 2.98 74.91
9 15210 90528 5.95 85.62
10 0 101257 0 100.00
110
Figure 4.3: Calibration curve for %conversion.
The % conversion was measured by using the equation 4.2, then plot between %
conversion and ratio, showed the linearity with a regression of 0.994 and linear
equation of y=9.3175x+3.4177, Figure 4.3.
As the substrate and product of angiotensin converting enzyme maintain a linearity
between their different ratios, it shows that the MALDI-MS instrument can be used
as a tool for the enzymatic reaction analysis for ACE.
4.3.3 MALDI MS Analysis
4.3.3.1 Method Parameters
Data of peptides sample spotted on MALDI Target Plate (MTP) was acquired by
maintaining all parameters constant; like analysis method, laser intensity and
number of shots added, for this purpose default peptide mixture analysis method
was used and few tweks were made to specify and enhance the analysis. To get
the reproducible results laser intensity was kept constant at 30%. All parameters
of methods are given in the Figure 4.4.
The sample carrier was set on random walk to get an accumulated spectrum of
nearby crystals rather than shooting on a specific crystal, it can help in enhancing
111
the reproducibility. Detection range was selected only to get AI and AII peptides,
all other settings in that tab were used as of default. In spectrophotometer all
voltages were set by default and suppression was turned on to hide the peak prom
matrix of solvents. In instrument specific setting all were used as of default.
Laser frequency was set to 100 Hz and in click 200 shots was set, sample was
irradiated 3 times on different location of spot and total 600 shots of laser was
saved as a single sum spectra. Only those spectra were summed which have
sufficient resolution of AI (i.e. >8000).
After acquisition, data was interpreted to collect the isotope cluster areas for the
peptide peak area of iAngiοtensin-l (substrate) iand iAngiοtensin-ll (product), and
ithe ratio iοf both was used for further calculation.
112
Figure 4.4: Parameters of analysis method. (A) sample carrier, (B) detection parameters, (C) Spectrophotometer settings and (D) instrument digitizer and detector settings.
113
After collection of analysis data, it was processed by using Bruker FlexAnalysis,
for which a default method was used along with minor tweaks, as shown in Figure
4.5.
Figure 4.5: MALDI-MS peaks detection and processing parameters.
4.3.4 Enzymatic Reaction
Enzymatic assays were performed in well plate, which contain one well as blank
or control and other contain reaction mixtures. Control or blank well contain only
enzyme and substrate, but reaction mixture contains enzyme, substrate and
inhibitors of different concentrations. All the wells contain same volume of each
enzyme, substrate and inhibitor.
114
In method optimization procedure, the total reaction volume was 13.5 μL which
was comprised of 1 μL of 0.5 μM ACE, 10 μL of 4 μM AI and 2.5 μL of buffer or
inhibitor. As it was already established that ACE performed better at 37 oC
therefore reaction mixture was incubated at 37 oC and aliquots of 1 μL were taken
out and spotted on MALDI Target Plate (MTP) in triplicates after a time interval of
5, 10, i20, i30, i40 iand i50 iminutes. iThe ireactiοn iwas iquenched iby mixing with 1 μL
of α-CHCA (premix method) and analyzed with optimized method.
The ipercent icοnversion i(%C) of isubstrate itο iprοduct was caIculated by using ithe
equation 4.2, and given in Table 4.2 and then plotted against the different time
intervals as shown in Figure 4.6.
Table 4.4: ACE reaction without any inhibitor.
Time (min)
Average peak area of triplicate run
% Conversion
Angiotensin I Angiotensin II
5 132360 12820 8.83
10 143016 37384 20.72
15 182265 76609 29.59
20 155086 91138 37.01
30 121964 101493 45.01
40 118814 128814 52.02
50 94180 134543 58.82
60 84912 133422 61.11
115
Figure 4.6: Graph for ACE reaction without any inhibitor.
The reaction of ACE enzyme without any inhibitor showed the 45% conversion of
substrate into product in 30 minutes. This time was then selected for further
inhibition studies.
4.3.5 Determination of IC50 of Standard Inhibitors
After establishing the linearity parameters of peptides, enzyme assay and
optimizing instrument analysis conditions, standard drugs were checked for their
inhibitory potential. For concentration dependent inhibition studies, inhibition can
be measure by calculating the %maximal activity (%MA). iThus, ithe iconcentration
dependent iinhibition idata can be pIotted ias i%MA vs log of concentration of
inhibitor, iwhere ithe maximaI iactivity iis ithe i%C imeasured in icontrol ireactions
(without iinhibitor) divided iby ithe i%C measured in a reaction with inhibiter, as
represented in equation 4.3 (Greis et al., 2006). But here % inhibition activity was
calculated by using the equation 3.5, rather them %MA. The %MA or %IA iis ithe
imeasurement iof ithe idegree iof iinhibition iand it is equivalent to IC50.
116
Figure 4.7: Scheme for analysis of inhibitor samples and control in well plate.
All well contains same volume of enzyme and substrate i.e. 1 µL of enzyme and
10 µL of substrate. Blank well contain 2.5 µL of milli-Q water and all other well
contain 2.5 µL of inhibitor (Captopril or Lisinopril) of different concentration. Schem
for samples of of inhibitors is given in Figure 4.7. All reaction mixtures and blank
were incubated for a standard time of 30 minutes and 1 μL οf sample frοm each
well was then spotted οn MALDI Target Plate (MTP) by quenching the reaction
with premix of 1 μL of α-CHCA and analyzed with standard method. For the
determination of %IA, all inhibitor’s reactions were compared with the control.
117
Figure 4.8: Inhibition of ACE by Captopril and Lisinopril.
A graph of % Maximal activity vs. different concentrations of Captopril and
Lisinopril was plotted and used to determine their IC50 values. iAs ithe iconcentration
iof captopriI or lisinopriI iincreased ithe conversion iof substrate to product
idecreased, even at higher concentration of both inhibitors there was no any
significant conversion. The point where %IA is 50% at y-axis the corresponding
concentration of inhibitor at x-axis is the IC50 value of that inhibitor. From the
graphs in Figure 4.8, the IC50 values of Captopril and Lisinopril iwere ifound ito ibe
8.256 iμM iand 1.962 iμM, irespectively.
4.3.6 Comparison of Captopril and Lisinopril Inhibitory Potential
By comparing the IC50 values of different inhibitors it is possible to indicate that
which inhibitor is more potent than the other and different inhibitors can be ranked
according to their inhibitory potential. Here Lisinopril is more potent than the
Captopril. This trend can also see in the spectra of MALDI-MS, in Figure 4.9 that,
in a row as the cοncentration of any inhibitοr increase, the relative intensity οf
product decrease, and in columns the relative intensity of product in Lisinopril is
less than that of Captopril.
118
Figure 4.9: Comparative MALDl-MS spectra of different concentration of Captopril and Lisinopril.
4.4 Conclusion
MALDl-TOF iMS οffers ia ivaIid readout ifor icοmpound iscreening iassays. The
ispeed, isensitivity, ireduced icοst, and reprοducibiIity of ithis iapproach imake it ian
iideaI assay ifοr idose-respοnse icurves for icοmparative iIC50 imeasurements.
MALDI-MS enzyme inhibitions assays give high sensitivity, better lower limit of
quantification and wider linear range.
ImportantIy, the idirect inature of ithe iMS measurement iensures ithe iminimization iof
ithe faIse ipositive iand faIse inegative iresults and compounds ican be screened
iwithout ithe ineed for Iabels or additionaI internaI standards, which can plague
other screening approaches. As well as fast analysis of MALDI-MS make it more
possible to screen hundreds of samples per day.
119
Chapter 5: Screening of Different Synthetic
Drugs, Compounds and Natural Extracts for
Inhibitory Potential Against ACE
120
5.1 Introduction
Angiotensin converting enzyme is an important enzyme which work in RAAS and
control the hypertensive activity of the body. There are many marketed available
drugs for the inhibition of ACE, but the prolonged use of some drugs may cause
the drug resistance. It is necessary to identify new inhibitors of clinically important
enzymes. Working mechanism and inhibition of ACE has already been discussed
in Chapter 1, in detail.
Different types and classes of ACE inhibitors has already been discussed in
Chapter 1 in section ‘1.4 Enzyme Inhibition’. Many marketed available standard
inhibitors which are used as drugs are listed in Table 1.1. Many studies have
already performed the screening of compounds against ACE, from various natural
sources including plant extracts, peptides, and Flavonoids etc. as listed in Table
1.2, Table 1.3 and Table 1.4, respectively.
There are many online databases are available with the information of kinetic
parameters and inhibitors data of ACE, two of them (Brenda-Enzyme, 2019) and
(The Binding Database) are very useful and easy to use, link of both databases
are provided in references.
With the availability of these developed rapid assays in previous chapters, it has
become easier to screen-out potent drugs in a larger number of compounds for
their potential as inhibitors. Therefore, in this study a large number of different
drugs, synthetic compounds and natural products & extracts were screened
against ACE to find out their activity and many of the compounds were found to
be active. These finding may help to tackle the ACE’s diseases in future, by
utilizing new inhibitors.
5.2 Experimental
5.2.1 Reagents and Chemicals
Angiotensin iconverting ienzyme, its substrate iangiotensin iI, product iangiotensin iII,
and MALDI imatrix α -Cyanο -4-hydr οxyc innamic iacid (α-CHCA or iHCC A) were
purchased ifrom iSigma AIdrich (USA). Two standard inhibitors Captopril and
121
Lisinopril were purchased from Tokyo Chemical Industries Co, Ltd. (Japan), both
were >98% pure. Tris buffer (research grade) was purchased from Serva
(Germany). The concentration of this buffer was prepared 20 mM with 3 mM
dithiothreitol (DTT) for the pH 7.5 at 37 oC. Acetonitrile was purchased from Fisher
Scientific (Leicestershire, UK). All other solvents used, were of spectroscopic
grade, MiIIi-Q wa ter wa s u sed fresh fr om MiIIi-Q water assembIy (Bed ford, U S A)
which had 16.5 MΩ resistance
For inhibition study standard drugs were collected from the compound bank,
synthetics compounds were prepared in-house facility and natural compounds
were purified in lab and extracts were collected in lab.
5.2.2 Enzymatic Reactions for Inhibition Study
Enzymatic assays for inhibition study were performed in well plate in different
batches, each batch contained one well as blank or control and other contain
reaction mixtures with different drugs or compounds. Control or blank well contain
only enzyme and substrate, but reaction mixture contains enzyme, substrate and
inhibitors of different concentrations. All the wells contain same volume of each
enzyme, substrate and inhibitor.
Each ireaction iweII contained 1 iµL of iangiotensin converting ienzyme (0.5 μM), 10
iµL iof iangiotensin iI (4 iμM) iand i2.5 iµL of compound or drug of idifferent
concentration in idifferent well, iboth iACE iand isubstrate iwere iprepared iin iTRIS
ibuffer iof i20 imM iwith i3 imM iDDT iof ipH i7.5 iand standard drug were prepared in milli-
Q water. Reactions were perfοrmed in incubatοr iat i37 oC for a standard time of 30
minutes.
5.2.3 Preparation of Inhibitors
Most of the compounds and drugs were soluble in water therefore their solutions
were prepared in water. Some drugs were only soluble in DMSO, their solubility
was checked in different percentages of DMSO in water and 20% DMSO in water
was selected. Those drug’s solutions were prepared and diluted in the 20% DMSO
solvent. All of the synthetic compounds were also prepared in 20% DMSO. Few
of the natural compounds and extracts were soluble in only methanolic
122
environment therefore those were dissolved and diluted in 10% methanol in water.
Standard inhibitors, Captopril and Lisinopril were prepared in water as well as 10%
DMSO separately. All the concentrations were prepared in micro molar (µM) and
milli molar (mM) except natural extracts, which were prepared in µg/mL (ppm)
concentration.
5.2.4 Sample Preparation and Analysis
All samples were preapared as per standard developed method. Those samples
in which inhibitors were prepared or diluted in DMSO were let for ~24 hours for
complete dry after spotting on MALDI plate. While water and methanolic samples
were analyses within few hours of spotting on MALDI plate.
5.3 Results and Discussion
5.3.1 Inhibition AnaIysis of Drugs/Compounds
Repurposing of standard drugs is a valid method in which marketed available
standard drugs are treated and checked for treatment for the other purposed /
diseases, if a drug can provide multi function it can be used more widely. For this
purpose, inhibition study of 77 drugs was performed against ACE, all drugs were
selected randomly from the drug data bank and prepared according to their
solubility as given in the Table 5.1.
123
Table 5.1: All drugs or cοmpounds used for the inhibitions istudy.
S. No.
Code Name Molecular
weight
Structure Chemical class Solvent
used
1 DB-000 Acyclovir 225.205
Purine Water
2 DB-001 Amlodipine Besylate
567.051
Pyridine Water
124
3 DB-002 Amoxicillin trihydrate
365.404
Penicillin Water
4 DB-003 Ampicillin Trihydrate
349.405
Penicillin Water
5 DB-005 Atorvastatin Calcium
Trihydrate
597.71
Statin DMSO
125
6 DB-007 Azithromycin dihydrate
748.984
Macrolide DMSO
7 DB-009 Ciprofloxacin HCl monohydrate
386.826
Quinoline Water
126
8 DB-010 Clarithromycin HCl
784.414
Macrolide DMSO
9 DB-012 Diltiazem hydrochloride
450.979
Benzothiazepine Water
127
10 DB-016 Indomethacin 357.788
Indoleacetic acid DMSO
11 DB-017 Itopride Hydrochloride
394.892
Benzamide Water
12 DB-020 Lidocaine Hydrochloride monohydrate
288.813
Acetamide Water
13 DB-022 Lisinopril Dihydrate
405.488
Peptide DMSO
128
14 DB-027 Ofloxacine 361.368
Quinoline DMSO
15 DB-031 Prednisolone Acetate
402.481
Pregnane Steroids DMSO
16 DB-033 Ropinirole HCl 296.836
Indole DMSO
129
17 DB-035 Sodium valproate 166.193
Fatty acid DMSO
18 DB-040 Gamma-Aminobutyric Acid
103.12
Alkanoic acid DMSO
19 DB-043 Bromazepam 316.153
Benzodiazepine DMSO
20 DB-045 Celecoxib 381.372
Benzenesulfonamide DMSO
130
21 DB-046 Chloroquine Phosphate
319.872
Quinoline Water
22 DB-052 Diclofenac Sodium
318.13
Phenylacetic acid Water
23 DB-053 Diphenhydramine Hydrochloride
291.816
Benzothiazepine Water
131
24 DB-054 Doxycycline Hyclate
480.896
Tetracycline Water
25 DB-056 Enalapril Maleate 492.519
Pyrrolidine Water
26 DB-069 Mefenamic Acid 241.285
Benzoic acid Water
27 DB-070 Mesterolone 304.467
Androstan DMSO
132
28 DB-073 Nabumetone 228.286
Naphthalenes DMSO
29 DB-076 Oxaprozin 293.317
Oxazole DMSO
30 DB-077 D-Penicillamine 149.211
Amino acid Water
31 DB-079 Ramipril 416.511
N
O O
H O
N
CO2H
H
H
Pyrrole-carboxylic acid DMSO
32 DB-082 Tranexamic acid 157.21
Cyclohexanecarboxylic acid Water
133
33 DB-083 Valsartan 435.519
Biphenyl DMSO
34 DB-084 Epinephrine Bitartrate/Adrenali
ne Bitartrate
333.291
Benzene Water
35 DB-086 Cefadroxil monohydrate
381.404
Cephalosporin Water
134
36 DB-089 Ceftriaxone Sodium 3.5 H2O
598.544
Cephalosporin Water
37 DB-091 Dextromethorphan Hydrobromide
Monohydrate
370.324
Morphinan Water
38 DB-093 Gliclazide 323.411
Thiazole DMSO
135
39 DB-094 Hydrocortisone Sodium Succinate
484.514
Pregnan Water
40 DB-096 Mirtazapine 265.353
benzazepine DMSO
41 DB-098 Norethisterone 298.419
Pregnane DMSO
136
42 DB-099 Clavulanic acid 199.161
Beta-lactam Water
43 DB-100 Clioquinol 305.5
Quinolin DMSO
44 DB-101 Cloxacillin Sodium Hydrate
475.878
Penicillin Water
45 DB-104 Lysine Hydrochloride
182.649
Amino acid Water
137
46 DB-105 Montelukast Sodium
608.165
Quinolin DMSO
47 DB-106 Quinine Dihydrochloride
324.417
Cinchonan Water
48 DB-107 Salbutamol Sulfate
339.405
Ethanolamine Water
138
49 DB-108 Sulfadoxine 310.329
Sulfonamide Water
50 DB-110 Bupropion Hydrochloride
276.202
Phenone Water
51 DB-113 Diclofenac Potassium
334.239
Phenylacetate DMSO
139
52 DB-114 Domperidone 425.911
Benzimidazole DMSO
53 DB-117 Gabapentin 171.237
γ-Aminobutyric acid Water
54 DB-120 Levetiracetam 170.209
Pyrrolidin DMSO
55 DB-123 Sertraline Hydrochloride
342.691
Naphthalen DMSO
140
56 DB-125 Beclomethasone Dipropionate
521.042
Pregnan DMSO
57 DB-127 Cefazolin Sodium 476.489
Cephalosporin Water
58 DB-128 Crotamiton 203.28
Toulidin DMSO
141
59 DB-129 Folic Acid 441.397
Pteridin Water
60 DB-132 Cefotaxime Sodium
477.447
Cephalosporin Water
61 DB-133 Cholecalciferol / Vitamin D3
384.638
Secosterol DMSO
142
62 DB-137 Metronidazole 171.154
Imidazol DMSO
63 DB-141 Venlaflaxine HCl 313.863
Cyclohexanol Water
64 DB-142 Aminophylline 240.262
Purine Water
143
65 DB-143 Fluoxetine HCl 345.787
Phenylpropylamines DMSO
66 DB-145 Pantoprazole Sodium
405.352
Benzimidazoles Water
67 DB-146 Pyridoxine HCl / Vitamin B6
205.639
Pyridine Water
68 DB-147 Rabeprazole Sodium
381.424
Benzimidazoles DMSO
144
69 DB-149 Terbutaline sulfate 325.379
Resorcinols Water
70 DB-150 (±)-Alpha-Tocopherol
acetate / Vitamin E
472.743
Chroman DMSO
71 DB-152 Enoxacin sesquihydrate
320.319
Fluoroquinolone Water
72 DB-156 Suxamethonium HCl
326.86
Quaternary ammonium compound
Water
145
73 DB-157 Thiamine HCl / Vitamin B1
337.269
Thiopyrimidine Water
74 DB-158 Topiramate 339.36
Dioxolopyrans DMSO
75 DB-191 Permethrin 391.288
Cyclopropanecarboxylate DMSO
146
76 DB-214 Caffeine 194.191
Xanthenes DMSO
77 DB-221 Papaverine (HCl) 375.846
Alkaloid DMSO
147
A total of 77 drugs were analyzed for their inhibitory potential against ACE among
them 40 were prepared in water and 37 were prepared in DMSO. Three of these
drugs were standard inhibitors of ACE. All drugs were analyzed of a high
concentration of 50 mM, those drugs whose showed the inhibition were again
analyzed with low concentrations. IC50 values of all drugs were calculated as given
below in Table 5.2.
Table 5.2: Screening results of drugs/compounds, IC50 values of active drugs.
S.No. Code Name IC50 (μM)
1 DB-000 Acyclovir Inactive
2 DB-001 Amlodipine Besylate 288
3 DB-002 Amoxicillin trihydrate 364
4 DB-003 Ampicillin Trihydrate 319
5 DB-005 Atorvastatin Calcium Trihydrate Inactive
6 DB-007 Azithromycin dihydrate 3957
7 DB-009 Ciprofloxacin HCl monohydrate 272
8 DB-010 Clarithromycin HCl Inactive
9 DB-012 Diltiazem hydrochloride 490
10 DB-016 Indomethacin Inactive
11 DB-017 Itopride Hydrochloride 296
12 DB-020 Lidocaine Hydrochloride monohydrate 645
13 DB-022 Lisinopril Dihydrate 2.663
14 DB-027 Ofloxacine 2691
15 DB-031 Prednisolone Acetate Inactive
16 DB-033 Ropinirole HCl Inactive
17 DB-035 Sodium valproate 1147
18 DB-040 Gamma-Aminobutyric Acid 3766
19 DB-043 Bromazepam 7231
20 DB-045 Celecoxib 5711
21 DB-046 Chloroquine Phosphate 342
148
22 DB-052 Diclofenac Sodium Inactive
23 DB-053 Diphenhydramine Hydrochloride 338
24 DB-054 Doxycycline Hyclate 329
25 DB-056 Enalapril Maleate 10.026
26 DB-069 Mefenamic Acid 10959
27 DB-070 Mesterolone Inactive
28 DB-073 Nabumetone 3279
29 DB-076 Oxaprozin Inactive
30 DB-077 D-Penicillamine Inactive
31 DB-079 Ramipril 8.125
32 DB-082 Tranexamic acid Inactive
33 DB-083 Valsartan Inactive
34 DB-084 Epinephrine Bitartrate/Adrenaline Bitartrate Inactive
35 DB-086 Cefadroxil monohydrate Inactive
36 DB-089 Ceftriaxone Sodium 3.5 H2O Inactive
37 DB-091 Dextromethorphan Hydrobromide Monohydrate 5258
38 DB-093 Gliclazide ~50000
39 DB-094 Hydrocortisone Sodium Succinate ~20000
40 DB-096 Mirtazapine Inactive
41 DB-098 Norethisterone 3839
42 DB-099 Clavulanic acid Inactive
43 DB-100 Clioquinol Inactive
44 DB-101 Cloxacillin Sodium Hydrate Inactive
45 DB-104 Lysine Hydrochloride 2905
46 DB-105 Montelukast Sodium Inactive
47 DB-106 Quinine Dihydrochloride 8104
48 DB-107 Salbutamol Sulfate Inactive
49 DB-108 Sulfadoxine Inactive
50 DB-110 Bupropion Hydrochloride ~55000
149
51 DB-113 Diclofenac Potassium ~46000
52 DB-114 Domperidone 1491
53 DB-117 Gabapentin Inactive
54 DB-120 Levetiracetam Inactive
55 DB-123 Sertraline Hydrochloride 581
56 DB-125 Beclomethasone Dipropionate Inactive
57 DB-127 Cefazolin Sodium Inactive
58 DB-128 Crotamiton 4383
59 DB-129 Folic Acid Inactive
60 DB-132 Cefotaxime Sodium Inactive
61 DB-133 Cholecalciferol / Vitamin D3 4691
62 DB-137 Metronidazole 2238
63 DB-141 Venlaflaxine HCl 1722
64 DB-142 Aminophylline Inactive
65 DB-143 Fluoxetine HCl 962
66 DB-145 Pantoprazole Sodium ~73000
67 DB-146 Pyridoxine HCl / Vitamin B6 15980
68 DB-147 Rabeprazole Sodium Inactive
69 DB-149 Terbutaline sulfate Inactive
70 DB-150 (±)-Alpha-Tocopherol acetate / Vitamin E 9800
71 DB-152 Enoxacin sesquihydrate 5782
72 DB-156 Suxamethonium HCl Inactive
73 DB-157 Thiamine HCl / Vitamin B1 Inactive
74 DB-158 Topiramate 3296
75 DB-191 Permethrin 1814
76 DB-214 Caffeine 846
77 DB-221 Papaverine (HCl) 2699
150
Among these drugs, 33 were inactive even a high concentration of those drugs
didn’t show any inhibition at aII. DB-93, DB-94, DB-110, DB-113 and DB-145
showed very Iow inhibition their lC50 is ranges from 20000-73000 µM (20-73 mM)
in comparison to the standard inhibitor these vaIues are very high. Other 36 drugs
showed moderate to good inhibitions with as low as lC50=272 µM (DB-9). Remining
three drugs DB-22, DB-56 and DB-79 were the standard inhibitors of ACE and
they have lC50 vaIue 2.663 µM, 10.026 µM and 8.125 µM, respectiveIy. List of top
10 most active drugs aIong with their aIready used purposes is given beIow in
Table 5.3.
Table 5.3: Ten most active drugs along with their purposes.
Code Name Purpose of Drug lC50 (μM)
DB-009 CiprofIoxacin HCI monohydrate Antiobiotic 272
DB-001 AmIodipine BesyIate CaIcium channeI bIocker, CVS
288
DB-017 ltopride HydrochIoride AChE lnhibitor 296
DB-003 AmpiciIIin Trihydrate Antiobiotic 319
DB-054 DoxycycIine HycIate Antiobiotic 329
DB-053 Diphenhydramine HydrochIoride antihistamine mainIy used to treat aIIergies
338
DB-046 ChIoroquine Phosphate antimaIariaI 342
DB-002 AmoxiciIIin trihydrate Antiobiotic 364
DB-012 DiItiazem hydrochIoride CaIcium channeI bIocker, CVS
490
DB-123 SertraIine HydrochIoride anxity, depression, panic attacks
581
5.3.2 Inhibition Study of Synthetic Compounds
Synthetic compounds were included the derivatives of tryptamine and indole.
Tryptamine is a monoamine alkaloid. It contains an indole ring structure and is
similar to the amino acid tryptophan. While indole is an aromatic heterocyclic
151
compound which is synthesized by the fusion of a benzene ring with pyrrole ring.
Structures of both compounds are shown below in Table 5.4 A and B.
Four different cIasses of above compounds were used for screening incIuding
“Schiff bases of tryptamine” which were synthesized by treating tryptamine with
different substituted benzaIdehydes in methanoI. The second cIass of “Urea and
thiourea derivatives of tryptamine” were prepared by treating tryptamine with
dichIoromethane and triethyIamine then different substituted isocyanates or
isothiocyanates were added to the reaction mixture. The third cIass of “IndoIe
acryIonitriIes derivatives” were prepared by reacting cyanoacetic acid and indoIe
aIong with acetic anhydride to produce cyanoacetyI indoIe which then react with
corresponding benzaIdehydes in presence of ethanoI and cataIytic amount of
trimethyIamine. The fourth cIass of “Bis(indoIyI) methanes” were synthesized by
reaction of indoIe with various aryI substituted aIdehydes in the presence of grape
juice which was used as cataIyst. GeneraI structures of four cIasses of derivatives
are shown beIow in Table 5.4 C-F.
Table 5.4: Structures of base compounds and general structures of derivatives classes.
A) Tryptamine
B) IndoIe
C) Schiff bases of tryptamine
D) Urea and thiourea derivatives of tryptamine
NH
HN
HN
X
R
152
E) IndoIe acryIonitriIes
F) Bis(indoIyI) methanes
All compounds of different chemical class and functional groups were prepared in-
house and checked for their inhibitory potential, for this purpose 40 different
compounds were chosen randomly among many other compounds. All
compounds as given in Table 5.5 were prepared and diluted in DMSO and
analysed with standard method.
153
Table 5.5: Synthetic compounds used for inhibition study.
S.No. Code /
na me
MoIecuIar
formuIa
MoIecuIar
weight
Stru cture Chemical cIass Solvent
us ed
1 KHA-I-35 C17H14Cl2N2O 333
Tryptamine Schiff Bases DMSO
2 KHA-I-39 C18H17BrN2O2 373
Tryptamine Schiff Bases DMSO
154
3 KHA-I-40 C17H15ClN2O 298
Tryptamine Schiff Bases DMSO
4 KHA-I-51 C18H18N2S 294
Tryptamine Schiff Bases DMSO
5 KHA-I-53 C19H20N2O2 308
Tryptamine Schiff Bases DMSO
155
6 KHA-I-58 C18H18N2O2 294
Tryptamine Schiff Bases DMSO
7 KHA-I-90 C17H15N3O3 309
Tryptamine Schiff Bases DMSO
8 KHA-II-8 C17H15BrN2O 342
NH
N
H
OH
Br
Tryptamine Schiff Bases DMSO
156
9 KHA-I-69 C17H17N3S 295
Urea and Thiourea
derivatives
DMSO
10 KHA-I-71 C17H16N4O3 324
Urea and Thiourea
derivatives
DMSO
11 KHA-I-76 C18H16F3N3O 347
Urea and Thiourea
derivatives
DMSO
12 KHA-II-29 C17H16BrN3S 374
Urea and Thiourea
derivatives
DMSO
157
13 KHA-II-43 C17H16BrN3S 374
Urea and Thiourea
derivatives
DMSO
14 KHA-II-69 C18H19N3OS 325
Urea and Thiourea
derivatives
DMSO
15 KHA-II-73 C19H21N3S 323
Urea and Thiourea
derivatives
DMSO
16 KHA-II-76 C17H16BrN3S 374
Urea and Thiourea
derivatives
DMSO
158
17 KHA-I-100 C21H18N2O3 346
Indole acrylonitriles DMSO
18 KHA-II-19 C18H11N3O4 333
Indole acrylonitriles DMSO
19 KHA-II-38 C20H16N2O3 332
Indole acrylonitriles DMSO
159
20 KHA-II-39 C20H15BrN2O3 410
Indole acrylonitriles DMSO
21 KHA-II-51 C22H19BrN2O3 438
Indole acrylonitriles DMSO
22 KHA-II-56 C19H14N2O2 302
Indole acrylonitriles DMSO
160
23 KHA-II-61 C20H14BrFN2O 396
Indole acrylonitriles DMSO
24 KHA-III-6 C20H16N2O 300
Indole acrylonitriles DMSO
25 KHA-III-10 C24H18N2O 350
Indole acrylonitriles DMSO
161
26 KHA-IV-39 C22H19BrN2O3 438
Indole acrylonitriles DMSO
27 KHA-III-59 C23H18N2 322
KHA Bis indole DMSO
28 KHA-III-66 C24H20N2O2 368
KHA Bis indole DMSO
162
29 KHA-III-71 C24H19IN2O2 494
KHA Bis indole DMSO
30 KHA-III-78 C23H17N3O2 367
KHA Bis indole DMSO
31 KHA-III-80 C23H17BrN2 400
KHA Bis indole DMSO
163
32 KHA-III-90 C23H17BrN2O 416
KHA Bis indole DMSO
33 KHA-III-97 C23H17ClN2 356
KHA Bis indole DMSO
34 KHA-IV-7 C24H20N2 336
KHA Bis indole DMSO
35 KHA-IV-45 C29H30N2O2 438
KHA Bis indole DMSO
164
36 KHA-IV-47 C29H29BrN2O2 516
KHA Bis indole DMSO
37 KHA-IV-55 C27H25BrN2 456
KHA Bis indole DMSO
38 KHA-IV-62 C29H30N2O2 438
KHA Bis indole DMSO
165
39 KHA-IV-72 C28H27FN2O 426
KHA Bis indole DMSO
40 KHA-IV-81 C25H24N2S 384
KHA Bis indole DMSO
166
Total 40 compounds were selected for screening, 8 from tryptamine schiff bases,
8 from urea and thiourea derivatives, 10 from indole acrylonitriles and 14 from KHA
bis indole. All compounds were prepared of a high concentration of 50 mM in
DMSO, those compounds which showed the inhibition were again analyzed with
the low concentration. IC50 value of all compounds is given in Table 5.6.
Table 5.6: Screening results of synthetic compounds, IC50 of active compounds.
S. No. Code / Name IC50 (µM)
1 KHA-I-35 4395
2 KHA-I-39 Inactive
3 KHA-I-40 Inactive
4 KHA-I-51 2936
5 KHA-I-53 933
6 KHA-I-58 441
7 KHA-I-90 522
8 KHA-II-8 1442
9 KHA-I-69 3439
10 KHA-I-71 Inactive
11 KHA-I-76 2116
12 KHA-II-29 1334
13 KHA-II-43 Inactive
14 KHA-II-69 1030
15 KHA-II-73 Inactive
16 KHA-II-76 1703
17 KHA-I-100 673
18 KHA-II-19 5123
19 KHA-II-38 782
20 KHA-II-39 737
21 KHA-II-51 622
22 KHA-II-56 566
167
23 KHA-II-61 ~50000
24 KHA-III-6 1207
25 KHA-III-10 2785
26 KHA-IV-39 Inactive
27 KHA-III-59 1336
28 KHA-III-66 859
29 KHA-III-71 353
30 KHA-III-78 534
31 KHA-III-80 643
32 KHA-III-90 320
33 KHA-III-97 445
34 KHA-IV-7 990
35 KHA-IV-45 1259
36 KHA-IV-47 1340
37 KHA-IV-55 1449
38 KHA-IV-62 Inactive
39 KHA-IV-72 1397
40 KHA-IV-81 2031
Among these compounds 7 were inactive, 1 compound KHA-II-61 showed very
low inhibition while all other 32 compounds showed significant inhibition activity
ranged from IC50 = 320-5123 µM
5.3.3 Inhibition Study of Natural Compounds and Extracts
Many natural compounds have already been discovered as ACE inhibitors infact
the first inhibitor of ACE was from the snake venom as already discussed in the
introduction of chapter 1. This developed method was also discovered for its
potential for screening of natural products and extracts of plants. For this purpose,
4 natural compounds (Table 5.7) and 3 methanolic extract of plants (Table 5.8),
were investigated for their potential against ACE.
168
Table 5.7: Natural compounds used for inhibition study.
S.No. Code Name Mοlecular formula
Mοlecular weight
Structure Sοlvent used
1 SC-1 7-Hydroxy Vasicine (Vasicinol)
C11H12N2O2 204.229
Water
2 SC-2 Vasicine C11H12N2O 188.0950
Methanol
3 SC-3 Quercetin C15H10O7 302.24
Methanol
4 SC-4 Vasicinone C11H10N2O2 202.0742
Methanol
169
Table 5.8: Natural extracts of plants used for inhibition study.
S.No. Code Extract name Name of plant Extract type Solvent used
5 SC-5 Piper longum MeOH
defatted (in 1% MeOH)
Long pepper or
Piper longum
Extract
defatted with
hexane
Methanol
6 SC-6 Fabago MeOH Zygophyllum
fabago Methanolic Methanol
7 SC-7 AVU-M Artemisia
vulgaris Methanolic Methanol
Among total 7 samples, 4 were natural compounds purified in-house and 3 were
natural extracts of plants also extracted in-house.
Natural compounds used in this study are the purified products of Adhatoda vasica
or Justicia adhatoda (SC-1 to 4). It is a medicinaI important pIant and it beIοngs to
ithe famiIy iAcanthaceae (Aslam et al., 2013). It iis a icοmmon imedicinaI pIant iwhich
is widely iused iin iUnani iand iAyurvedic traditiοnaI imedicines ifor irespiratοry itract
idisοrders ilike icοugh, iasthma, irheumatism, iexpectοrant iand ichrοnic ibronchitis
(Singh et al., 2017).
Lοng ipepper or iPiper Iοngum (SC-5) sοmetimes caIIed as lndian Iοng pepper or
pipli, is a fIοwering ivine and belοngs to ithe ifamiIy iPiperaceae. It is cultivated ifοr iits
ifruit, iwhich iis generally idried iand iused ias ispice. It also iused iin ithe itreatment οf
irespiratοry and idigestive disοrders.
Zygophyllum fabago (SC-6) is commonly iknown ias iSyrian ibean-caper. iIt iis
iconsidered ias a inοxious iweed of ieconοmic iimpοrtance in imany regions iof the
iwestern iUnited States. iIt iis inative to iAsia iand iEurοpe.
Artemisia vulgaris (SC-7) commonly know as mugwort is mostly found in
temperate Europe, Asia and northern Africa, where it is considered as invasive
weed. It is used for pain relif, treamt of fever and it is also used as diuretic agent.
Compounds 1-4 were prepared in a high concentration of 50 mM and extracts 5-
7 were prepared in high concentration of 5000 µg/mL (ppm).
170
Table 5.9: Results of natural compounds and extracts, IC50 of active samples.
S.No. Code Name IC50 Value
1 SC-1 7-Hydroxy Vasicine 9705 µM
2 SC-2 Vasicine 496 µM
3 SC-3 Quercetin 7124 µM
4 SC-4 Vasicinone 2990 µM
5 SC-5 Piper longum Inactive
6 SC-6 Zygophyllum fabago Inactive
7 SC-7 Artemisia vulgaris 10779 µg/mL
(ppm)
All four natural compounds showed moderate activity, with IC50 value 496 µM -
9705 µM; while in plant extracts only SC-7 of “Artemisia vulgaris” showed some
inhibitory activity with IC50 of 10779 µg/mL (ppm) while other did not showed any
activity. Results are tabulated in Table 5.9.
5.4 Conclusion
Developed method of MALDI-MS had proved to be very effective for screening of
different types of compounds or drugs. In this work, a total of 124 different
compounds/samples from different classes of compounds, including 77
commercial drugs, 40 synthetic compounds, 4 natural products and 3 extracts of
plants were investigated for their inhibitory potential. Among these compounds /
samples a range of activity was founds in different compounds.
In commercial drugs, 36 drugs showed moderate to good inhibition of as low as
IC50 = 272 µM. In drugs analysis, 3 ACE standard inhibitors were also screened
blind folded, which further proved the authenticity and reproducibility of the assay.
Those drugs which found to be active against ACE can be further studied for their
repurposing as ACE inhibitors. In synthetic compounds, 32 compounds showed
significant inhibition ranged from IC50 = 320-5123 µM. Further derivatives of those
171
drugs and standard inhibitors of ACE can be synthesized for “structural activity
relationship study”, to find more potent inhibitors of ACE. In the last category, all 4
natural compounds were moderately active with IC50 = 496-9705 µM; while only
one plant extracts showed some inhibition with IC50 = 10779 µg/mL (ppm).
Among screened compounds most οf them were found to be good to moderate
inhibition activity against ACE. This methodology ifurther ican ibe iused ifοr ithe
iscreening iοf different drug libraries itο iidentify the Iead icοmpounds as more potent
inhibitors of ACE.
172
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