Drug Discovery and Molecular Modeling Drug Discovery and Molecular Modeling LaboratoryLaboratory

Pavel A. PetukhovPavel A. Petukhov

Department of Medicinal Chemistry and PharmacognosyDepartment of Medicinal Chemistry and PharmacognosyCollege of PharmacyCollege of Pharmacy

University of Illinois at ChicagoUniversity of Illinois at Chicago

Office 304, College of PharmacyOffice 304, College of Pharmacy

ee--mail: mail: pap4@uic.edupap4@uic.eduhttp://medchem.pharm.uic.edu/http://medchem.pharm.uic.edu/

Research interestsResearch interests

Drug discovery using a combination of computerDrug discovery using a combination of computer--aided molecular design, aided molecular design, medicinal chemistry, and biologymedicinal chemistry, and biology

Molecular modeling of the targets of interest and design of new Molecular modeling of the targets of interest and design of new inhibitors inhibitors de novode novo

Photoaffinity labeling and its application in drug designPhotoaffinity labeling and its application in drug design

Development of new computerDevelopment of new computer--aided molecular design methodsaided molecular design methods

Current therapeutic projects/ Current therapeutic projects/ targetstargets

Current projectsCurrent projectsHistone deacetylase Histone deacetylase ––HDACHDAC-- inhibitors inhibitors (cancer) (cancer) –– funded by Breast Cancer funded by Breast Cancer Research Program, Department of Research Program, Department of DefenseDefenseBetaBeta--secretase 1 inhibitors secretase 1 inhibitors ––BACE1 BACE1 --(Alzheimer(Alzheimer’’s disease) s disease) –– funded by NIH, funded by NIH, National Institute on AgingNational Institute on AgingPantothenate synthetase inhibitors Pantothenate synthetase inhibitors (Tuberculosis) (Tuberculosis) –– funded by NIH, National funded by NIH, National Institute of Allergy and Infectious Institute of Allergy and Infectious DiseasesDiseasesPhotoaffinity labeling probes for Photoaffinity labeling probes for development of isoform selective HDAC development of isoform selective HDAC inhibitors (cancer, neurological inhibitors (cancer, neurological diseases) diseases) –– funded by NIH, National funded by NIH, National Cancer InstituteCancer InstituteMalate synthase (Tuberculosis) Malate synthase (Tuberculosis) ––submission pendingsubmission pending

CollaborationsCollaborationsDesign of Calpain inhibitors Design of Calpain inhibitors ––AlzheimerAlzheimer’’s disease, in collaboration s disease, in collaboration with Dr. Greg Thatcher (UIC) and with Dr. Greg Thatcher (UIC) and Ottavio Arancio (Columbia University)Ottavio Arancio (Columbia University)

Method developmentMethod developmentDevelopment of automated Development of automated system for in silico screening of system for in silico screening of available chemical compoundsavailable chemical compoundsDevelopment of advanced Development of advanced scoring/ranking procedures for scoring/ranking procedures for various targetsvarious targetsExpert, knowledgeExpert, knowledge--based systems based systems for computerfor computer--aided drug designaided drug design

Molecular modeling and drug Molecular modeling and drug design laboratorydesign laboratory

Graduate Students:Boobalan Pachaiyappan (CAMD + medicinal chemistry)Kelvin Bai He (CAMD + molecular biology)Isaac Schiefer (CAMD + medicinal chemistry), joint with Dr. Greg Thatcher)

Visiting Fellows:Reaz Uddin

Postdocs:Dr. Michael Brunsteiner (computer-aided drug design)Dr. Gilles PieffetDr. Subash Velaparthi (synthetic organic chemistry)

Former postdocs:Dr. Fero Bizik, FDADr. Ae Nim Pae (Assistant Professor Korean Institute of Science and Technology)Dr. Dahua Pan (industry)Dr. Hongbin Yuan (Burnham Institute, San Diego, CA)Dr. Srinivas Reddy Chirapu (Scripps Research Institute, San Diego, CA)

Where are we in the food chain ?Where are we in the food chain ?

Overview of the facilitiesOverview of the facilities

softwareSybyl (Tripos)InsightII, DS Modeling (Accelrys)OpenEye MOE (CCG)Academic software

Medicinal chemistryMedicinal chemistry

wet labs with all wet labs with all necessary equipmentnecessary equipment

ComputerComputer--aided drug designaided drug design

Research projectsResearch projects

Computer aided molecular designComputer aided molecular designMedicinal chemistryMedicinal chemistry

Development of new medications for cancer, Development of new medications for cancer, AlzheimerAlzheimer’’s disease and neurological s disease and neurological diseases, tuberculosisdiseases, tuberculosis

Development of new drug discovery toolsDevelopment of new drug discovery toolsDecision trees, artificial intelligence Decision trees, artificial intelligence algorithms, data mining for Quantitative algorithms, data mining for Quantitative StructureStructure--Activity Relationship (QSAR)Activity Relationship (QSAR)

IsoxazoleIsoxazole--33--carboxamide Derivatives as Novel carboxamide Derivatives as Novel Potent Inhibitors of Pantothenate Synthetase: Potent Inhibitors of Pantothenate Synthetase:

Initiating a Quest for New AntiInitiating a Quest for New Anti--tubercular tubercular DrugsDrugs

TuberculosisTuberculosis

Mtb is the slow growing bacteria and can survive in the human body for many years in the dormant state, in which case people are infected but show no signs of the disease

Tuberculosis most commonly attacks the lungs (as pulmonary TB) but can also affect the central nervous system, the lymphatic system, the circulatory system, the genitourinary system, bones, joints and even the skin

Tuberculosis (abbreviated as TB for tubercle bacillus) is a common and deadly infectious disease caused by mycobacteria, mainly Mycobacterium tuberculosis

Rifampin (RMP)

Rifapentine

Current TB Drugs Current TB Drugs

N

OHN

NH2

Isoniazid(INH)

NH

HNMe

Me

OH

HO

Ethambutol

N

NNH2

O

PyrazinamideNH2

OH

O OH

ONH

OH2N

CycloserineN

S NH2

Me

Ethionamide (ETA)

OH

HO OOH

N

H2N

H2N N

H2NNH2

OO

O

HN

OH

OH

OH

OH

Streptomycin

p-aminosalicylic acid (PAS)

SignificanceSignificanceOne third of the worldOne third of the world’’s human population is thought to be infected withs human population is thought to be infected with Mycobacterium Mycobacterium tuberculosis tuberculosis ((MtbMtb) and there are 8 million new cases of tuberculosis (TB) each ye) and there are 8 million new cases of tuberculosis (TB) each year.ar.

Strains of Strains of MtbMtb resistant to existing drugs are found in nearly every country aresistant to existing drugs are found in nearly every country and a percentage of nd a percentage of these are resistant to multiple drugs making effective treatmentthese are resistant to multiple drugs making effective treatment extremely expensive and in extremely expensive and in many cases impossible.many cases impossible.

One of the hallmarks of One of the hallmarks of Mtb Mtb is the persistent phase of infection, when the bacteria are not is the persistent phase of infection, when the bacteria are not actively growing and overall metabolic activity is down regulateactively growing and overall metabolic activity is down regulated, often termed nond, often termed non--replicating replicating persistence (NRP).persistence (NRP).

Most currently available drugs are not effective against NRPMost currently available drugs are not effective against NRP--MtbMtb thus requiring a minimum of 6 thus requiring a minimum of 6 months of therapy to prevent relapse. Longmonths of therapy to prevent relapse. Long--term chemotherapy inevitably increases the risk of term chemotherapy inevitably increases the risk of drug resistance. Therefore the discovery and development of drugdrug resistance. Therefore the discovery and development of drugs effective against NRPs effective against NRP--MtbMtb is is considered the highest priority among TB drug discovery efforts.considered the highest priority among TB drug discovery efforts.

Target validationTarget validationRecent publications suggested that pantothenate synthetase (PS) Recent publications suggested that pantothenate synthetase (PS) involved in lipid biosynthesis may, indeed, be a potential involved in lipid biosynthesis may, indeed, be a potential antimicrobial target.antimicrobial target.

Pantothenate synthetase catalyzes amide bond formation of Pantothenate synthetase catalyzes amide bond formation of pantothenate from Dpantothenate from D--pantoate and pantoate and ββ--alanine accompanied by alanine accompanied by hydrolysis of Mghydrolysis of Mg--ATP into AMP and MgATP into AMP and Mg--PPPPii. Pantothenate is a key . Pantothenate is a key precursor of coenzyme A and acyl carrier protein, essential for precursor of coenzyme A and acyl carrier protein, essential for many intracellular processes including fatty acid metabolism, cemany intracellular processes including fatty acid metabolism, cell ll signaling and synthesis of polyketides and nonsignaling and synthesis of polyketides and non--ribosomal peptides.ribosomal peptides.

A gene (panC) knock out of PS in A gene (panC) knock out of PS in Mtb Mtb results in a highly attenuated results in a highly attenuated phenotype in immunophenotype in immuno--compromised SCID mice and in immunocompromised SCID mice and in immuno--competent BALB/c mice. The PS pathway is not present in humans. competent BALB/c mice. The PS pathway is not present in humans.

Pantothenate synthetasePantothenate synthetase

HO CO2-

OHH

HO

OHH

O

OP

O-

O O

OHHO

N

N

N

N

NH2

HO

OHH

O

NH

CO2-O H2N

COOHATP

Pantoate Pantoyl adenylate Pantothenate

N

NN

N

NH2

O

OHHO

OPO

OO

O

OHOH

ONH2

Gln72O

NH2

Gln164

Met40Phe67Val139Leu146Pro38Val142Val143

Ile168Phe157

Tyr82

Ser197

His47 His44 O

Met195

H N Val187

Leu50

Lys160

Val184

multiple interactionswith the backbone CO andNH are not shown

Asp161

pantoatesubpocket

adeninesubpocket

sugarsubpocket

entrance

Structures of active compounds 1a and 1b Structures of active compounds 1a and 1b and their inactive analogs 2aand their inactive analogs 2a--ff

O

ONH

N

N

N

1a, R = ortho-Me1b, R = para-F

Ractive

O

ONH

N

N

N

2a, R1= H, R2, R3 = H, R4 = 2, 4-dichlorophenyl2b, R1= H, R2, R3 = CH3, R4 = 2-methylphenyl2c, R1= t-butyl, R2, R3 = CH3, R4 = 2-methylphenyl2d, R1= meta-methoxyphenyl, R2 = phenyl2e, R1= 2-furanyl, R2 = phenyl

R2

R3

inactive

R1

R4

O

ONH

N

N

N

R1

R2

O

ONH

N

N

N

2a-c 2d,e 2f

Two plausible poses suggested by Two plausible poses suggested by docking and MDdocking and MD

Green - hydrophobic, light-purple - polar, blue ring - basic, red ring - acidic

Tetrahydrobenzoisoxazole ring of 1a occupies either Tetrahydrobenzoisoxazole ring of 1a occupies either the position of the adenine ring (shown below) or the the position of the adenine ring (shown below) or the

position of the pantoyl portion of the reaction position of the pantoyl portion of the reaction intermediate cointermediate co--crystallized with PS in PDB:1N2Hcrystallized with PS in PDB:1N2H

Newly designed compoundsNewly designed compounds

O

ONH

N

N

N

R

3a, R = Ph3b, R = 2-BrC6H43c, R = 4-ClC6H43d, R = 2-IC6H43e, R = 2-naphthyl3f, R = 4-(CO2CH3)C6H43g, R = 2, 4-F2C6H33h, R = 4-(CO2H)C6H43i, R = 4-(CO2C(CH3)3)C6H43j, R = 4-(CONHCH2Ph)C6H43k, R = 4-(CONH(CH2)2Ph)C6H4

3a-k

O

ONH

N

N

N

R

O

ONH

N

NO

R

3l, R = 2,4,6-Trichlorobenzoyl 8, R = H

3m, R = 4-(OCH3)C6H4

H2NOC

Synthesis of the novel ligandsSynthesis of the novel ligands

NO

CO2Et

O

(CO2Et)2

O

CO2Et

O

NO

CO2H4 5 6 7

a b c

RCH2X

9a, R = Ph, X = Br9b, R = 2-BrC6H4, X = Br9c, R = 4-ClC6H4, X = Cl9d, R = 2-IC6H4, X = Cl9e, R = 2-naphthyl, X = Br9f, R = 4-(CO2CH3)C6H4, X = Br9g, R = 2, 4-F2C6H3, X= Br

NH

N

H2N

aReagents and conditions: (a) NaOEt, EtOH, 4h, reflux, 52 %; (b)NH2OH.HCl, EtOH, 1h, reflux, 78%; (c) 2N NaOH, MeOH, 1h, 89%0 °C-rt; (d) EDC, HOBT, DIPEA, CH2Cl2, 6h, 62%; (e) NaH, DMF 0°C-rt; (f) 2,4,6 trichlorobenzoyl chloride, NaH, THF, 0 °C, 1h, 61%.

d

9a-h

O

ONH

HN

N

N

O

ONH

N

N

N

R

3a, R = Ph (53%)3b, R = 2-BrC6H4 (63%)3c, R = 4-ClC6H4 (71%)3d, R = 2-IC6H4 (69%)3e, R = 2-naphthyl (57%)3f, R = 4-(CO2CH3)C6H4 (70%)3g, R = 2, 4-F2C6H3 (61%)

3a-h

e

8

O

O

NH

N

NN

RO

f

3l, R = 2,4,6-Trichlorophenyl

Inhibition of Pantothenate Synthetase Inhibition of Pantothenate Synthetase (IC(IC5050, nM) by 1a,b, 2a, 3a, nM) by 1a,b, 2a, 3a--m, 7 and 8m, 7 and 8

Compd % inhibition at 100 μM

IC50

1a 89.17 120 ± 6a,c

1b 98.36 150 ± 9a,c

2a 18.25 NDa,b,d

3a 100.00 97 ± 4c

3b 93.54 140 ± 9c

3c 98.41 140 ± 9c

3d 84.34 160 ± 8c

3e 97.45 90 ± 1c

3f 100.00 160 ± 1c

3g 99.95 130 ± 10c

3h 99.14 460 ± 42c

3i 79.34 250 ± 8c

3j 81.79 210 ± 10c

3k 78.06 140 ± 8c

3l 17.43 NDb,d

3m 78.74 7130 ± 297c

7 12.06 NDb, d

8 43.24 61000 ± 5650c

The assays were conducted by the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) through a research and development contract with the U.S. National Institute of Allergy and Infectious Diseases (NIAID) of the NIH.

SARSAR

These studies identified These studies identified tt--butyl and pyrazole portions of the PS inhibitors butyl and pyrazole portions of the PS inhibitors as the two areas containing the key pharmacophore elements.as the two areas containing the key pharmacophore elements.On the other hand, the substituents in the aryl moiety of the pyOn the other hand, the substituents in the aryl moiety of the pyrazole razole portion are well tolerated, suggesting that this part of the scaportion are well tolerated, suggesting that this part of the scaffold is an ffold is an auxophore and, thus, it may be used to fineauxophore and, thus, it may be used to fine--tune ADMET profile.tune ADMET profile.Further more drastic modifications of the scaffold would be requFurther more drastic modifications of the scaffold would be required to ired to determine the binding pose of the inhibitors.determine the binding pose of the inhibitors.These findings are an important step in development of PS inhibiThese findings are an important step in development of PS inhibitors and tors and validation of PS as a therapeutic target for NRPvalidation of PS as a therapeutic target for NRP--TB.TB.

O

O

NH

N

N N

pharmacophores

auxophore

Future directions: FollowFuture directions: Follow--up on in silico up on in silico screening for novel scaffoldsscreening for novel scaffolds

Applied FILTER 2.0 software to select druglike compounds only

In silico screeningIn silico screening

AcknowledgmentAcknowledgment

Reaz Uddin Dr. Michael Brunsteiner (computer-aided drug design)Dr. Subash Velaparthi (synthetic organic chemistry)Dr. Baojie WanDr. Scott G. Franzblau

This research was supported by the NIAID/NIH grant R21 AI070997 and the Institute for Tuberculosis Research at the University of Illinois at Chicago.

PS inhibition assays were funded by the Division of Acquired Immunodeficiency Syndrome of the NIAID/NIH.

The financial support for R.U. was provided by the Higher Education Commission of Pakistan.

The authors thank OpenEye Scientific Software for providing academic license for modeling software.