NATURAL PRODUCTS DRUG
DEVELOPMENT OF BIOACTIVE
COMPOUNDS FROM
INDONESIAN BIODIVERSITY
Umar Anggara Jenie,
Muhammad Hanafi, Leonardus B.S. Kardono
Indonesian Institute of Sciences, Kawasan PUSPIPTEK,
Serpong 15314, Indonesia
INTRODUCTION
Several Approach on Natural Products Drug
Discovery in Indonesian Research
Institute of Sciences
�� Bioactive Natural Products from Indonesian Biodiversity,Bioactive Natural Products from Indonesian Biodiversity,
�� SemisynthesisSemisynthesis/Structure Modification based on /Structure Modification based on rational rational
drug design.drug design.
�� Genetic Engineering using Genetic Engineering using molecular farmingmolecular farming techniquetechnique..
��Considered as the oldest, and Considered as the oldest, and
��The most intensive area of research in The most intensive area of research in
Natural Products Drug Discovery
��The most intensive area of research in The most intensive area of research in Indonesia, since Indonesia rich in biodiversityIndonesia, since Indonesia rich in biodiversity
��Natural selection and interspecies Natural selection and interspecies competition resulted the various chemical and competition resulted the various chemical and bioactive compound diversity potential for bioactive compound diversity potential for drug discovery. drug discovery.
� Biodiversity screening selecting the starting materials to search the biological active constituents, isolation dan purification of the active constituents, and determintation of chemical structure,
Natural Product Drug Development
In Indonesian Institute of Sciences:
chemical structure,
� Calculation of structure activity relationship, drug design and analog synthesis
� Study the drug interaction of the selected molecule and target protein (patologic protein),
� Synthesis desain for the bioactive molecule and increasing the production process through synthesis, fermentation and genetic engineering, ,
� Available Instruments in Indonesian Institute of
Sciences: UV-Vis Spectrometer, FT-IR, FT-
Structure determination is
important in natural products drug
discovery and development.
Sciences: UV-Vis Spectrometer, FT-IR, FT-
NMR (500 MHz), dan Mass Spectrometer
(GC/LC-MS)
� X-ray crystalograph: not available.
Indonesian Biodiversity Based Indonesian Biodiversity Based
Natural Product Development Natural Product Development
database /genes
protein targets
chemical
diversity identify ‘hit’
optimize ‘hit’ structure test safety/efficacy
animal human
Diagram 1. Natural Product Drug Development from new
information to new therapy (Guo et al., 2006)
Bioactivity Screening
from Natural Products
Endophytic fungi from T. sumatrana
(31 Strains) for anticancer screening
Cell Morfologi T47D
(a) sel tanpa perlakuan (b) perlakuan dgn TsC3
[22µg/mL]
CYTOTOXIC ASSAY of TsC3to T47DCYTOTOXIC ASSAY of TsC3to T47D
sel hidup, tampak seperti helaian daun yang menempel pada dasar sumuran
sel yang telah mengalami perubahan morfologis, tampak membulat dan mengapung
5 active mycroorganisme :
• 3 Endogenic mycrobe (S49, TT 41 dan TT 10)
• 2 Endofitic mycrobe (TsC2 dan TsC11) .
Antibiotic Discovery
Test Organism:
Escherichia coli, Staphylococcus aureus, Bacillus subtilis,
Pseudomonas aeruginosa Salmonella typhi , Salmonella
para typhi A, Mycrosforum gypseum, Thricopyton sp.
Aspergillus niger, Candida albican.
Mikroba endofitikMikroba endogenik
BIOACTIVITIES OF WATER AND ETHANOL EXTRACTS OF BIOACTIVITIES OF WATER AND ETHANOL EXTRACTS OF Indonesian Mistletoes (Indonesian Mistletoes (DendrophthoeDendrophthoe pentandrapentandra L. L. MiqMiq and and
MacrosolenMacrosolen cochinchinensiscochinchinensis ((Lour van Lour van TieghTiegh))
Macrosolens cochinchinensis
(Lour) van Tiegh.Dendrophthoe pentandra L. MIQ
In vitro In vitro antIcancerantIcancer assay assay
using using cancercancer cell linecell line
OOH
HO O
OH
OH
OH
12
345
6
78
9
10
1'
2'
6'
3'4'
5'
Quersetin
OOH
HO O
OH
O
OH
OH
OH
O
OH
12
345
6
78
9
10
1'
2'
6'
3'4'
5'
Quersitrin (Quersetin-3-ramnosida)
+ M. cochinchinensis - D. pentandra
In vivo In vivo antIcancerantIcancer assay using mice assay using mice
induced with carcinogeninduced with carcinogen
Isolation and
Structure Elucidation
of Active Compoundof Active Compound
O OO
O
COOH
12
4a10a
6
7 9
5
14
11
17
19
2
21
27
28
20
3133
35
34
H
13
O OO
O
COOH
CYTOTOXIC COMPOUNDS FROM
Garciniaa gaudichaudii,
Xu, Y.J., S. Kosela, Enny F., Hanafi, M., et al., Org. Lett., 2000, 2, 3945
OHO
178
911 21
23
25
30
HHOH
36
37
1
OHO
H
2
O O
OHO
O
O
COOH
8
5
H
3.R=CH34.R=CH2CH3
RO H
Gaudichaudiic acids F - I (1 - 4) showedcytotxic to Artemia salina brine shrimp at LC50
value of 2.6; 6.55; 4,32 and 11,3 µg/ml,
Cytotoxic to the P388 cell line at IC50 of 4.6,3.4, 2.0, 1.7 µg/ml, rtespectively.
O
OOH
OOH
HO
HO
HO
HO
O
OH
LEAD COMPOUNDS FOR ANTIDIABETES (a-Glucosidase inhibitor)
from plant and fungiIC50 10 – 20 µg/ml
OHO
OH
OH
O
HO
HO
O HO
OHO
OO
Sulochrin (A) IC50 8,5 µg/ml
N
HO
HO
OH
OH
nojirimisin (IC50 5 µg/ml)
From Koji Extract of A. terreus
150
200
250
Kontrol
Acarbose
Glucose tolerant test (GTT) of Sulochrin
0
50
100
H0 H 30
menit
H 60
menit
H 90
menit
H 120
menit
H 180
menit
0.41 mg
0.82 mg
1.64 mg
H H
H
OOH
HO
OH
Lead Compounds for Cardiavascular from Artocarpus
HO O
O
OH
OH
HO
O
HO
OH
OH
O
OH
Umar A. Jenie, et al., P00200700707, 2007
Structure –Activity Studies
and Synthesis of analogs
�� CompoundCompound activityactivity:: lipophyliclipophylic,, electronicelectronic andand stericstericgroupsgroups
� Studi in silico: Activity improvement :: QSAR,QSAR, dockingdocking viaviaHMGHMG CoACoA reductasereductase
Development of LOVASTATIN
AS ANTICHOLESTEROL
HMGHMG CoACoA reductasereductase
�� SemisyntheticSemisynthetic StudyStudy fromfrom isolatedisolated compoundcompound fromfrom AA..terreusterreus solidsolid statestate fermentationfermentation intointo Dehidrolovastatin
�� SimvastatinSimvastatin isis usedused asas referencereference compoundcompound asas selectedselectedanticholesterolanticholesterol statinstatin duedue toto itsits abilityability forfor increasingincreasing thetheHDLHDL
�� ToxicityToxicity evaluationevaluation :: acuteacute andand subsub--chronicchronic..
DEHIDROLOVASTATIN DERIVED FROM
LOVASTATIN ISOLATED FROM
Aspergillus terreus
Sintesis
QSAR Parameter Identification
O
O
O
O
Activity evaluation
In vivo
Drug Design
Hyperchem &Docking
Active
Anticholesterol
compound
Activity evaluation Activity evaluation
111.79
156.66
112.03 106.64 105.54
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
1 2 3 4 5
Kelompok
Evaluation Results Total cholesterol(mg/dl) Evaluation Results: HDL (mg/dl)
Properties of orally Available Properties of orally Available
DrugDrug--like Compoundslike Compounds
The Lipinski : Rule of five criteriaThe Lipinski : Rule of five criteria
��Molecular weight 500 Molecular weight 500 DaDa
�� Log Log PP ≤ 5 ≤ 5 �� Log Log PP ≤ 5 ≤ 5
��Hydrogen bond donors (OH and NH) ≤5 Hydrogen bond donors (OH and NH) ≤5
��Hydrogen bond acceptors (loneHydrogen bond acceptors (lone--pairs of heteropairs of hetero--atoms, like O and N) atoms, like O and N)
��Number of heavy atoms 10Number of heavy atoms 10––70 70
CH3
O
H3C
H3C
O
HO O
O
H
CH3
O
H3C
H
H3C
O
H
HO O
O
H3C CH3
Log P 3.77Log P 5.73
Log P 5.68
SIMVASTATIN & LOVASTATIN DERIVATIVES AND LOG P
H
CH3
O
H
CH3
H3C
O
H
O O
O
O
10
C30H46O6Exact Mass: 502.33
H3C
Lovastatin (1) Simvastatin(2)
CH3
O
H3C
O
O
O
18
Log P 4.8
H
OH
H
OH
H
HO O
O
Log P 4.6
H3C
10
Interaction Dehydrolovastatin
(grey)
and the active site of HMG-CoA reductase
(dark)
ArgusLab 4.0
HyperChem 7.0
(dark)
NONO CompoundsCompounds InteractionInteraction EnergyEnergy ((kcalkcal / mol)/ mol) Log PLog P
11 SubstratSubstrat (HMG(HMG--CoACoA)) -- 10,505510,5055
22 DehydrolovastinDehydrolovastin -- 9.959.95 4.804.80
33 Lovastatin (1)Lovastatin (1) -- 9,489,48 3.773.77
44 Simvastatin (2)Simvastatin (2) -- 8,868,86 5.735.73
55 Buthyl ester (Lovastatin)Buthyl ester (Lovastatin) -- 9,919,91 4,924,92
INTERACTION ENERGY WITH HMG CoA REDUCTASE AND LOG P
Synthesis Synthesis DehydrolovastatinDehydrolovastatin
CH3
OH3C
O
O
O
CH3
OH3C
O
O
O
HO
pTsOH, Cyclohehane
CH3
H3C
CH3
H3C
Lovastatin Dehydrolovastaton
88,3 % (EtOH)
Heksan:EtOAC (4:1)
Lovastatin
BIOLOGICAL ACTIVITIES OF DEHYDROLOVASTATINBIOLOGICAL ACTIVITIES OF DEHYDROLOVASTATIN
106.29
172.53
102.28 103.8594.79
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
trigliserida
1 2 3 4 5
1
111.79
156.66
112.03 106.64 105.54
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
1. Normal control, 2. Induced Cholesterol, 3.Simvastatin 7.2 mg/200g bw/day.
4.Dehydrolovastatin 7.2 mg/200 g bw/day, 5. Dehydro lovastatin 14.4 mg/200gbw/day,
1 2 3 4 5
Kelompok
Cholesterol Total (mg/dl) Triglyceride (mg/dl)
HDL (mg/dl)LDL (mg/dl)
0.001 2 3 4 5
Kelompok
Parameter Normal
control
Hiperlipi-
demic
Simvastatin
(7,2 mg/
200 g bw)
Dehydrolova
statin
(7,2 mg/
200 g bw)
Dehydrolova
statin
(14,4 mg/
200 g bw)
Total
cholesterol
(mg/dl)
(%)
111,79 156,66 112,03
(28,49%)
106,64
(31,93 %)
105,54
(32,55 %)
Trigliseride 106,29 172,53 102,28 103,85 94,79
Evaluation Results of Antihiperlipidemic Activity on Mice
for Dehydrolovastatin and Simvastatin
Trigliseride
(mg/dl)
(%)
106,29 172,53 102,28
(40,72%)
103,85
(40,0%)
94,79
(45,06%)
LDL-
cholesterol
(mg/dl)
(%)
32,34 72,99 30,23
(58,58%)
25,00
(65,75%)
28,77
(60,58%)
HDL-
cholesterol
(mg/dl)
(%)
58,20 49,16 61,34
(24,77%)
60,87
(23,82%)
57,81
(17,60%)
Acute Toxicity of Dehydrolovastatin
Body weight development of average female mice
Liver Parameter (Subchronic toxicity)
28.91
30.66 35.8731.77
0
10
20
30
40
Aktivitas ALT
Rata-rata (U/L)
I II III IV
Kelompok Perlakuan
�Gambar 1. Diagram rata-rata aktivitas ALT plasma pada
tikus putih jantan setelah diberi perlakuan selama 60 hari.Gambar 2. Diagram rata-rata aktivitas ALT plasma pada
431.02 434.24 437.46 433.78
0
100
200
300
400
500
Aktivitas ALP
Rata-rata
(U/L)
I II III IV
Kelompok Perlakuan
Keterangan: ALT = SGPT ; ALP= Alkali fosfatase
Keterangan: kelompok I = kelompok dosis 0,9 mg/200 g bb; kelompok II = kelompok dosis 1,8 mg/200 g bb
kelompok III = kelompok dosis 3,6 mg/200 g bb; kelompok IV = kelompok kontrol normal.
�tikus putih jantan setelah diberi perlakuan selama 60 hari.Gambar 2. Diagram rata-rata aktivitas ALT plasma pada
tikus putih betina setelah diberi perlakuan selama 60 hari.
Gambar 3. Diagram rata-rata aktivitas ALP plasma pada tikus
putih jantan setelah diberi perlakuan selama 60 hari. Gambar 4. Diagram rata-rata aktivitas ALP plasma pada
tikus putih betina setelah diberi perlakuan selama 60 hari.
Diameter of Hystology Glomerulus &
Glomerulus - Capsula bowman In Rat
Male 1,8 mg/200 g bw Male 3.6 mg/200 g bw
Male 7,2 mg/200 g bw Control
Vena Centralis Liver Diameter in Rat
Jantan 1,8 I Jantan 3,6 I
Male 1,8 mg/200 g bw Male 3.6 mg/200 g bw
Jantan 1,8 I Jantan 3,6 I
Male 7,2 mg/200 g bw Control
Development of UK-3A analog potential for
Breast cancer treatment
Lipinski Rule
Hyperchem Program
Virtual Interaction
(molecular docking)
Structure Analog Design UK3A
in silico
Hyperchem Program
MW < 500 g/mol; log P < +5
(molecular docking)
ArgusLab program
HyperChem Pro & ArgusLab 4.0 Results
HyperChem Pro 7.0
34
HyperChem Pro 7.0
ArgusLab 4.0
Interaction of Protein BcL-xL & Analog UK-3
UK-3A Analog Development
N
OH
NH
O
O
O
DEVELOPMENT OF ANALOG UK-3A
POTENTIAL FOR BREAST CANCER TREATMENT
PSMOEPSMOE
N
OH
NH
O
O
O
O
A
B C
O
PSMOE
H O
O
O
OUK-3A
UK-3A Ring opening (Analog UK-3A)
HN
O
O O
N
OOH
O
BcL-xL Protein
QSAR Parameter & Cytotoxic
Test Results N
OH
HN
O
OH
O O
CH3
O
O
O
OOR
HN
N
OOH
O
UK-3A
Log P -1.18Ebinding = -7.1 kcal/mol
Log P 1.61Ebinding = -11.65 kcal/mol Ebinding = -7.1 kcal/mol
IC50 = >100 µµµµg/mlEbinding = -11.65 kcal/molP388 : IC50 = 38 µµµµg/ml
O
O
OOH
OO
O
OOHO
O
O
OH
NH
OTaxol
Log P 1.67Ebinding = -10.39 kcal/mol
O
OHN
OOH
HN
O
O
OH O
OAntimycin A3
Log P 1.30, Ebinding = -10.24 kcal/mol
KB :IC50 = 0.23 mg/mlYMB-1:IC50 = 0.015 mg/ml
Cytotoxic Test Results to P388, KB and YMB-1
NHN
O
O
OH
O
OO
PDBGE : R = ButylIC50 34 µµµµg/ml (P388)IC50 2.28 µµµµg/ml (KB)IC50 1.83 µµµµg/ml (YMB-1)
Ebinding=-9.66 kcal/mol),
Log P 1.5
N
HN
O
O
O
OO
OH
NDBGE : R = ButylIC50 38 µµµµg/ml (P388)
IC50 1.92 µµµµg/ml (KB)IC50 5.46 µµµµg/ml (YMB-1)
Ebinding=-10.29 kcal/mol);
Log P 1.62
Cytotoxic Test Results to P388, KB and YM-1
N
OH
HN
O
O
O OCH3
O
Log P 1.61, Ebinding = -11.93 kcal/molP388 :IC50 = 15.4 µµµµg/mlKB :IC = 69.6 µµµµg/ml
OH
HN
O
O
O O
CH3
O
KB :IC50 = 69.6 µµµµg/mlYMB-1:IC50 = 5.28 µµµµg/ml
Log P 1.40; Ebind -12.33 kcal.molP388 :IC50 >40 µµµµg/ml
KB :IC50 =26.6 µµµµg/ml
YMB-1:IC50 = 2.69 µµµµg/ml
Metabolite Secundar from Microbial Soil
Pseudomonas pycocyanea
N
H
H
H
H
8,53(dd)
8,04(dd)
8,35(dd)
8,02(dd)
139,91 137,45
135.14
130,29125,08
143,46
144,16
131,73
133,22
130,15
MIC 4,8 µg/ml (E. coli); 0,07 µg/ml ( S. aureus)
IC50 : 5,20 µg/ml (L1210)
Erythromycin : MIC 5,08 (E.coli), 4,06 (S. aureus) dan 3,36 µg/ml (B. subtillis)
N
HO O
H
H
H 8,98(dd)
7,98(dd)
15,55(s)
165,88
140,12
8,28(dd)
128,03
δH15,5 ppmp-Carboxyl-phenazine
SALYCIL ANILIDE DERIVATIVES (PHENAZINES ANALOGS)
NH
O
OH
NH
O
OH
OCH3
L1210: IC50 = 4.8 mg/ml
L1210 IC50 5,5 µg/ml
L1210 IC50 7,0 µg/ml
L1210 IC50 5,2 µg/ml
NH
N
O
OH
NH
O
Cl
M. Hanafi, Paten P00200200449, 2002
SAR Parameter & Cytotoxic Test
Results P388, KB and YMB-1
OH
HN
O
P388 :IC50 = 7.75 µµµµg/mlKB :IC50 = 0.6 µµµµg/mlYMB-1:IC =2.97 µµµµg/ml
Log P -0.17Ebinding = -10.21 kcal/mol
OH O
2-Hydroxy-N-phenyl-benzamide (SA)
50
YMB-1:IC50 =2.97 µµµµg/ml
Calanone derivatives and Its
Cytotoxic Activity
O
O OHO
O
O OHO
O
O OHO
O O
HO
HO
O
Calanone Ester Calanol
Log P 2.32
Against colon cancer cells
HCT116: IC50 = 1.29 µg/mL
P388 : IC50 = 7,5 µg/mL
Log P 0.43
Against colon cancer cells
HCT116: IC50 > 20 µg/mL
L1210 : 59.4 µg/mL
P388 : IC50 = 15
Cisplatin IC50 = 1.02 µg/ml
R
Log P -0.42
Against colon cancer cells
HCT116: IC50 > 20 µg/mL
L1210 : 70.0 µg/mL
P388 : IC50 = 15
InulinInulin
fructotransferasefructotransferase
（（（（（（（（depolymerizingdepolymerizing））））））））
O
O
O
O
HOCH2
CH2OH
HOCH2
OH
HO
OH
OHCH2
CH2
HO
HO
O
O
CH2
O
HOCH2
OH
OH
（（（（（（（（depolymerizingdepolymerizing））））））））
DFAIII Production from DFAIII Production from inulininulin by by InulaseInulase II from II from ActomycetesActomycetes
InulinInulinOH
O
O
O
n
HOCH2
HOCH2 HO
OH
OH
CH2
CH2HO
((DFADFAⅢⅢⅢⅢⅢⅢⅢⅢ))
OH
CH2OH
OHOCH2
OH
DiDi--DD--fructofranosylfructofranosyl 1,2’: 2,3’ anhydride1,2’: 2,3’ anhydride
Arthrobacter, Corynebacterium
20% - Root Chicory
interferon
red blood cell kidney
INTERFERON
Drug on DNA Recombinat Technology
Base
Gen interferon dan erythropoietin diisolasi
Gen dimasukkan kedalam yeast
interferon
Erytrhopoetin
interferon
New hope forhepatitis C
INTERFERON:
Antivirus Anti tumor
ERYTRHOPOIETIN: Anti anemia
Prototype Lab Production
erythropoietin
DNA RNA Protein
EPO
IFN
Cleaner?Mass Production?Cheaper?
Based on Indonesian Biodiversity the research activity entering the Post Genomic Program
Yeast Animal Cell Plant Biodiversity
EPO
Bacteria animal
Biodiversity
1 2 3
1. Infected leaf
Evidence for successful infection/expression
Western analysis
1. Infected leaf2. Infected leaf3. Non-infected leaf
Molecular Farming on N. tabacum
Activity of Research Center for Biotechnology: Dr. Arief Budiwitarto
Tembakau
marker
promoter
cDNA
RB
RK2 ori
bla
ColE1 ori
LB
cDNA
penyandi
protein
Vektor ekspresiHSA,
IFN-α2, anti-
MUC-1 m12 mAb
InsertedDitransfor-
masikan
Potongan daun
Incubated;
cut
Extraction &
Protein
Purification Regenertion;
Selection
Tembakau
MUC-1 m12 mAbGen transformed
Potongan daun
Tunas
Plant identification with high level protein
expresion
Characterizati
on
1 2
3 4
The growth of N. tabacum
Cultured N. tabacum
Height: 135 cm
Number of leaves : 15
Natural N. tabacum
Height: 123 cm
Number of leaves: 14
No significant different on
fenotype/morphology.
For differentiating, tobacco resulted from
biotechnology cultured red color stained by
expression of red fluoresence of DsRed
coral
Tembakau hasil bioteknologiTembakau biasa
Protein DsRed Discosoma coral as
protein DsRed source
CONCLUSION CONCLUSION
�� Natural Products Drug Development based on Indonesian Natural Products Drug Development based on Indonesian Biodiversity conducted in Indonesian Institute of Sciences Biodiversity conducted in Indonesian Institute of Sciences using several approaches: using several approaches:
� (1) Chemical screening , isolation and purification of the Chemical screening , isolation and purification of the bioactive constituentsbioactive constituents, , bioactive constituentsbioactive constituents, ,
� (2) Computational Chemical Calculation Computational Chemical Calculation ((Molecular dockingMolecular docking, , QSAR) for improving the activity of the isolates, QSAR) for improving the activity of the isolates,
� (3) Semisyntheses Semisyntheses of improved compounds based on the of improved compounds based on the above calculation, above calculation,
� (4) BiotechnologyBiotechnology based on Classical Fermentation or Genetic based on Classical Fermentation or Genetic
Engineering (Engineering (molecular farming techniquesmolecular farming techniques))
�DEHYDROLOVASTATIN, is an initial step for natural product development study for commercial purpose whenever possible.
�UK-3A analog is underdevelopment for in vivo activity �UK-3A analog is underdevelopment for in vivo activity evaluation.
� Sulochrin, as molecular hit for antidiabetic compound.
�HUMAN ERYTHROPOETIN and INTERFERON 2A, is further studied for basic long term purpose in the area of molecular farming.
Expression of recombinant hEPO in barley stripe mosaic virus (BSMV)
E P O
Study on Human Erythropoetin (hEPO) production and
Interferon using Molecular Farming Technique
Activity of Research Center for Biotechnology: Dr. Adi Santosa et al.
mosaic virus (BSMV) and Pichia pastoris
Cloning and expression of hIFN alpha 2a in Pichia pastoris
E P O
I F N
Yeast
Bakteria
PRODUKSI OBAT BERBASIS MOLECULAR PHARMING
DNA
DNA
Contoh: LIPI memasukkanDNA EPO dari ginjal manusia ke yeast
Sel hewan
Tanaman
Hewan RNA
Protein
Yeast
LIPI bisa panen EPO manusia
dari yeast
Expression of hEPO on plant and yeast
In vivo analysis of LIPIpoetin
180
APPRLICDSRVLERYLLEAKEAEITTGCAEH
CSLNEITVPDTKVNFYAWKRMEVGQQAVEVW
QGLALLSEAVLRGQALLVSSQPWEPLQLHVD
KAVSGLRSLTTLLRALRAQKEAISPPDAASA
APLRTITADTFRKLFRVYSNFLRGKLKLYTG
EACRTGDR
0
20
40
60
80
100
120
140
160
180
PBS 20 80 160
Dose IU/ml
Reticulocytes (%)
Purification and characterization ofprotein of the tobacco leaves
3 4 5 10 St.
kDa
70
55
40
35
Daun Stan-#1 #2 #3 #4 dard
WESTERN BLOT
ELISA
Leaves Standard
1 2 3 1 2 3kDa
705540
35
25
15
10
100
kDa
Leaves1 2 3 4 5 6
70
55
40
35
25
100
H chain
L chain
Human serum albumin
(HSA) for stabilizer in
pharmaceutical product.Interferon αααα2 (IFN αααα2) for
hepatitis.Anti MUC-1 M12 monoclonal
antibody (Anti MUC-1 M12 mAb)
diagnostic and cancer therapy.
ELISAExpression level: 2 µµµµg/g leaf (yield0,02 % of water soluble total protein)
10
Expression 2,5 µµµµg/g leaf
25
Expression level 480 µµµµg/g leaf
Antioxidant activity and toxicity of water and ethanol extractsAntioxidant activity and toxicity of water and ethanol extracts
of jackfruit extracts and its mistletoe extractsof jackfruit extracts and its mistletoe extracts
DPPH DPPH ICIC5050(Ppm)(Ppm)
DPPH free radical DPPH free radical scavenging activity scavenging activity at at 50 ppm (%)50 ppm (%)
BSLT (LDBSLT (LD50, 50, Ppm)Ppm)
SampleSample WaterWaterextractextract
EtOHEtOHextractextract
WaterWaterextractextract
EtOHEtOHextractextract
WaterWaterextractextract
EtOHEtOHextractextract
JackfruitJackfruit branchbranch woodwood > 100 > 100 24.7 ± 2.2 44.1 ± 0.1 1.4 x 1017 91JackfruitJackfruit branchbranch woodwood > 100 > 100 24.7 ± 2.2 44.1 ± 0.1 1.4 x 1017 91
JackfruitJackfruit barkbark > 100 > 100 16.3 ± 1.9 79.8 ± 1.3 2.4 x 1013 103
JackfruitJackfruit leavesleaves 75 > 100 79.8 ± 0.8 13.9 ± 4.4 2.2 x 1033 9.7 x 104
M. M. cochincochin--chinensischinensisleavesleaves
23 > 100 90.3 ± 0.8 87.1 ± 0.7 2,2 x 1016 1.1 x 104
M. M. cochinchinensiscochinchinensistwigstwigs
21 > 100 88.1 ± 1.2 65.4 ± 2.2 ~ 6.1 x 107
Development of Lead Compound
� Virtual docking
� Lab Scale Sinthesis
� In vitro and in vivo Evaluation
Structure Activity Relationship (SAR)� Structure Activity Relationship (SAR)
� Pharmacopore Identification
Identification of lead compound
pharmacophore
�Molegro Virtual Docking (MVD)
�HyperChem Pro-6.0, Argus Lab-4 �HyperChem Pro-6.0, Argus Lab-4
�Compared to data base on line
(http://pubchem.ncbi.nlm.nih.gov).
Molegro Virtual Docking (MVD)
Alignment of analog compound to ligand
Determination of binding site “pocket” in the enzyme
Calculation of docking energy value of compound Calculation of docking energy value of compound
candidate to fill the “pocket”
Compound candidate synthesis
O
HO
OH
H3C
O
OH
HO
HO
OH
OH
HN
S
HO
HO
CH2OH
CH2 C
H
OH
C
H
CH2OH
OSO3
Salacinol
Inhibitor αInhibitor α--GlukosidaseGlukosidase
O
O
HO
OHO
HO
OH
OH
OH
O
N
HO
HO
OH
OH
O
HO
OCH3
OH
H3C
HO
HO
NH
OH
OH
1-deoksi-nojirimicin
akarbose
nojirimisin
Salacinol
OCOOCH3 OHOCOOCH3 OH
OCOOCH3 OH
HO OCH3
HO
CH3
5 (sulochrin)
OH O COOCH3
OH
H3CO
H3C
OH
Br
Br
OH O COOCH3
OCH3
H3CO
H3C
OCH3
O
OCH3
OH3COOC
OOH
Br
H3C
Br
O
OCH3
OH3COOC
OOH
I
H3C
I C
B
A
Sulochrine Derivatives
OCOOCH3 OCOCH3
H3CO OCH3
H3COCO
CH3
H3CO OCH3
HO
CH3H3CO O CH3
OCOOCH3 OH
H3CO OCH3
H3CO
CH3
OCOOCH3 OH
HO O CH3
OCOOCH3 OH
H3CO OH
HO
CH3
7
3
21
6
4
O
OCH3
OH3COOC
OOH
Cl
H3C
Cl
D
E
OH
O OH
OCH3
OH
O OH
OCH3
H3CO
O OH
OH
dioxybenzene
benzophenone-6
oxybenzone
No Ligan Similarity Score
1 Salacinol -494.341
2 B -420.861
3 C -420.769
4 E -420.347
5 S3 -407.934
Similarity Calculation Score of the ligan to MVD
6 1 -399.824
7 Sulochrin -385.956
8 4 -377.17
9 5 -357.712
10 2 -370.041
11 7 -369.389
12 6 -366.136
13 Benzophenone-6 -362.692
14 dioxybenzene -359.89
Visualisasi enzim αα--GlukosidaseGlukosidase
Binding site prediction
Positon of ligand in enzym target
BIOLOGICAL ACTIVITIES OF DEHYDROLOVASTATIN
55.35
82.39 79.8874.21
61.01
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5
KADAR KOLESTEROL TOTAL
(mg/dl)
56.62
91.32 88.99
73.1477.35
0
20
40
60
80
100
1 2 3 4 5
KELOMPOK
KADAR TRIGLISERIDA
1. Normal control, 2. Induced Cholesterol, 3. Dehydrolovastatin 1.8 mg/200 g bw/day, 4. Dehydrolovastatin 3.6 mg/200g bw/day, 5. Simvastatin 1.8 mg/200g bw/day.
1 2 3 4 5
KELOMPOKKELOMPOK
37.5533.4
40.46
45.85
39.21
0
10
20
30
40
50
1 2 3 4 5
KELOMPOK
KADAR HDL (mg/dl)
Cholesterol Total (mg/dl)
6.47
30.72
21.62
13.74
6.32
0
5
10
15
20
25
30
35
1 2 3 4 5
KELOMPOK
KADAR LDL (mg/dl)
Triglyceride (mg/dl)
HDL (mg/dl)LDL (mg/dl)
StatinStatin GroupsGroups
O
O
O
OHO
Simvastatin
O
O
O
OHO
H
O
O
HO
H
O
O
N
COOH
HO
F
OH
Fluvastatin
Simvastatin
Lovastatin
O
O
OH
COOH
HO
HO
Pravastatin
N
HN
O
COOH
HO
OH
F
Atorvastatin
(Lipitor)
Mevastatin
15
20
25
30
Kadar glukosa darah
(mmol/ml)
(a-Glucocidase Inhibition of Extracct koji Fraction
(IC50=1.05 ug/ml )
0
5
10
15
H0 H 15
menit
H 30
menit
H 60
menit
H 120
menit
H 180
menit
Waktu
Kadar glukosa darah
(mmol/ml)
Control ; Glucobay; dose 1; dose 2; dose 3
Stages of New Drug
Development
Drug Design
And SynthesisBasic
Pharmacolog
y
Scale-up
Synthesi
s
Pharmacology
Biochemistry
IND ClinicalI, II, III
NDA Clinical
IV
Basic
Pharmacokinetics
Analysis :
Physicochemical
Properties:Formulation
Formulation
Process
Formulation
Process
Acute Toxicology
Mutagenicity
Production
Process:
Mid-term toxicity
Pharmacology:
Pharmacokinetics
Pharmacology:
Pharmacokinetics; Chronic toxicity
Preclinical Stage Clinical Stage
1-3 yrs 3-4 yrs 6-7 yrs
How do How do statinsstatins work?work?
�� StatinsStatins reduce the liver's production of cholesterol, and reduce the liver's production of cholesterol, and increase the ability of the liver to remove LDL cholesterol ("bad" increase the ability of the liver to remove LDL cholesterol ("bad" cholesterol) from the blood. They also have a moderate ability cholesterol) from the blood. They also have a moderate ability to reduce triglyceride levels, and increase levels of HDL to reduce triglyceride levels, and increase levels of HDL cholesterol ("good" cholesterol.) cholesterol ("good" cholesterol.)
�� SimvastatinSimvastatin appears to increase HDL cholesterol to a greater appears to increase HDL cholesterol to a greater extent than other extent than other statinsstatins..extent than other extent than other statinsstatins..
�� SimvastatinSimvastatin Increases HDLIncreases HDL--C And Apo AC And Apo A--I More Than I More Than AtorvastatinAtorvastatin in Patients With Hypercholesterolemiain Patients With HypercholesterolemiaAmerican Heart Journal, 11/10/2003, By Emma American Heart Journal, 11/10/2003, By Emma HittHitt, PhD, PhD
