Ruoying Gong
Department of Chemistry
March 12, 2009
What Is A Drug?
 Drug is any substance used in the treatment,
prevention, or diagnosis of disease
 The earliest drugs were natural products
 Currently, more drugs are synthesized or semi-
synthesized
Collins Essential English Dictionary 2nd Edition, HarperCollins Publishers, 2006
2
Drug Discovery
 Trial and error testing
 Random screening
 Rational drug design
 Structural information of a drug receptor
 Information of a ligand
Twyman, R., The Human Genome, 2002
Greer, J., et. al. J Med Chem. 1994, 37, 1035–1054
3
Rational Drug Design Process
Genomics/Proteomics
Cloning/Protein Expression
Bioinformatics
Modeling
Docking
X-ray crystallography
NMR spectroscopy
Domain Architecture
Prediction
Crystal Structure
High Throughput Screen
200,000 compound/week
Potential Ligand Class
Breinbauer, R., et. al. Angew. Chem. Int. Ed. 2002, 41, 2878 - 2890
4
Rational Drug Design Process
Potential Ligand Class
Synthesize Library of
Similar Compounds
Formulation
Biological tests
Pharmacological tests
Clinical tests
Hits
Structure-activity relationship
Bioavailability
Lead
Drug
Breinbauer, R., et. al. Angew. Chem. Int. Ed. 2002, 41, 2878 - 2890
5
Drawback of Rational Drug Design
 Time consuming
 Costly
 Limited understanding of drug receptors
 Labour intensive
 Low hit rate generated
6
New Approach to Drug Design
 Novel approach
 Biology oriented synthesis
 Created by Waldmann group
 Max Planck Institute, Germany
7
Drug and Drug Receptor
Catalytic core
Protein
Ligand binding site
 Knowledge of 3D structure of protein can assist in the
design of drug scaffold
8
Protein Structure
N+
TACEVAEISYKKFRQLIQVN
P
D
VKESTVQLRRAMQASLRMLI
G
NALFLDVTGRIAQTLLNLAKQ
Petsko, G. A. et. Al. Protein Structure and Function New Science Press Ltd., 2004
C-
9
Protein Classification
Protein
2
Protein
3
Protein
1
Similar 3D structure,
function, and
primary structure
Protein family
10
Proteins In the Same Family
 Similar mechanism
 Similar primary structure
 Similar 3D structure
 Similar amino acid residues
11
Process of Ligand Discovery
Target
Protein
Model
Protein
12
Waldmann Approach
 Compare proteins with their 3D structure
 Use a natural inhibitor as guiding structure for
compound library development
13
Protein Domain and Fold
 Protein Domain
 Tertiary structure folded independently as functional
units
 Protein fold
 Conformational arrangement of protein secondary
structures into tertiary structure
Alberts, B. et. al. The Shape and Structure of Proteins. New York and London: Garland Science, 2002
14
Protein Structure Architecture
Proteins
(100,000 – 450,000)
Domains
(4,000 – 50,000)
Folds
(800 – 1,000)
SCOP databank: Murzin, A. G., Brenner, S. E., J. Mol. Biol. 1995, 247, 536 - 540
15
Superfold and Supersite
 Superfold: highly populated folds
 Supersite: common ligand binding sites within a
superfold
Alberts, B. et. al. The Shape and Structure of Proteins. New York and London: Garland Science, 2002
16
Classification Comparison
Protein Family
 Similar primary
structure
 Similar ligand binding
site
Protein Fold
 Not related to primary
structure
 Similar ligand binding
site
17
Biology Oriented Synthesis
• Biology
 Protein Structure Similarity Clustering (PSSC)
• Chemistry
 Compound library synthesized according to guiding
structure of natural inhibitor
Koch, M. A. et al Drug Discovery Today. 2005, 10, 471 - 483
18
Grouping Proteins Together
 Protein Structure Similarity Clustering (PSSC)
3D similarity of ligand binding sites
Ignore the amino acid sequence identity
19
Computation Tools Used
 Structural Classification of Proteins (SCOP)
 Dali/Fold Classification Based on Structure-Structure
Alignment of Proteins (FSSP) Database
 Combinatorial Extension (CE) superimposition
algorithm
20
Protein Clustering Process
1
Protein of Interest
Structural Alignment
2
Dali/FSSP
Interesting cases
3
Sequence identity (SI) < 20%
Superimposition of Catalytic Cores
4
Root mean square deviation (RMSD) < 5Å
Grishin, N.V., et al J. Struct. Biol. 2001, 134, 167 - 185
21
1.Protein of Interest - Cdc25A
 Phosphatase family
 Rhodanese fold
 Catalytic site contains Cys-430, Glu-431
 Regulates progression of cell division
 A potential antitumor drug target
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
22
2.Structure Alignment
Cdc25A
AChE
11βHSD1,2
23
3.Acetylcholinesterase (AChE)
 α/β-hydrogenase family
 α/β-hydrogenase fold
 Catalytic site contains Ser-200
 Terminate synaptic transmission
 Target protein in the treatment of myasthenia gravis,
glaucoma, and Alzheimer’s disease
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
24
4.Superimposition
Cys-430 (Cdc25A)
Ser-200 (AChE)
Super-site
Cdc25A
AChE
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
25
2.Structure Alignment
Cdc25A
AChE
11βHSD1,2
26
3. Isoenzymes 11βHSD1,2
 Tyrosine-dependent oxidoreductase family
 Rossmann fold
 Tyrosine residue located at catalytic site
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
27
11βHSD1
 Reduces cortisone to the active hormone cortisol
 Potential target for treatment of obesity, the metabolic
syndrome, and type 2 diabetes
OH
OH
O
OH 11ßHSD1
11ßHSD2
O
H
O
H
HO
H
Cortisone
O
OH
O
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
H
H
H
Cortisol
28
11βHSD2
 Catalyzes the oxidation of cortisol into the inactive
cortisone
 Inhibition causes sodium retention resulting in
hypertension
OH
OH
O
OH 11ßHSD1
11ßHSD2
O
H
O
H
HO
H
Cortisone
O
OH
O
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
H
H
H
Cortisol
29
4.Superimposition
Super-site
Cys-430 (Cdc25A)
Tyr-183 (11βHSD1)
Tyr-232 (11βHSD2)
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
Cdc25A
11βHSD1
11βHSD2
30
Structure Alignment
Cdc25A
AChE
11βHSD1,2
31
Superimposition
Cys-430 (Cdc25A)
Tyr-183 (11βHSD1)
Ser-200 (AChE)
Super-site
Cdc25A
11βHSD1
AChE
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
32
Cluster Member Comparison
Cdc25A
AChE
11βHSD1,2
Protein family
Phosphatase
α/β-hydrogenase
Hydroxysteroid
dehydrogenase
Sequence identity
-
17%
6%
RMSD
-
2.6Å
4.9Å
AChE
11βHSD1,2
Sequence identity
-
6%
RMSD
-
3.9Å
33
Compound Library Discovery
34
Dysidiolide: Natural Inhibitor of
Cdc25A
O
O
OH
OH
Dysidiolide, IC50=9.4μM
Natural inhibitor of Cdc25A
γ-hydroxybutenolide
Brohm, D., et. al. Angew. Chem. Int. Ed. 2002, 41, 307 - 311
35
Dysidiolide: Natural Inhibitor of
Cdc25A
O
O
O
O
Br
OH
γ-hydroxybutenolide
O
O
OTMS
O
α,β-Unsaturated lactone
Brohm, D., et. al. Angew. Chem. Int. Ed. 2002, 41, 307 - 311
36
Representative Synthesis
Br
Li
n-BuLi
O
Et2O, -78°C
O
O
H3CO
(0.12eq)
C11H23
49%
O
HO
C11H23
HO
O
O
O
OH
O2, hv, (iPr)2NEt, O
Rose Bengal
DCM, -78°C, 2h
37%
HO
C11 H23
O
37
γ-Hydroxybutenolides Synthesis
O
OH
R1
H
OH
R1
O
O
LDA
THF, -78°C
O
Br
O
O Li
O
1
R
1
R
O
Li
n-BuLi
Et2O, -78°C
Cl
2
2
R MgBr or R Li
Et2O, -78°C,1h
O
H3CO
O2, hv, (iPr)2NEt,
Rose Bengal
DCM, -78°C, 2h
OH 1
R
2
O R
O
O
IBX, DMSO/THF,
r.t., 12h
IBX, DMSO/THF
r.t., 12h
O
R1
OH
OH1
R
R1
O
O
O
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
O
HO
O
R1
R2
OH
O
O
HO
O
O
R1
OH
O
O
R1
OH
OH
38
α,β-Unsaturated Lactones
Synthesis
O
R3 H
BF3 Et2O, CH2Cl2, -78°C
TMSO
O
R3
O
O
O
O
R4
H
BF3 Et2O, CH2Cl2, -78°C
OH
O
H
O
O
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
HO
O
4
R
O
O
R4
39
Results
 147 compounds synthesized
 Contains γ-hydroxybutenolide or α,β-unsaturated
lactone
Hits (rate)
Cdc25A
AChE
11βHSD1
11βHSD2
42(28.5%)
3 (2%)
3(2%)
4(2%)
 Inhibitors with these structures have never been
reported
40
Best Compounds
O
Natural inhibitor of Cdc25A
Dysidiolide, IC50=9.4μM
O
OH
OH
MeO
O
O
OH
OH
O
O
O
HO
OH
OH
Cdc25A, IC50=0.35μM
AChE, IC50>20μM
11βHSD1, IC50=14μM
11βHSD2, IC50=2.4μM
HO
OH
O
O
F
Cdc25A, IC50=45μM
AChE, IC50>20μM
11βHSD1, IC50=10μM
11βHSD2, IC50=95μM
O
O
O
Cdc25A, IC50=1.8μM
AChE, IC50>20μM
11βHSD1, IC50=19μM
11βHSD2, IC50=11μM
Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726
O
O
Cdc25A, IC50>100μM
AChE, IC50>20μM
11βHSD1, IC50=19μM
11βHSD2, IC50=5.3μM
41
Take Home Message
 PSSC group proteins together regardless of primary
structure identity
 High hit rate achieved from small library size
 Compound library was designed to mimic the structure
of natural products (NPs)
42
Second Approach
O
O
OH
OH
Natural inhibitor of Cdc25A
Dysidiolide, IC50=9.4μM
 Structure of NP dictates the way it binds to proteins
 Structural classification of natural products (SCONP)
43
Structural Classification of
Natural Products (SCONP)
 Method
 Chose compounds in the Dictionary of Natural Products
containing ring structures
 Create scaffold map
 Properties of SCONP
 Structural relationships between different NP classes
 Tool for NP derived compound library development
44
Computational Simulation to
Generate SCONP
 Deglycosylation prior to running simulation
 Neglect stereochemistry
 Reduce structural complexity of multi-ring systems
 Choose heterocyclic substructures as parent scaffolds
45
N-Heterocycles
Scaffolds of
Natural products
O-Heterocycles
Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277
Carbocycles
46
Implications of SCONP
 Parent scaffold represents a substructure of a
respective offspring scaffold
 Two to four-ring-containing NPs are the most common
scaffolds
 Scaffolds include the structural information of how NPs
bind to proteins
47
11βHSD1
 Potential target for treatment of obesity, the metabolic
syndrome, and type 2 diabetes
 Inhibition of isoenzyme 11βHSD2 causes sodium
retention resulting in hypertension
OH
OH
O
OH 11ßHSD1
11ßHSD2
O
H
H
O
H
Cortisone
O
OH
HO
H
H
O
H
Cortisol
48
Glycyrrhetinic Acid
O2C
O
H
HO
Glycyrrhetinic Acid (GA)
Natural inhibitor of Cdc25A
49
Glycyrrhetinic Acid
O2C
O
H
HO
Glycyrrhetinic Acid (GA)
Natural inhibitor of Cdc25A
50
Glycyrrhetinic Acid
O2C
O
H
HO
Glycyrrhetinic Acid (GA)
Natural inhibitor of Cdc25A
51
Scaffolds of Natural Products
Carbocycles
O
Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277
O
O
52
Scaffolds of Natural Products
O2C
O
O
O
OH
Dysidiolide
Natural inhibitor of Cdc25A
OH
H
HO
Glycyrrhetinic Acid (GA)
Natural inhibitor of Cdc25A
O
O
O
?
Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277
53
Compound Library Synthesis
R3
O
O
+
R
R3
R1
O
R2
R2
HO
O
HO
R
3
R
R
HO
R
R2
R3
R3
6
R5
1
pTSA
CuI, iPr2NEt
R2
R1
R2
O
O
R5CCH, Pd(PPh3 )4
1
O
O NaBH4, EtOH
1
O
R3
R3
Ph3P,
O
R6CH2 Br
BuLi
R4
O
O
O
R1
R2
R4
O
LDA, R4 CHO,THF
O
O
O
R1
R2
R3
O
10%TFA
HO
R1
R4
R2
DCM
Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277
54
Library General Structure
R
X1
X2
OH
R
X1 = X2 = O, CH2, CHR'
X1 = H, X2 = OH,OR',NHR'
R,R' = Alkyl, Aryl, etc
Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277
55
Results
 162 members synthesized with the simple bicycle ring
scaffold
Hits (rate)
R
11βHSD1
11βHSD2
30(18.5%)
3(2%)
OH
X1
X2
R
 28 compounds selectively inhibit 11βHSD1
 Inhibitors with this bicycle ring scaffold have never
been reported
56
Best Compounds
O2C
O
HO
Glycyrrhetinic Acid (GA)
Natural inhibitor of Cdc25A
H
O
O
OH
OH
OH
F
11βHSD1, IC50=0.31μM
11βHSD2, IC50=6.6μM
11βHSD1, IC50=0.74μM
11βHSD2, IC50>30μM
Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277
11βHSD1, IC50=0.35μM
11βHSD2, IC50>30μM
57
Combined With PSSC and SCONP
Natural
inhibitor
PSSC
CO2
O
H
OH
Target
Biology
Oriented
Synthesis
(BIOS)
Biology
Nören-Müller, et. al. PNAS. 2006, 103, 10606-10611
Compound
Library
X1
X2
OH
R
Chemistry
58
Conclusion
 PSSC classifies proteins together by 3D similarity of
ligand binding site
 SCONP is a guiding tool for NP derived compound
library development
 Small compound libraries synthesized generate high
hit rates for proteins from different families
 The chemical and biological approaches of BIOS were
useful for the synthesis of drug-like compounds
59
Acknowledgement











Dr. Robert Ben
Dr. Mathieu Leclere
Roger Tam
Jennifer Chaytor
Elisabeth von Moos
Pawel Czechura
John Trant
Wendy Campbell
Sandra Ferreira
Taline Boghossian
Jackie Tokarew
60