Biology Oriented Synthesis A New Approach to Drug Design 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

Drug Discovery Trial and error testing Rational drug design 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

Rational Drug Design Process Genomics/Proteomics Cloning/Protein Expression Bioinformatics Modeling Docking X-ray crystallography NMR spectroscopy Crystal Structure Domain Architecture Prediction High Throughput Screen 200,000 compound/week Potential Ligand Class Breinbauer, R., et. al. Angew. Chem. Int. Ed. 2002, 41, 2878 - 2890

Rational Drug Design Process Potential Ligand Class Synthesize Library of Similar Compounds Structure-activity relationship Bioavailability Hits Formulation Biological tests Pharmacological tests Clinical tests Lead Drug Breinbauer, R., et. al. Angew. Chem. Int. Ed. 2002, 41, 2878 - 2890

Drawback of Rational Drug Design Time consuming Costly Limited understanding of drug receptors Labour intensive Low hit rate generated

New Approach to Drug Design Novel approach Biology oriented synthesis Created by Waldmann group Max Planck Institute, Germany

Drug and Drug Receptor Knowledge of 3D structure of protein can assist in the design of drug scaffold Catalytic core Protein Ligand binding site

Protein Structure Petsko, G. A. et. Al. Protein Structure and Function New Science Press Ltd., 2004

Protein Classification Similar 3D structure, function, and primary structure Protein family

Proteins In the Same Family Similar mechanism Similar primary structure Similar 3D structure Similar amino acid residues

Process of Ligand Discovery Target Protein Model Protein

Waldmann Approach Compare proteins with their 3D structure Use a natural inhibitor as guiding structure for compound library development

Protein Domain and Fold 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

Protein Structure Architecture (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

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

Classification Comparison Protein Family Similar primary structure Similar ligand binding site Protein Fold Not related to primary structure Similar ligand binding site

Biology Oriented Synthesis 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

Grouping Proteins Together Protein Structure Similarity Clustering (PSSC) 3D similarity of ligand binding sites Ignore the amino acid sequence identity

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

Protein Clustering Process 1 Protein of Interest 2 Structural Alignment Dali/FSSP 3 Interesting cases Sequence identity (SI) < 20% 4 Superimposition of Catalytic Cores Root mean square deviation (RMSD) < 5Å Grishin, N.V., et al J. Struct. Biol. 2001, 134, 167 - 185

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

2.Structure Alignment Cdc25A AChE 11βHSD1,2

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

4.Superimposition Cys-430 (Cdc25A) Ser-200 (AChE) Super-site Cdc25A Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726

2.Structure Alignment Cdc25A AChE 11βHSD1,2 11βHSD1,2

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

11βHSD1 Reduces cortisone to the active hormone cortisol Potential target for treatment of obesity, the metabolic syndrome, and type 2 diabetes Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726

11βHSD2 Catalyzes the oxidation of cortisol into the inactive cortisone Inhibition causes sodium retention resulting in hypertension . Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726

4.Superimposition Super-site Cys-430 (Cdc25A) Cdc25A Tyr-183 (11βHSD1) Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726

Structure Alignment Cdc25A AChE 11βHSD1,2 11βHSD1,2

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

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

Compound Library Discovery

Dysidiolide: Natural Inhibitor of Cdc25A Dysidiolide, IC50=9.4μM Natural inhibitor of Cdc25A γ-hydroxybutenolide Brohm, D., et. al. Angew. Chem. Int. Ed. 2002, 41, 307 - 311

Dysidiolide: Natural Inhibitor of Cdc25A α,β-Unsaturated lactone γ-hydroxybutenolide Brohm, D., et. al. Angew. Chem. Int. Ed. 2002, 41, 307 - 311

Representative Synthesis

γ-Hydroxybutenolides Synthesis Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726

α,β-Unsaturated Lactones Synthesis Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726

Results 147 compounds synthesized Contains γ-hydroxybutenolide or α,β-unsaturated lactone Inhibitors with these structures have never been reported Cdc25A AChE 11βHSD1 11βHSD2 Hits (rate) 42(28.5%) 3 (2%) 3(2%) 4(2%)

Natural inhibitor of Cdc25A Best Compounds Natural inhibitor of Cdc25A Dysidiolide, IC50=9.4μM Cdc25A, IC50=0.35μM AChE, IC50>20μM 11βHSD1, IC50=14μM 11βHSD2, IC50=2.4μM Cdc25A, IC50=45μM AChE, IC50>20μM 11βHSD1, IC50=10μM 11βHSD2, IC50=95μM Cdc25A, IC50=1.8μM AChE, IC50>20μM 11βHSD1, IC50=19μM 11βHSD2, IC50=11μM Cdc25A, IC50>100μM AChE, IC50>20μM 11βHSD1, IC50=19μM 11βHSD2, IC50=5.3μM Koch, M. A., Wittenberg, L. O., et. al. PNAS 2004, 101, 16721 - 16726

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)

Natural inhibitor of Cdc25A Second Approach Structure of NP dictates the way it binds to proteins Structural classification of natural products (SCONP) Natural inhibitor of Cdc25A Dysidiolide, IC50=9.4μM

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

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

Scaffolds of Natural products N-Heterocycles Carbocycles O-Heterocycles Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277

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

11βHSD1 Potential target for treatment of obesity, the metabolic syndrome, and type 2 diabetes Inhibition of isoenzyme 11βHSD2 causes sodium retention resulting in hypertension

Glycyrrhetinic Acid Glycyrrhetinic Acid (GA) Natural inhibitor of Cdc25A

Glycyrrhetinic Acid Glycyrrhetinic Acid (GA) Natural inhibitor of Cdc25A

Glycyrrhetinic Acid Glycyrrhetinic Acid (GA) Natural inhibitor of Cdc25A

Scaffolds of Natural Products Carbocycles Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277

Scaffolds of Natural Products Dysidiolide Natural inhibitor of Cdc25A Glycyrrhetinic Acid (GA) Natural inhibitor of Cdc25A ? Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277

Compound Library Synthesis Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277

Library General Structure Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277

Results 162 members synthesized with the simple bicycle ring scaffold 28 compounds selectively inhibit 11βHSD1 Inhibitors with this bicycle ring scaffold have never been reported 11βHSD1 11βHSD2 Hits (rate) 30(18.5%) 3(2%)

Best Compounds Glycyrrhetinic Acid (GA) Natural inhibitor of Cdc25A 11βHSD1, IC50=0.31μM 11βHSD2, IC50=6.6μM 11βHSD1, IC50=0.74μM 11βHSD2, IC50>30μM 11βHSD1, IC50=0.35μM 11βHSD2, IC50>30μM Waldmann, H., et. al. PNAS. 2005, 102, 17272-17277

Combined With PSSC and SCONP Natural inhibitor Compound Library Target Biology Oriented Synthesis (BIOS) Biology Chemistry Nören-Müller, et. al. PNAS. 2006, 103, 10606-10611

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

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