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Lecture 8: Computational Drug Target Identification Y.Z. Chen Department of Computational Science National University of Singapore E-mail: [email protected] Web-page: http://www.cz3.nus.edu.sg/~yzchen/ Outline Potential Application: Unknown and secondary therapeutic targets of drugs, leads, natural products. Targets related to toxicity, side-effect, delivery. Ligand-protein interactions involved in pathways. Methodology: Ligand-protein inverse docking Tests: Known targets for therapeutics, toxicity and side effect. Y.Z. Chen, National University of Singapore E-mail: [email protected] Web-page: http://www.cz3.nus.edu.sg/~yzchen/ Why protein targets? Novel or Unknown Therapeutic Targets Nature 1998, 396:15 Why protein targets? Toxicity, side effect, pharmacokinetics and pharmacogenetics Annu. Rev. Pharmacol Toxicol 2000, 40:353-388 1997, 37:269-296 Pharmacological Rev. 2000, 52:207-236 Why protein targets? Towards the prediction of side effect, toxicity, pharmacokinetics in early stages of drug discovery Most drug candidates fail to reach market Pharmacokinetics, side effect and toxicity are the main reason. Drug Candidates in Different Stages of Development Majority of Them Fail to Reach Market Clin Pharmacol Ther. 1991; 50:471 Large portion of money ($350 million per drug) and time (6-12 years for a drug) has been wasted on failed drugs. Drug Discov Today 1997; 2:72 Why protein targets? Drugs from Natural Products From natural products to therapeutic drugs TIPS, May 1999, 20:190 Traditional medicines Pharmacology & Therapeutics 2000, 86:191 Screening or extraction of bioactive compounds Mechanism and standardization Further development Drug targets and new drug discovery Drugs from Traditional Medicines Mixture of multiple herbs etc. Therapeutic action + maintaining and restoring balance: Mutual accentuation, mutual enhancement, mutual counteraction, mutual suppression, mutual antagonism, mutual incompatibility Multiple targets: therapeutic effect, symptom treatment, toxicity modulation, drug delivery, harmonization Pharmacology & Therapeutics 2000, 86:191 Why searching protein targets of a molecule? Applications in pathways EGF Pathway From Signaling Pathway Database http://www.grt.kyushu-u.ac.jp/spad/ Strategy Existing Methods: Given a Protein, Find Potential Binding Ligands From a Chemical Database New Method: Given a Ligand, Find Potential Protein Targets From a Protein Database Compound Database Protein Database Compound 1 ... Compound n Protein 1 ... Protein n Protein Ligand Successfully Docked Compounds as Putative Ligands Successfully Docked Proteins as Putative Targets Science 1992;257: 1078 Proteins 2001;43:217 Feasibility Proteins Database: >12,000 3D structures in PDB. Protein diversity: 17% in PDB with unique sequence. Advances in structural genomics: 10,000 unique proteins within 5 years. Ann. Rev. Biophys. Biomol. Struct. 1996; 25:113 Nature Struct. Biol. 1998; 5:1029 Method Ligand-protein docking docking algorithms capable of finding binding conformations. Proteins. 1999; 36:1 Proteins 2001; 43:217 Additional information Rapid accumulation of knowledge in proteomics, pathways, protein functions (functional genomics). Computer resources Increasing power and decreasing cost (Linux PC, Multi-processor Machine) Automated Protein Targets Identification Software INVDOCK Ligand \|/ Automated Process to inversely dock the Lignad to each entry in a Built-In Biomolecular Cavity Database (10,000 Protein and Nucleic Acid Entries) \|/ Step 1: Vector-based docking of a ligand to a cavity Step 2: Limited conformation optimization on the ligand and side chain of biomolecule Step 3: Energy minimization for all atom in the binding site Step 4: Docking evaluation by molecular mechanics energy functions and comparison with other ligands Successfully Docked Proteins and Nucleic Acids as Putative Targets of a Ligand | \|/ Potential Applications: \|/ Protein function, Proteomics, Ligand transport, Metabolism Therapeutic Targets, Side-Effects, Metabolism, Toxicity Function in Pathways INVDOCK Cavity Models HIV-1 Protease INVDOCK Cavity Models Estrogen Receptor Energy Functions Chemical bonds Hydrogen bonding van der Waals interactions Electrostatic interactions Empirical solvation free energy V = Vbonds + H bonds [ V0 (1-e-a(r-r0) )2 - V0 ] + non bonded [ Aij/rij12 - Bij/rij6 + qiqj /r rij] + atoms i Dsi Ai INVDOCK Testing Results Molecule Docked Protein PDB Id RMSD Energy Description of Docking Quality Indinavir HIV-1 Protease 1hsg 1.38 Match -70.25 Xk263 Of Dupont Merck HIV-1 Protease 1hvr 2.05 Match -58.07 Vac HIV-1 Protease 4phv 0.80 Match -88.46 Folate Dihydrofolate Reductase 1dhf 2.41 5-Deazafolate Dihydrofolate Reductase 2dhf 1.48 Match -65.49 Estrogen Estrogen Receptor 1a52 1.30 Match -45.86 4-Hydroxytamoxifen Estrogen Receptor 3ert 0.97 Guanosine-5'-[B,GMethylene] Triphosphate H-Ras P21 121p 0.94 Glycyl-*L-Tyrosine Carboxypeptidase A a 3cpa 2.19 One end match, the other in slightly different orientation Match Match Match -63.02 -55.15 -80.20 -44.84 INVDOCK Testing Results Compound Potential Targets Identified Experimentally Confirmed Experimentally Implicated 4H-Tamoxifen 17 4 4 Aspirin 52 4 16 Vitamin C 46 4 9 Vitamin E 26 2 11 INVDOCK Identified Protein Targets For an Anticancer Drug Tamoxifen PDB Putative Protein Target Experimental Finding 1a52 Estrogen Receptor 1akz Uracil-DNA Glycosylase 1ayk Collagenase 1az1 Aldose Reductase 1bnt Carbonic Anhydrase 1boz Dihydrofolate Reductase 1dht, 1fdt 17b -Hydroxysteroid Dehydrogenase 1gsd , 3ljr Glutathione Transferase A1-1, Glutathione S-Transferase Target Status Clinical Implication Ref Drug target Confirmed Treatment of breast cancer 36 Inhibited activity Confirmed Tumor cell invasion and cancer metastasis 38 Decreased level Inhibitor Suppressed enzyme and activity Combination therapy for cancer Confirmed Promotion of tumor regression 43 39 41 Genotoxicity and carcinogenicity INVDOCK Identified Protein Targets For an Anticancer Drug Tamoxifen 1mch Immunoglobulin l Light Chain Temerarily enhanced Ig level 44 Modulation of immune response 1p1g Macrophage Migration Inhibitory factor 1ulb Purine Nucleoside Phosphorylase 1zqf DNA Polymerase b 2nll Retinoic Acid Receptor 1a25 Protein Kinase C 1aa8 D-Amino Acid Oxidase 1afs 1pth Inhibition 3a -Hydroxysteroid Dehydrogenase Effect on androgen induced activity Prostaglandin H2 Synthase-1 Direct inhibition Confirmed Confirmed Anticancer 37 Hepatic steroid metabolism 42 Prevention of vasoconstriction 40 INVDOCK Testing on Toxicity Targets Compound Number of experimentally confirmed or implicated toxicity targets Number of toxicity targets predicted by INVDOCK Number of toxicity targets missed by INVDOCK Number of toxicity targets without 3D structure or involving covalent bond 4 Number of INVDOCK predicted toxicity targets without experimental finding 2 Aspirin 15 9 2 Gentamicin 17 5 2 10 2 Ibuprofen 5 3 0 2 2 Indinavir 6 4 0 2 2 Neomycin 14 7 1 6 6 Penicillin G 7 6 0 1 8 Tamoxifen 2 2 0 0 4 Vitamin C 2 2 0 0 3 Total 68 38 5 25 29 Toxicity and side effect targets of Aspirin identified from INVDOCK search of protein database PDB Protein 1a42 Carbonic anhydrase II 1a6a HLA-DR3 1a7c Plasminogen activator inhibitor 1d6n Hypoxanthine-guanine phosphoribosyltransferase 1hdy Alcohol dehydrogenase Experimental Finding Target Status Toxicity/Side Effect Ref Activate enzyme activity that may lead to increase in plasma bicarbonate concentration. Change in HLA level Implicated Metabolic alkalosis (hypoventilation). Puscas I Implicated Aspirin-induced asthma Dekker JW Tissuedependent response of protein. Implicated Hypertension, thrombolysis Smokovitis A Excess uric acid in serum* Inhibition of activity Confirmed Increased blood alcohol level Gentry RT Toxicity and side effect targets of Aspirin identified from INVDOCK search of protein database 1hdy Alcohol dehydrogenase Inhibition of activity Confirmed Increased blood alcohol level Gentry RT 1hiq Insulin Tissue insensitivity to insulin Implicated Impaired glucose metabolism in insulin-sensitive cells. Newmann WP 1hmr Fatty acid binding protein Increased binding capacity and protein content. Implicated Effect on peroxisomal beta oxidation activity. Kawashima Y 1mch Immunoglobulin lambda light chain Ig reactivities Implicated Allergic reaction to aspirin. Zhu DX 1pah Phenylalanine hydroxylase 2ant Antithrombin Irreversibly acetylate antithrombin. Confirmed Blood coagulation, thrombolysis. Villanueva GB 2hdh L-3-hydroxyacyl CoA dehydrogenase Reversible inhibition of enzyme activity. Confirmed Effect on Reye's syndrome patients. Glasgow JF Phenylketonurea* Molecular targets of Chinese natural products Chinese Natural Product Number of Identified Therapeutic Targets Number Confirmed or Implicated Therapeutic Targets by experiment Number of Identified Toxicity/Side effect Targets Number Confirmed or Implicated Toxicity/Side Effect Targets by experiment Acronycine 3 1 4 - Allicin 5 2 1 1 Baicalin 14 4 6 - Catechin 17 12 5 - Camptothecine 9 6 3 2 Dicoumarin 7 1 3 1 Emodin 6 3 5 1 Genistin 22 7 12 1 Putative and known therapuetic targets of Camptothecine identified from INVDOCK search of human and mammalian proteins PDB Protein Experimental Finding Target Status Theraputic Effect Ref 1ads Aldose Reductase 2gss Glutathione STransferase p1-1 7ice DNA Polymerase Beta 1a25 Protein Kinase C 1cdk CAMP-Dependent Protein Kinase Anti-cancer 3bct Beta-Catenin Anti-cancer 1dvi Calpain Inhibition of calpain activities. Implicated Induces apoptosis in leukemic cells. Eymin 1yfo Receptor Protein Tyrosine Phosphatase Causes elevation of PTPase in the cytosol and the nucleus which play a critical role in the induction of the differentiation of IW32 erythroleukemia cells. Implicated Anti-cancer Wang MC 1a35 Topoisomerase I Inhibitor Confirmed Anti-cancer Wang MC Diabetes treatment Increases intracellular glutathione Implicated Enhance radical scavenging activities that may useful in cancer treatment Matsumoto Anti-cancer Inhibitor Confirmed Induction of apoptosis in tumor. Martelli Nieves-Neira Putative and known therapuetic targets of Camptothecine identified from INVDOCK search of human and mammalian proteins PDB Protein Experimental Finding Target Status Theraputic Effect Ref 1ads Aldose Reductase 2gss Glutathione STransferase p1-1 7ice DNA Polymerase Beta 1a25 Protein Kinase C 1cdk CAMP-Dependent Protein Kinase Anti-cancer 3bct Beta-Catenin Anti-cancer 1dvi Calpain Inhibition of calpain activities. Implicated Induces apoptosis in leukemic cells. Eymin 1yfo Receptor Protein Tyrosine Phosphatase Causes elevation of PTPase in the cytosol and the nucleus which play a critical role in the induction of the differentiation of IW32 erythroleukemia cells. Implicated Anti-cancer Wang MC 1a35 Topoisomerase I Inhibitor Confirmed Anti-cancer Wang MC Diabetes treatment Increases intracellular glutathione Implicated Enhance radical scavenging activities that may useful in cancer treatment Matsumoto Anti-cancer Inhibitor Confirmed Induction of apoptosis in tumor. Martelli Nieves-Neira