Serine Proteases and their Inhibitors

ugo Kubinyi, www.kubinyi.de Serine Proteases and their Inhibitors ugo Kubinyi Germany E-Mail kubinyi@t-online.de omepage www.kubinyi.de ugo Kubinyi, www.kubinyi.de Serine Proteases of Physiological Importance Protease Cleavage Site Function Trypsin Arg-X, Lys-X Digestion Chymotrypsin Tyr-X, Phe-X, Trp-X Digestion Elastase Val-X Tissue degradation Thrombin Arg-Gly Blood coagulation Factor Xa Arg-Ile, Arg-Gly Blood coagulation ther Important Serine Proteases: Tryptase, Lipases, Phospholipases, Subtilisin

ugo Kubinyi, www.kubinyi.de Catalytic Mechanism of Serine Proteases Asp is Ser Substrate substrate "oxygen anion hole" Ser tetraedric transition state ugo Kubinyi, www.kubinyi.de Catalytic Mechanism of Serine Proteases Ser 2 acyl-enzyme complex Ser 2 products

ugo Kubinyi, www.kubinyi.de Dissection of the Catalytic Triad of Subtilisin (substrate: -succinoyl-l-ala-l-ala-l-pro-l-phe-p-nitroanilide). P. Carter and J. A. Wells, ature 332, 564-568 (1988) Subtilisin, wild type and mutants K m k cat/k m AA 32 AA 64 AA 221 (µm) (s -1 mol -1 ) Asp is Ser 220 250 000 Ala is Ser 480 5 Asp Ala Ser 390 0.1 Asp is Ala 420 0.1 Ala Ala Ala 330 0.1 uncatalysed reaction: k vs. k Ala,Ala,Ala 0.0003 Why is the Ala-Ala-Ala mutant still enzymatically active? ugo Kubinyi, www.kubinyi.de Substrate-Assisted Catalysis in a Subtilisin is64ala Mutant Asp32 - R is64ala mutant X Ser221 R Subtilisin cleaves structurally different peptide substrates. The is64ala mutant cleaves XXX-is-XXX peptides 4 orders of magnitude slower than wild-type subtilisin but about 200 times faster than substrates without is. P. Carter and J. A. Wells, Science 237, 394-399 (1987)

ugo Kubinyi, www.kubinyi.de ugo Kubinyi, www.kubinyi.de

ugo Kubinyi, www.kubinyi.de Divergent Evolution ugo Kubinyi, www.kubinyi.de is Ser Asp

ugo Kubinyi, www.kubinyi.de Convergent Evolution ugo Kubinyi, www.kubinyi.de is Asp Ser

ugo Kubinyi, www.kubinyi.de Convergent Evolution of Serine Proteases subtilisin chymotrypsin www.new-science-press.com/browse/protein/1/16 ugo Kubinyi, www.kubinyi.de Convergent Evolution of Serine Proteases Asp, is and Ser are the amino acids of the catalytic triad. Ser, Leu/Trp and Gly interact with the substrate. J. D. Robertus et al., Biochemistry 11, 2439-2449 (1972)

ugo Kubinyi, www.kubinyi.de Protease Binding Pockets for a Peptide Substrate cleavage site S 3 S 1 S 2 ' R 3 P 3 P 2 R 2 R 1 P 1 P 1 ' R 1 ' R 2 ' P 2 ' P 3 ' R 3 ' S 2 S 1 ' S 3 ' amino-terminal peptide carboxy-terminal peptide ugo Kubinyi, www.kubinyi.de Comparison of the P 1 Pockets of Trypsin, Chymotrypsin and Elastase Gly 216 2 Gly 216 Thr 216 Val 226 Asp 189 Gly 226 Gly 226 Ser 189 Trypsin Chymotrypsin Elastase The binding pockets of trypsin and thrombin accommodate positively charged amino acid side chains by the negatively charged Asp 189. The P 1 pocket of chymotrypsin is designed for large, lipophilic side chains. Elastase has a relatively small lipophilic P 1 pocket; it binds only small hydrophobic amino acids, like alanine and valine.

ugo Kubinyi, www.kubinyi.de Specificity of a Trypsin Asp189Lys Mutant Trypsin, wild-type enzyme Trypsin Asp189Lys Mutant Gly 216 Gly 226 + 2 2 Gly 216 Gly 226 -??? Asp 189 Lys 189 What is the reason for this surprising selectivity? ugo Kubinyi, www.kubinyi.de Specificity of a Trypsin Asp189Lys Mutant Trypsin, wild-type enzyme Trypsin Asp189Lys Mutant Gly 216 Gly 226 + 2 2 - Gly 216 Gly 226 + 3 Asp 189 Lys 189

ugo Kubinyi, www.kubinyi.de Trypsin and Thrombin Show Different Substrate Selectivity open pocket, non-selective closed pocket, selective ugo Kubinyi, www.kubinyi.de Blood Coagulation Intrinsic pathway F XII F XI F IX F X Thrombin Fibrin F XIII Thrombus Extrinsic pathway F VIII Fibrinolysis Plasmin Plasminogen

ugo Kubinyi, www.kubinyi.de Cyclooxygenase Arachidonic acid Prostaglandines, ASS Thromboxanes GP IIb/IIIa antagonists Vitamin K antagonists (coumarins) Blood platelet aggregation Blood Coagulation: Antithrombotic Principles Factor X Factor Xa Factor Xa inhibitors Prothrombin Thrombin Thrombin inhibitors, AT3 Ancrod Fibrinogen Clot (soluble Fibrin) soluble Factor XIIIa eparin degradation Thrombus products Fibrinolysis (Plasmin, tpa) ugo Kubinyi, www.kubinyi.de Relative Inhibitory Activities of Tripeptide Aldehydes vs. Thrombin Peptide Relative inhibitory activities Gly-Val-Arg- 1 Gly-Pro-Arg- 9 Phe-Pro-Arg- 57 D-Ala-Pro-Arg- 469 D-Val-Pro-Arg- 1 273 D-Phe-Pro-Arg- 7 370 Arg- = arginine aldehyde Why is the D-Phe analog more active than the L-Phe analog?

ugo Kubinyi, www.kubinyi.de Binding Mode of Fibrinogen to Thrombin ugo Kubinyi, www.kubinyi.de fibrinopeptide A (green) APAP (white) thrombin surface

ugo Kubinyi, www.kubinyi.de Affinity of Inhibitors to Thrombin and Trypsin 2 2 K i (thrombin) = 220 µm K i (trypsin) = 35 µm K i (thrombin) = 150 µm K i (trypsin) = 360 µm Benzamidine binds specifically to trypsin, whereas -amidino-piperidine has a slightly higher specificity for thrombin.! Thrombin 189-200: DACEG DSGGP FV Trypsin 189-200: DSCQGDSGGPVV ugo Kubinyi, www.kubinyi.de 2 C - Ser195 2 + 2 Cys191 p-amidinophenylpyruvic acid (1AT) emiketal Formation of Phenylpyruvic Acid Ser195 Asp194 Gly193 Lys60F - - + 3 is57 W757

ugo Kubinyi, www.kubinyi.de Groups that Covalently Interact With the Catalytically Active Serine Inhibitor Type Functional Groups Irreversible Chloromethylketones -CC2Cl Inhibitors Sulfonylfluorides -S2F Esters Boronic Acids -CR -B(R)2 Reversible Aldehydes -C Inhibitors Arylketones -C-Aryl Trifluoromethylketones Ketocarboxylic acids -CCF3 -CC ugo Kubinyi, www.kubinyi.de Classical Thrombin Inhibitors R = K d = 180 nm 2 R = CC 2 Cl (irreversible covalent inhibitor) 1DWE, 1PPB, (cf. 1AI8, 1AIX, 1AI) R R = C D-Phe-Pro-Arg- K d = 1.8 nm (reversible covalent inhibitor) 2

ugo Kubinyi, www.kubinyi.de Classical Thrombin Inhibitors D-APAP (1DWD, 1ETS) K i = 2.1 nm 3 C S S meta: 3-TAPAP para: 4-TAPAP (1ETT) 2 2 C 3 S C 3 Argatroban, MD-805 (1DWC, 1ETR) K i = 19 nm C 2 ugo Kubinyi, www.kubinyi.de APAP- Thrombin Complex 1DWD

ugo Kubinyi, www.kubinyi.de Difference Electron Density of the APAP- Thrombin Complex (1DWD) ugo Kubinyi, www.kubinyi.de 1DWD

ugo Kubinyi, www.kubinyi.de Binding Mode of APAP (1DWD) hydrophobic P3 pocket hydrophobic P2 pocket S Gly-216 Gly-219 P1 pocket + 2 2 Asp-189 ugo Kubinyi, www.kubinyi.de Binding Modes of Thrombin Inhibitors ( hydrophobic collapse ) APAP (1DWD) Argatroban (1DWC)

ugo Kubinyi, www.kubinyi.de Binding Modes of Different Thrombin Inhbitors ugo Kubinyi, www.kubinyi.de Binding Modes of Different Thrombin Inhbitors (coordinates from X-ray structure analyses and from docking)