Serine Proteases and their Inhibitors

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ugo Kubinyi, www.kubinyi.de Serine Proteases and their Inhibitors ugo Kubinyi Germany E-Mail kubinyi@t-online.de omepage www.

1 ugo Kubinyi, Serine Proteases and their Inhibitors ugo Kubinyi Germany omepage ugo Kubinyi, 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

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

3 ugo Kubinyi, 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, (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 Ala is Ser Asp Ala Ser Asp is Ala Ala Ala Ala uncatalysed reaction: k vs. k Ala,Ala,Ala Why is the Ala-Ala-Ala mutant still enzymatically active? ugo Kubinyi, 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, (1987)

4 ugo Kubinyi, ugo Kubinyi,

5 ugo Kubinyi, Divergent Evolution ugo Kubinyi, is Ser Asp

6 ugo Kubinyi, Convergent Evolution ugo Kubinyi, is Asp Ser

Proteases Asp, is and Ser are the amino acids of the catalytic

7 ugo Kubinyi, Convergent Evolution of Serine Proteases subtilisin chymotrypsin ugo Kubinyi, 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, (1972)

8 ugo Kubinyi, 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, Comparison of the P 1 Pockets of Trypsin, Chymotrypsin and Elastase Gly 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.

9 ugo Kubinyi, Specificity of a Trypsin Asp189Lys Mutant Trypsin, wild-type enzyme Trypsin Asp189Lys Mutant Gly 216 Gly Gly 216 Gly 226 -??? Asp 189 Lys 189 What is the reason for this surprising selectivity? ugo Kubinyi, Specificity of a Trypsin Asp189Lys Mutant Trypsin, wild-type enzyme Trypsin Asp189Lys Mutant Gly 216 Gly Gly 216 Gly Asp 189 Lys 189

non-selective closed pocket, selective de Blood Coagulation Intrinsic pathway

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

11 ugo Kubinyi, 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, 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 D-Phe-Pro-Arg Arg- = arginine aldehyde Why is the D-Phe analog more active than the L-Phe analog?

12 ugo Kubinyi, Binding Mode of Fibrinogen to Thrombin ugo Kubinyi, fibrinopeptide A (green) APAP (white) thrombin surface

13 ugo Kubinyi, 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 : DACEG DSGGP FV Trypsin : DSCQGDSGGPVV ugo Kubinyi, 2 C - Ser Cys191 p-amidinophenylpyruvic acid (1AT) emiketal Formation of Phenylpyruvic Acid Ser195 Asp194 Gly193 Lys60F is57 W757

14 ugo Kubinyi, 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, 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.

15 ugo Kubinyi, 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, APAP- Thrombin Complex 1DWD

16 ugo Kubinyi, Difference Electron Density of the APAP- Thrombin Complex (1DWD) ugo Kubinyi, 1DWD

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

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

CHROMOGENIC SUBSTRATE TECHNOLOGY. Giovanni Russi

CHROMOGENIC SUBSTRATE TECHNOLOGY Giovanni Russi What is a chromogenic substrate? A peptide linked to a chromophore The peptide is formed by 3-5 residues The chromophore is p-nitroaniline (p-na) The residues