Crystallographic determination of the structures of human α‐thrombin complexed with BMS‐186282 and BMS‐189090

Abstract

The crystallographic structures of the ternary complexes of human a-thrombin with hirugen (a sulfated hirudin fragment) and the small-molecule active site thrombin inhibitors BMS-186282 and BMS-189090 have been determined at 2.6 and 2.8 A. In both cases, the inhibitors, which adopt verysimilar bound conformations, bind in an antiparallel P-strand arrangement relative to the thrombin main chain in a manner like that reported for PPACK, D-Phe-Pro-Arg-CH,Cl. They do, however, exhibit differences in the binding of the alkyl guanidine moiety in the specificity pocket. Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the binding pocket. Although PPACK forms covalent bonds to both serine and the histidine of the catalytic triad of thrombin, neither BMS-186282 nor BMS-189090 bind covalently and only BMS-186282 forms a hydrogen bond to the serine of the catalytic triad. Both inhibitors bind with high affinity (K, = 79 nM and 3.6 nM, respectively) and are highly selective for thrombin over trypsin and other serine proteases.Keywords: crystallography; human a-thrombin-inhibitor complexes; structure-based drug designThe serine protease thrombin occupies a central position in the blood coagulation cascade. It cleaves circulating fibrinogen to fibrin monomers, which then polymerize and are covalently linked by thrombin-activating factor XIIIa, which is also a prodReprint requests to: Mary E Malley, Department of Solid State Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New Jersey 08543-4000; e-mail: malley@bms.com.Present address: Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, Indiana 47907. uct of thrombin activation. In addition, thrombin activates several other coagulation and plasma factors, such as factors V, VIII, protein C, and protein S. Thrombin also induces platelet aggregation and promotes the proliferation of endothelial cells, the liberation of tissue plasminogen activator, and the contraction and dilation of blood vessels. Selective inhibition of thrombin may result in efficient control of various pathophysiologic states, such as thrombosis and arteriosclerosis, and aid in the prevention of myocardial infarction (Berliner 1992; Fenton, 1986; Fenton et al., 1991). Abbreviations: BMS-186282, [S-(R*,R*)]-4-[Aminoiminomethyl)-amino]-N-[[l-[3-hydy-2-[(2-naphthalenyls~fony~)~no]-l-oxopropyl]- 2-pyrrolidinyl]methyI]butanamide; BMS-189090, [S-(R*,R*)]-l-(Aminoiminomethyl)-N-[[l-[N-[(2-naphthalenylsulfonyl)-~-seryl]-2-pyrrolidinyl]-Human a-thrombin consists of two polypeptide chains, A and B, connected through a single disulfide bond. The A chain has 36 residues (after the loss of a tridecapeptide during activation with prothrombin) and the B chain has 259 residues. The B chain is glycosylated at Asn 60G and contains the active site residues His 57, Asn 102, and Ser 195. The numbering scheme for the thrombin residues in this paper are assigned by homology with chymotrypsin as d...

Structural basis for chemical inhibition of human blood coagulation factor Xa

“…In contrast to the abundance of crystal structures of complexes between thrombin and chemical inhibitors (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45), our structure is only the second structure of the inhibitor-bound factor Xa. The structure of the DX-9065a-bound factor Xa was the first inhibitor complex structure published (13).…”

Human α-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-d-phe-pro-α-azalys p-nitrophenyl ester and N-carbobenzoxy-pro-α-azalys p-nitrophenyl ester: A kinetic, thermodynamic and x-ray crystallographic study

“…From a second viewpoint, it should be considered that the azaLys substructure has proved rather¯exible (compare A and B in Figure 4) in allowing proper occupation of the active site by the rest of the inhibitor molecule, which can exploit its binding interactions as ef®ciently as in the case of the irreversible inhibitor PPACK. This observation suggests that the azaLys (or azaArg) fragment may be suitable to support rather different synthetic inhibitor moieties, such as that of BMS-189090 (Malley et al, 1996), achieving improved thrombin af®nity and/or selectivity against homologous serine proteinases.…”

“…One parent compound, in this series, is PPACK (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone; Bode et al, 1989Bode et al, , 1992, an irreversible thrombin inhibitor which forms a tetrahedral hemiketal with the Ser195 OG atom and a covalent linkage with His57 NE2. Other notable competitive inhibitors of thrombin, such as BMS-189090 Malley et al, 1996), achieve K values in the nanomolar range.…”

Progress in the Development of Synthetic Thrombin Inhibitors as New Orally Active Anticoagulants

“…A similar central alkylpyrrolidine as replacement for the P2 Pro was the key in an inhibitor series developed by Bristol-Myers Squibb. Due to the reversed P2-P1 peptide bond these compounds, such as 126 (BMS-189,090, K i = 3.44 nM) and 127 (IC 50 = 13 nM) are named as retro-binding inhibitors [165,166]. BMS-189,090 showed a suitable pharmacological overall profile that was comparable to that of argatroban but no information regarding oral bioavailability was given.…”