Lewis Sd

Inhibition of thrombin and other trypsin-like serine proteinases by cyclotheonamide A.

Cyclotheonamide A (CA), a cyclic peptide isolated from the marine sponge of the genus Theonella was shown to be a slow-binding inhibitor of several trypsin-like serine proteinases. Values of 4.6 x 10(4), 4.8 x 10(4), 9.3 x 10(3), 2.1 x 10(3) and 2.7 x 10(2) M-1 s-1 were determined for the second-order rate constants for formation of CA complexes with thrombin, trypsin, plasmin, 2-chain t-PA and factor Xa, respectively. The equilibrium constant (Ki) was measured for dissociation of CA from the CA complex with human thrombin (Ki = 1.0 nM), bovine trypsin (Ki = 0.2 nM), human plasmin (Ki = 12 nM), human factor Xa (Ki = 50 nM) and human 2-chain tissue plasminogen activator (t-PA) (Ki = 40 nM). CA produces dose dependent increases in clotting time assays. The clotting time in the thrombin time, activated partial thromboplastin time and prothrombin time assays, were doubled by 1.5, 0.9 and 48 microM CA, respectively. A model for the binding of CA to the active site of thrombin is proposed.

Synthesis, evaluation, and crystallographic analysis of L-371,912: A potent and selective active-site thrombin inhibitor.

Abstract Removal of the β-ketoamide functionality from L-370,518 (Ki = 0.09 nM) provided a 5 nM Ki inhibitor of thrombin: L-371,912. Comparison of the enzyme-inhibitor crystal structures suggests a possible explanation for the relatively small change in affinity for thrombin. L-371,912 is selective for thrombin over related serine proteases and is efficacious in an animal model of arterial thrombosis.

Amide and α-keto carbonyl inhibitors of thrombin based on arginine and lysine: Synthesis, stability and biological characterization

We report structure-activity investigations in a series of tripeptide amide inhibitors of thrombin, and the development of a series of highly potent active site directed α-keto carbonyl inhibitors having the side chain of lysine at P1. Compounds of this class are unstable by virtue of reactivity at the electrophilic carbonyl and racemization at the adjacent carbon (CH). Modifications of prototype α-keto-ester 8a have afforded analogs retaining nanomolar Ki. Optimal potency and stability have been realized in α-keto-amides 11b (Ki = 2.8 nM) and 11c (Ki = 0.25 nM)

Selective Inhibition of Factor Xa in the Prothrombinase Complex by the Carboxyl-terminal Domain of Antistasin

Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112. The decreased reactivity of ATS-119 suggests that the carboxyl-terminal domain of ATS-119 stabilizes an ATS conformation with a reduced reactivity toward factor Xa. The observation that calcium ion increases the reactivity of ATS-119 but not that of ATS-112 suggests that calcium ion may disrupt interactions involving the carboxyl terminus of ATS-119. Interestingly, ATS-119 inhibited factor Xa in the prothrombinase complex 2–6-fold more potently and 2–3-fold faster than ATS-112. These differences in affinity and reactivity might well account for the greater effectiveness of ATS-119 in prolonging the APTT and suggest that the carboxyl-terminal domain of ATS-119 disrupts interactions involving phospholipid, factor Va, and prothrombin in the prothrombinase complex. The peptide RPKRKLIPRLS, corresponding to the carboxyl domain of ATS-119 prolonged the APTT and inhibited prothrombinase-catalyzed processing of prothrombin, but it failed to inhibit the catalytic activity of isolated factor Xa. Thus, this novel inhibitor appears to exert its inhibitory effects at a site removed from the active site of factor Xa.