A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond

Abstract

INTRODUCTIO'NThrombin is the principal enzyme that acts in concert with other activated factors of the hemocoagulative system to regulate clot formation in response to vascular tissue clamage. Although cu.thrombin is highly specific toward fibrinogen as its natural substrate, its szruccurai homology to other serine proteases may preclude the development of specific and effective active-site directed inhibitors which could be useful to treat thrombotic disorders, Indeed a large number of synthetic or substratemrelated inhibitors have been reported [l], but most of them suffer from vaeious drawbacks such as low potency, poor selectivity or potential toxicity. The most potent inhibitor of Mthrombin known is hirudin [2,3], a '7 kDa family of isoproteins isolated from the glandular extracts of the leech Wirudo medicinalis. I-Iirudin forms a high affinity non-covalent stoichiometric complex with a-thrombin (Ki = 10'" to lo-*' M), but does not inhibit trypsin, kaltikrein or other hcmocoagulant swine proteases (41. It is now known that the high affinity is due to the multiple interactions of hirudin with cu-thrombin while the specificity stems from the interaction with at least one complementary binding site that is unique to CYthrombin [5,6]. This putative 'anion' exosite may also be required for fibrinogen binding. We have previously demonstrated that desulfohirudin'5'65 corresponding to the carboxyl termincrl fragment of recombinant hirudin variant 1 (r-HVl) can inhibit the amidolytic activity of human cr-thrombin towards synthetic substrates [7]. Although the inhibition by this native fragment was weak and the mechanism partially competitive compared to rhirudin, a modification of the NHZ-terminal residues by substituting D-Phe and Arg in positions 45 and 47 respectively, afforded an inhibitor with a Ki = 2.8 ZL 0.9 nM against human a-thrombin. The enhancement of enzyme affinity compared to the corresponding native fragment was attributed to favored interaction with the S1 and S3 subsites of the catalytic center while the G-terminal region comprising residues 55-65 could serve as an anchor that binds to the putative 'anion' exosite.tlbbreviu&ms: NOE, nuclear Qvsrhaussr enhancement; Boc-AOGO, SN-ten-butyloxycarbonyl-4-oxo-8.tosyl guanidinooctanoic acid; TFA, trifluoroacetic acid; AMC, 'I-amino-l-methyl coumarin; APTT, activated partial thromboplastin time In this communication, we report that this model was used to obtain a new class of reversible inhibitors prototyped by P49 (Pig. 1) with high enzyme affinity and whose in vitro plasma antithrombin activity approaches the level of r-hirudin despite the drastically reduced Published by Elsevier Science Publishers B. V.

Design of Potent Bivalent Thrombin Inhibitors Based on Hirudin Sequence: Incorporation of Nonsubstrate-Type Active Site Inhibitors

“…This substitution reduced the size of the inhibitors to 21 residues, which is more reasonable for conventional peptide synthesis; however, it also reduced the affinity 3-4-orders of magnitude due to the low affinity of the tetrapeptide to the thrombin active site. A further replacement of the Arg47-Pro48 scissile bond with a pseudo peptide bond (DiMaio et al, 1991 and or a replacement of the N-terminal D-Phe with D-Cha (Witting, et al, 1992b) restored some of the affinity lost (1.4…”

Rational design of hirulog-type inhibitors of thrombin

“…Some of these, the hirulogs and hirutonins, mimic the distinctive mechanism of hirudin by simultaneously occupying both the active site and the anion binding exosite [7,8]. The binding mode of some of these inhibitors to thrombin has been confirmed by X-ray structure determination, although in some complexes no or weak electron density was found for the linker peptides [9,10].…”

Design and synthesis of thrombin inhibitors

“…It yielded potent in vitro inhibitors of thrombin with concomitant activity in the thrombin time coagulation assay. Since the completion of this work, other reports of thrombin inhibitors containing ketomethylene isosteres have also appeared [7,8]. More recently, we have investigated other electrophilic ketone derivatives of arginine as thrombin inhibitors [9,10] and identified alkoxymethyl ketones [10,11] as potentially useful drug candidates (see Results and Discussion).…”