Synthesis of Novel Peptide Inhibitors of Thrombin‐induced Platelet Activation

Abstract

Inhibitors of the activation of platelet aggregation have promise as important therapeutic agents for the management of acute coronary syndrome (ACS). Platelet activation by thrombin, a serine protease, occurs by binding to and cleavage of the extracellular N‐terminal domains of protease‐activated receptors 1 and 4 (PAR1 and PAR4). The proteolysis of the PARs exposes new tethered ligands that then signal through transmembrane domains to initiate platelet activation as a downstream effect. A pentapeptide cleavage product of bradykinin with the sequence Arg‐Pro‐Pro‐Gly‐Phe serves as a thrombin inhibitor by blocking α‐ and γ‐thrombin‐induced platelet aggregation. Analogs of RPPGF have been prepared that result in improved inhibition of thrombin activation of platelets. Specific amino acid residues required for activity against platelet aggregation have been identified, and a lead compound, rOicPaPhe(p‐Me)‐NH2 (FM19), has been developed. FM19, which completely inhibits threshold γ‐thrombin‐induced platelet aggregation at a concentration of 16 ± 4 μm, represents an important lead compound in the development of inhibitors of thrombin‐mediated platelet aggregation for treatment of ACS.

Rational Design and Characterization of D-Phe-Pro-D-Arg-Derived Direct Thrombin Inhibitors

“…Peptides were synthesized using standard solid-phase fluorenylmethyloxycarbonyl (Fmoc) chemistry on a 432A Synergy Personal Peptide synthesizer (ABI) as previously described [19]. Amide Rink resin (Novabiochem) was used to produce all peptides as C-terminal amides.…”

“…More recent approaches include the study of several synthetic analogs of the angiotensin converting enzyme breakdown product of bradykinin (RPPGF) [17], [18], [19], [20] as well as 1,3,5-trisubstituted benzenes [21] as potent thrombin inhibitors.…”

Structure‐Based Design of Residue 1 Analogs of the Direct Thrombin Inhibitor Pentapeptide FM 19

“…Additionally, replacement of the guanidino group with an amino group on the side chain was explored (6)(7)(8), while maintaining the same linker length between the backbone carbonyl carbon of the residue and the positively charged moiety on the end of the side chain (guanidino or amino). Finally, one conformationally restricted analog was synthesized (9), in an attempt to lock in the eclipsed conformation observed in the X-ray structure. Analytical data for all nine compounds along with lead compound FM 19 are displayed in Table 1.…”

“…Thus, peptide RPPGF serves as a starting point for the development of a direct thrombin inhibitor, as well as an inhibitor of platelet activation. SAR studies were carried out to follow-up on RPPGF antithrombotic activity and culminated in the development of lead compound, FM 19, with the sequence D-Arg-Oic-Pro-D-Ala-Phe(p-Me)-NH 2 , where Oic is ((2S,3aS,7aS)-octahydroindole-2-carboxylic acid) ( Figure 1) (9). Although FM 19 is more potent than RPPGF (K i = 4.4 lM versus 1.75 mM), FM 19 is still only a modest inhibitor of thrombin activity (10).…”

Antiplatelet drugs

“…Both PAR4 and PAR3 knock‐out mice are protected against arterial thrombosis (Weiss et al , 2002) indicating that PAR inhibition might be explored for the prevention or treatment of thrombosis in humans. Several peptide and non‐peptide antagonists of PAR1 and some of PAR4 have been developed with proven antithrombotic potency (Burke et al , 2006). The PAR1 peptide mimetic antagonist RWJ‐58259 significantly prolonged the occlusion time in an electrolytic injury thrombosis model in cynomolgus monkeys (Derian et al , 2003) and in the same animal species, the PAR1 antagonist SCH205831, based on the natural product himbacine, inhibited the ex vivo platelet aggregation (Chackalamannil et al , 2005).…”