Synthesis and Characterization of Novel Unnatural Peptide Inhibitors of Thrombin Activation of Platelet Aggregation.

Abstract

Inhibitors of the activation of platelet aggregation have promise as important therapeutic agents for the management of acute coronary syndrome. Platelet activation by thrombin 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. A pentapeptide cleavage product of bradykinin with the sequence Arg-Pro-Pro-Gly-Phe (RPPGF) serves as an inhibitor of α- and g-thrombin-induced platelet aggregation. Analogs of RPPGF were 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, D-Arg-Oic-Pro-D-Ala-Phe(p-Me)-NH2 (27), has been developed. Compound 27, which completely inhibits threshold γ-thrombin-induced platelet aggregation at a concentration of 16 µM, represents an important lead compound in the development of inhibitors of thrombin-mediated platelet aggregation. The mechanism of action of compound 27 was studied, and there is a clear interaction with the active site of thrombin, as well as preliminary results that indicate analog 27 is binding to PAR1. In addition, due to its composition of unnatural amino acid residues, 27 is orally available with 12.7% bioavailability in rat models. In order to improve upon this bioavailability, a new series of peptides with modified N- and C-termini, di-substituted phenylalanine, and methylated arginine analogs designed to reduce the net charge and/or increase the lipophilicity of the peptide was prepared. These modifications were well tolerated and a new lead compound was discovered. Compound 48 (D-Arg-Oic-Pro-D-Ala-Phe(p-Me)-NH-Et) has the same amino acid sequence as analog 27, but possesses an ethyl group at the amide of the C-terminus. This compound is a 3-fold more potent inhibitor of thrombin and platelet aggregation than is 27 (Ki = 2.1 μM, IC100 = 5 μM). This dissertation has shown the benefits of substituting unnatural amino acid residues into the sequence of a naturally occurring peptide as a means of modulating activity and developing analogs with desirable pharmacokinetic profiles. Compounds 27 and 48 demonstrate promise to become the first orally administered direct thrombin inhibitor.