Unraveling the role of the homoarginine residue in antiplatelet drug eptifibatide in binding to the αIIbß3 integrin receptor.

Eptifibatide is an αIIbß3 inhibitor that is currently used in the clinic. More than 10 scientific communications indicate that eptifibatide has a Lys-Gly-Asp or Arg-Gly-Asp sequence, while it actually has a hArg-Gly-Asp sequence. We aimed to unravel the importance of the homoarginine residue in eptifibatide in platelet activation and aggregation. Arg- and Lys-eptifibatide were synthesized by solid-phase peptide synthesis and measured in light transmission aggregometry, flow cytometry and whole blood thrombus formation under flow. Interactions of eptifibatide and its variants with αIIbß3 integrin were studied using molecular dynamics simulations. Eptifibatide showed inhibition of collagen- and ADP-induced platelet aggregation, while Arg- and Lys-eptifibatide did not. Multiparameter assessment of thrombus formation showed suppressed platelet aggregate and fibrin formation upon eptifibatide treatment, in contrast to the other variants. Molecular dynamics simulations revealed that the hArg residue in eptifibatide is crucial to its activity, since the substitution of the hArg to Arg or Lys resulted in the inability to form double H-bonds with Asp224 in the αIIb chain of the αIIbß3 receptor. The hArg is pivotal for the interaction of eptifibatide for the αIIbß3 receptor and efficient inhibition of platelet aggregation.

Introduction of functional groups into peptides via N-alkylation.

An optimized protocol for the mild and selective Fukuyama-Mitsunobu reaction was used for mono- and di- N-alkylation on solid support. Thereby, nonfunctionalized aliphatic and aromatic residues are quickly introduced into transiently protected, primary amines of a linear peptide. N-Alkylation can also be used to implement alkyl chains carrying (protected) functionalities suited for subsequent modification. Applicability of this method is demonstrated by various N-alkylated analogues of a cyclic CXCR4 receptor antagonist originally developed by Fujii et. al.