CONFORMATIONAL-ANALYSIS OF THE THROMBIN RECEPTOR AGONIST PEPTIDES SFLLR AND SFLLR-NH2 BY NMR - EVIDENCE FOR A CYCLIC BIOACTIVE CONFORMATION

The conformational properties of the pentapeptide Ser-Phe-Leu-Leu-Arg (P5), a human thrombin receptor-derived sequence forming part of a tet hered ligand which activates the thrombin receptor, and its more activ e amide derivative Ser-Phe-Leu-Leu-Arg-NH2 (5S-NH2), have been studied by proton NMR spectroscopy in dimethylsulfoxide. Measurements of nucl ear Overhauser effects, performed using two-dimensional rotating frame nuclear Overhauser (ROESY) and one-dimensional nuclear Overhauser enh ancement (NOE) spectroscopy, revealed that P5 exists mainly in an exte nded conformation. However, proton-proton 1D-NOEs between Phe CalphaH and Ser CalphaH, Leu(3) CalphaH and Leu(3) NH, and Leu(4) CalphaH and Leu(4) NH, as well as between the Ser and Arg sidechains, also implica ted a minor conformer for P5 having a curved backbone and a near-cycli c structure. In contrast to P5, measurements of NOEs and ROEs for PS-N H2 revealed a more stabilized cyclic structure which may account for i ts higher biological potency. Thus strong interresidue sequential NH ( i)-NH (i + 1) interactions, as well as C-terminal carboxamide to N-ter minal side-chain interactions, i.e., Arg CONH2, to Phe ring and Arg CO NH2 to Ser C-alpha/C-beta beta, observed at lower levels of the ROESY spectrum, supported a curved backbone structure for SFLLR-NH2. Since t he higher potency P5-NH2 analogue adopts predominantly a cyclic struct ure, a cyclic bioactive conformation for thrombin receptor agonist pep tides is suggested.