Conformation of a novel tetrapeptide inhibitor NH2-D-Trp-D-Met-Phe(pCl)-Gla-NH2 bound to farnesyl-protein transferase

Farnesyl-protein transferase (FPTase) catalyzes the post-translational farn esylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers. A synthetic tetrapeptide library, which in cluded an expanded set of 68 L-, D- and noncoded amino acids, has been scre ened for inhibitors of FPTase activity. The tetrapeptide, NH2-D-Trp-D-Met-L -Phe(pCl)-L-Gla-NH2 was shown to be competitive with the isoprenyl cosubstr ate, farnesyl diphosphate (FPP) but not with the peptide substrate, the C-t erminal tetrapeptide of the Ras protein. The FPTase-bound conformation of t he inhibitor, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was determined by MMR sp ectroscopy. Distance constraints were derived from two-dimensional transfer red nuclear Overhauser effect (TRNOE) experiments. Ligand competition exper iments identified the NOEs that originate from the active-site conformation of the inhibitor. Structures were calculated using a combination of distan ce geometry and restrained energy minimization. The peptide backbone is sho wn to adopt a reverse-turn conformation most closely approximating a type I I beta-turn, The resolved conformation of the inhibitor represents a distin ctly different structural motif from that determined for Ras-competitive in hibitors. Knowledge of the bound conformation of this novel inhibitor provi des a template and future direction for the design of new classes of FPTase antagonists.