SELECTION OF POTENT INHIBITORS OF FARNESYL-PROTEIN TRANSFERASE FROM ASYNTHETIC TETRAPEPTIDE COMBINATORIAL LIBRARY

Inhibitors of farnesyl-protein transferase (FPTase) show promise as an ticancer agents, Based on the sequence of the protein substrates of FP Tase (the CAAX sequence), potent and selective peptidomimetic inhibito rs have been developed; these compounds share with the peptide substra te a free thiol and a C-terminal carboxylate, We have used a synthetic tetrapeptide combinatorial library to screen for new leads devoid of these features: the peptides were C-terminally amidated, and no free t hiol was included in the combinatorial building blocks, To compensate for this negative bias, an expanded set of 68 amino acids was used, in cluding both L and D as web as many non-coded residues, Sixteen indivi dual tetrapeptides derived from the consensus were synthesized and tes ted; all were active, showing IC50 values ranging from low micromolar to low nanomolar, The most active peptide, ne-D-4-chlorophenylalanine- L-gamma-carboxyglutamic acid (K-i = 2 nM), is also very selective show ing Little inhibitory activity against the related enzyme geranylgeran yl-protein transferase type I (IC50 > 50 mu M). In contrast to CAAX-ba sed peptidomimetics, ne-D-4-chlorophenylalanine-L-gamma-carboxyglutami c acid appeared to mimic the iso prenoid substrate farnesyl diphosphat e as determined by kinetic and physical measurements, ne-D-4-chlorophe nylalanine-L-gamma-carboxyglutamic acid was a competitive inhibitor of FPTase with respect to farnesyl diphosphate substrate and uncompetiti ve with respect to CAAX substrate, Furthermore, we demonstrated that F PTase undergoes ligand dependent conformational changes in its circula r dichroism spectrum and that ne-D-4-chlorophenylalanine-L-gamma-carbo xyglutamic acid induced a conformational change identical to that obse rved with farnesyl diphosphate ligand.