Design and structural requirements of potent peptidomimetic inhibitors of p21^ras farnesyltransferase

Cysteine farnesylation of the ras oncogene product, p21^ras, on its carboxyl-tenninal CA₁A₂X bOx (C = cysteine, A = aliphatic, and X = methionine or serine) is required for its transforming activity. p21^ras farnesyltransferase (FTase), the enzyme responsible for this important post-translational modification can be inhibited by simple CA₁A₂X peptides. We have synthesized a family of CA₁A₂X peptidomimetics where the central 2 aliphatic amino acids are replaced by a variety of spacer groups with different shapes and conformational characteristics to investigate the structural requirements of these inhibitors. The biological activities of CA₁A₂X peptidomimetics, where the dipeptide "A₁A₂" is replaced by 3- or 4-aminomethylbenzoic acid (AMBA) and 3- or 4-aminobenzoic acid (ABA), are evaluated in a p21ras FTase inhibitory assay. Peptidomimetics Cys-4-ABA-Met and Cys-3-AMBA-Met contain spacers that provide good distance correspondence of the carboxylate and ammonium separation with that of the parent K_B p21^ras tetrapeptide, Cys-Val-Ile-Met, and are as potent FTase inhibitors (IC₅₀ values of 50 and 100 nM, respectively). In contrast, replacing the central dipeptide with 4-AMBA reduced FTase inhibitory activity by 17-fold whereas replacement by 3-ABA reduces inhibitory activity of the peptidomimetics by 43-fold. Cys-4-ABA-Met (IC₅₀ = 50 nM) is 128 times more potent as a p21^ras FTase inhibitor than Cys-3-ABA-Met (IC₅₀ = 6400 nM), yet these two peptidomimetics differ only in the substitution pattern around the phenyl ring. These results coupled with computer modeling studies demonstrate that the interaction between FTase and the peptidomimetics requires precise structural and conformational characteristics; in particular, correct positioning of the Cys and Met must be respected. Furthermore, Cys-3-AMBA-Met and Cys-4-ABA-Met are true inhibitors of p21^ras FTase since they are not farnesylated by this enzyme, in contrast to Cys-Val-Ile-Met, which inhibits the enzyme by acting as alternative substrate. Computer modeling studies of the potent FTase inhibitor Cys-4-ABA-Met show that a folded conformation, where the thiol and carboxylate groups are close, is not possible. Therefore a β-turn conformation that would result in the simultaneous coordination of the Cys-thiol and Met-carboxylate to zinc ion is not important for inhibition of p21^ras FTase, as previously suggested.