Abstract
Investigation of the comparative activities of various inhibitors of farnesyl:protein transferase (FPTase) has led to the observation that the presence of phosphate or pyrophosphate ions in the assay buffer increases the potency of farnesyl diphosphate (FPP) competitive inhibitors. In addition to exploring the phenomenon of phosphate synergy, we report here the effects of various other ions including sulfate, bicarbonate, and chloride on the inhibitory ability of three FPP competitive compounds: Cbz-His-Tyr-Ser(OBn)TrpNH2 (2), Cbz-HisTyr(OPO42-)-Ser(OBn)TrpNH2 (3), and alpha-hydroxyfarnesyl phosphonic acid (4). Detailed kinetic analysis of FPTase inhibition revealed a high degree of synergy for compound 2 and each of these ions. Phosphorylation of 2 to give 3 completely eliminated any ionic synergistic effect. Moreover, these ions have an antagonistic effect on the inhibitory potency of compound 4. The anions in the absence of inhibitor exhibit non-competitive inhibition with respect to FPP. These results suggest that phosphate, pyrophosphate, bicarbonate, sulfate, and chloride ions may be binding at the active site of both free enzyme and product-bound enzyme with normal substrates. These bound complexes increase the potency of FPP competitive inhibitors and mimic an enzyme:product form of the enzyme. None of the anions studied here proved to be synergistic with respect to inhibition of geranylgeranyl transferase I. These findings provide insight into the mechanism of action of FPP competitive inhibitors for FPTase and point to enzymatic differences between FPTase and geranylgeranyl transferase I that may facilitate the design of more potent and specific inhibitors for these therapeutically relevant target enzymes.
Highlights
Since mutations rendering the Ras protein (p21) oncogene are prevalent in many human cancers [1] and farnesylation of the C-terminal region of the Ras protein is essential for activation of Ras function in vivo [2, 3], a potential therapeutic approach to tumor regression would be to inhibit the farnesylation reaction
Farnesyl:protein transferase (FPTase)1 catalyzes the transfer of a 15-carbon group to several cellular proteins containing the requisite C-terminal CAAX recognition sequence
We wanted to determine whether the phosphate enhancement of inhibitor binding was specific for FPTase compared with geranylgeranyl transferase I since distinguishing inhibition characteristics for these two similar enzymes may be critical in generating selective agents with distinctive cellular activities and further delineating the specificity by which these enzymes operate
Summary
Materials—Tritiated farnesyl pyrophosphate and geranylgeranyl pyrophosphate were obtained from American Radiolabeled Chemicals (St. Louis, MO). Thr-Lys-Cys-Val-Ile-Met and biotin-Aha-Thr-Lys-Cys-ValIle-Met were synthesized according to solid phase peptide chemistry techniques [21, 22]. ␣-Hydroxyfarnesyl phosphonic acid was synthesized as described [13]. Compounds 1, 2, and 3 were synthesized as described [20]. Potassium phosphate and sodium sulfate were obtained from Fisher and were the highest grade available. Sodium pyrophosphate and potassium chloride were obtained from Sigma. Potassium bicarbonate was obtained from Mallinckrodt and Hepes buffer was obtained from Life Technologies, Inc. Geranylgeranyl transferase I was
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