Anions modulate the potency of geranylgeranyl-protein transferase I inhibitors

We have identified and characterized potent and specific inhibitors of gera nylgeranyl-protein transferase type I (GGPTase I), as well as dual inhibito rs of GGPTase I and farnesyl-protein transferase. Many of these inhibitors require the presence of phosphate anions for maximum activity against GGPTa se I in vitro, Inhibitors with a strong anion dependence were competitive w ith geranylgeranyl pyrophosphate (GGPP), rather than with the peptide subst rate, which had served as the original template for inhibitor design. One o f the most effective anions was ATP, which at low millimolar concentrations increased the potency of GGPTase I inhibitors up to several hundred-fold. In the case of clinical candidate L-778,123, this increase in potency was s hown to result from two major interactions: competitive binding of inhibito r and GGPP, and competitive binding of ATP and GGPP, At 5 mM, ATP caused an increase in the apparent K-d for the GGPP-GGPTase I interaction from 20 pM to 4 nM, resulting in correspondingly tighter inhibitor binding. A subset of very potent GGPP-competitive inhibitors displayed slow tight binding to GGPTase I with apparent on and off rates on the order of 10(6) M-1 s(-1) an d 10(-3) s(-1) respectively, Slow binding and the anion requirement suggest that these inhibitors may act as transition state analogs. After accountin g for anion requirement, slow binding, and mechanism of competition, the st ructure-activity relationship determined in vitro correlated well with the inhibition of processing of GGPTase I substrate Rap1a in vivo.