Investigation of Candida albicans Prenyltransferases: Substrate Recognition and Enzyme Inhibition.

Abstract

Prenylation is a post-translational modification that is essential for the proper membrane localization of many cellular proteins. Protein prenylation is carried out by protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I), zinc-dependent sulfur alkyltransferases that catalyze attachment of either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid moiety to an invariant cysteine residue near the C-terminus of the substrate protein. Inhibitors targeting human FTase and GGTase-I are being developed as therapeutics for various diseases. Prenyltransferases from Candida albicans pathogen have been investigated as targets for anti-mycotic agents. Early studies with mammalian FTase and GGTase-I proposed a “CaaX” recognition motif where C is the modified cysteine, ‘a’ is often an aliphatic amino acid, and X determines enzyme specificity. Recent work indicates that this substrate recognition paradigm is too restrictive, and many “non-canonical” CaaX sequences are efficiently prenylated by both FTase and GGTase-I. In this study substrate specificities of C. albicans FTase and GGTase-I were evaluated to assess their substrate recognition overlap with each other and were compared to mammalian enzymes to determine whether small molecule inhibitors could be specifically targeted to these. Peptide library studies showed that C. albicans FTase has similar substrate specificity to the mammalian enzyme; however, C. albicans GGTase-I has a significantly broader substrate specificity, including recognition of substrates with large amino acids. C. albicans GGTase-I structure shows that it has a larger a2 binding pocket, providing rationale for the observed substrate preferences. C. albicans GGTase-I also lacks a product exit groove and appears to have a faster product dissociation rate. Docking studies identified a dipeptide derivative as a potent and selective C. albicans FTase inhibitor with an IC50 = 200 nM. This compound shows competitive mode of inhibition with FPP and mixed mode of inhibition with peptide substrate, indicating that it does not behave as a peptidomimetic. SAR studies with related compounds showed that addition of zinc-coordinating ligands enhances in vitro potency. Together, these studies propose a substrate recognition model for C. albicans prenyltransferases, identify similarities and differences between mammalian and yeast substrate interactions, and suggest possible lead compounds for the treatment of C. albicans infection.