Inhibitors of tubulin polyglycylases and isopentenyl diphosphate isomerase

Microtubules are principal components of the cytoskeleton, and are composed of alpha- and beta-tubulin heterodimers. Microtubules play important roles during mitosis and intracellular transport due to the dynamic instability of microtubule growth. These diverse microtubule functionalities are achieved through multiple post-translational modifications (PTMs) of tubulin. Polyglycylation is one of PTMs that involves the addition of one or more glycines to alpha- and beta-tubulin. This modification has been proven to be essential for proper sperm motility. Polyglycylation is catalyzed by enzymes that belong to the tubulin tyrosine ligase-like (TTLL) family. TTLL3 and 8 function as glycyl-initiases that catalyze the addition of the first glycine onto the modification sites. TTLL10 functions as a glycyl-elongase that adds polyglycine chains onto the monoglycylated modification sites. In this work, the first known inhibitors of both TTLL8 and TTLL10 are reported. This was achieved by synthesizing phosphinic acid-based analogs of the tetrahedral intermediate and embedding them within a pentapeptide. Peptide-embedded inhibitors of both enzymes were shown to inhibit tubulin glycylation in the low micromolar range. The ongoing studies with these inhibitors involve crystallographic and mechanistic studies of the enzymes. In the second project, inhibitors of type I isopentenyl diphosphate isomerase (IDI-1) were developed. IDI-1 is a metal-dependent enzyme that catalyzes the isomerization between isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) in the last step of the mevalonate (MVA) pathway. IPP and DMAPP are the precursors for various terpenoids such as cholesterol. To develop potential drug targets for lowering cholesterol levels, several mechanism-based inhibitors were designed and synthesized to mimic the tertiary carbocation intermediate of the isomerization step. A guanidinium with a planar geometry and a tertiary ammonium with a tetrahedral geometry act as carbocation mimics. Several non-hydrolyzable or less highly charged alternatives to the pyrophosphate moiety were also investigated. All inhibitors were tested IDI-1. The inhibitor bearing a guanidinium and a phosphinyl phosphate acted as a competitive inhibitor with a Ki of 129 nM. An inhibitor bearing the tertiary ammonium as the carbocation mimic was found to be 50-fold less potent, indicating that guanidinium is a more effective carbocation mimic.Science, Faculty ofChemistry, Department ofGraduat