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Transition-state structure analogues are among the most powerful chemical inhibitors discovered to date with picomolar efficacy for enzymes. The nucleoside analogue methylthioadenosine-DADMe-immucillin A (MTDIA) is an inhibitor of the enzyme methylthioadenosine phosphorylase (MTAP) in polyamine biosynthesis. The recently approved forodesine (Mundesine®) is an inhibitor of purine nucleoside phosphorylase (PNP) and purine synthesis. Although the targets of these drugs were known at the time of drug design, it is important to know the compendium of cellular perturbations resulting from use of these inhibitors. Several suspected mechanisms of MTDIA and forodesine in progression of apoptotic cell death have been identified but the underlying mechanisms initiating apoptosis remain elusive. We hypothesize that numerous cellular processes are affected in MTDIA and forodesine treatments given the importance of polyamine and purine synthesis in cancer cells. To elucidate the unsuspected mechanisms mediating anti-cancer activity, unbiased genomic analyses were employed using Saccharomyces cerevisiae. First, gene-gene interactions with MEU1 (the MTAP orthologue in yeast) were determined using Synthetic Genetic Array methodology followed by assessment of drug-gene interactions with MTDIA treatment under a MEU1 essential condition with MTA as the sole source of sulphur. Disruptions to suspected mechanisms of amino acid metabolism, carbohydrate metabolism, response to starvation, vesicle-mediated transport, vacuole fusion, lipid homeostasis, chromatin organisation, transcription, and translation were implicated well as unsuspected mechanisms of NAD+ dependent cellular processes, multi-vesicular body formation, endosomal transport, ion homeostasis, mitochondrion organisation, and cell cycle progression. Induction of autophagy was subsequently confirmed with MTDIA to validate the disruptions to vesicle-mediated transport, response to starvation, multi-vesicular body formation and va

The elucidation of drug targets and their biological effects can be aided by the identification of yeast deletion mutants that confer hypersensitivity to the drug. However, the biological activities of some compounds are reduced by the mechanisms of the pleotropic drug resistance (PDR) network. For this reason a PDR-deficient strain with deletions in the master transcriptional regulators of the PDR network, PDR1 and PDR3 was created. This double deletion mutant strain was robotically mass mated against the non-essential deletion mutant array (DMA) to create genome wide nonessential PDR-deficient DMA (PD-DMA). No phenotypic enhancements were observed with Δpdr1Δpdr3 double mutant and the various deletion strains of the DMA. Increased genome-wide sensitivity of the PDR-deficient mutants was demonstrated by screening pools of PD-DMA and the DMA against cycloheximide, a Pdr5p substrate and rapamycin which is not. Similar sensitivities were observed for the non-PDR substrate rapamycin for the two deletion mutant pools while sensitivity to Pdr5p substrate cycloheximide was significantly more sensitive in the PD-DMA. The genome-wide increased sensitivity of the PDR-deficient mutants was further assessed by screening the LOPAC library of pharmacologically active compounds against pools of PD-DMA and the wild-type background DMA. The DMA screen identified 5 of 1280 compounds having bioactivity whilst the PD-DMA screen identified 25 compounds including the 5 identified by the DMA. The 20 additional compounds identified in the PDR-deficient background were inactive at the concentrations used in the wild-type background. The PD-DMA was then used in chemical genetic profiling assays; namely solid-phase chemical genetic profiling and DNA barcode microarray experiments. The PD-DMA was screened against natural products, rapamycin and cycloheximide with well characterized chemical genetic profiles. The PDR-deficient strains hypersensitivity to rapamycin and cycloheximide were indica