Elucidating the anti-cancer mechanisms for transition-state structure inhibitors of nucleoside phosphorylases methylthioadenosine-DADMe-immucillin A and forodesine

Abstract

<p>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 vacuolar fusion. Reduction in ergosterol levels and disruptions to ergosterol biosynthetic proteins were confirmed with MTDIA and meu1Δ to validate disruptions to lipid homeostasis. To complement the genetic analyses, the abundance and localisation of proteins were evaluated in response to MTDIA or MEU1-deficiency. Disruptions to proteins implicated in carbohydrate metabolism, methionine salvage, transcription, translation, transmembrane transport, lipid homeostasis, cell cycle and DNA repair were identified with meu1Δ and MTDIA. Key findings from the analysis of protein abundance and localization were the relocalisation of plasma membrane proteins and disruptions to vesicle mediated transport proteins consistent with the induction of autophagy and disruptions to proteins in homeostasis of all major lipid classes, further corroborating the findings of screening gene deletion mutants for elucidating drug mechanisms. To investigate the mechanisms of forodesine toxicity, genetic interactions with PNP1 (the PNP orthologue in yeast) were determined using Synthetic Genetic Array methodology. Disruptions to amino acid metabolism, starvation responsive genes, vacuolar organisation and vesicle mediated transport, carbohydrate metabolism, lipid homeostasis, chromatin organisation, chromosome segregation, transcription, and translation were identified in response to PNP1-deficency. Despite the introduction of several human genes and supplementation of metabolites required for forodesine bioactivity in humans, forodesine was not sufficiently bioactive in yeast to evaluate sensitivity of gene deletion mutants to forodesine. Overall, chemical genomic analyses in yeast with transition-state structure analogues MTDIA and forodesine effectively highlight the vast number of cellular processes affected by inhibition of a single target. Moreover, genome-wide pre-screening should be carried out in yeast to identify side-effects and secondary effects from drug target inhibition prior to assessing desired and undesired outcomes of highly specific drugs in human cells.</p>

Characterising Changes in Lipid Homeostasis as an Anti-Cancer Mechanism of Action of Methylthio-DADMe-Immucillin-A

“…Since MTDIA targets SAM, a metabolite shown to regulate lipid homeostasis, this thesis aims to understand the depth of lipid metabolism-related effects caused by MTDIA (Lu et al 2012). This study expands on previous findings from our laboratory that identified dysregulation of proteins involved in glycerophospholipid, sphingolipid, and ergosterol metabolism caused by MTDIA treatment or MTAP knockout (Coorey 2017). SAM, polyamine, and lipid metabolism are highly conserved processes from yeast to humans (Heby et al 1990;Kurat et al 2006;Nielsen 2009).…”

“…Therefore, nucleoside transporters play a pivotal role in the efficacy and pharmacological activity of nucleoside based drugs such as MTDIA (J. D. Young et al 2013;Yao et al 2011). As wild type (WT) S. cerevisiae cells cannot uptake nucleosides (Gietz et al 2007), the human equilibrative nucleoside transporter 1 (hENT1) from the SLC29 family of nucleoside transporters was transformed into the yeast haploid deletion mutant library, thereby creating a library of yeast mutants bearing the hENT1 protein (Coorey 2017). The incorporation of hENT1 transporter facilitates the entry of MTDIA into yeast cells.…”

“…Solid-phase chemical genetic profiling methodology was used to identify deletion mutants that become hypersensitive with MTDIA treatment. The data obtained from this screen were extrapolated to generate a profile of cellular processes that were perturbed by MTDIA (Coorey 2017). Of the many processes identified, perturbation of deletion mutants associated with lipid metabolism and homeostasis were indicated.…”

“…Genes involved in glycerophospholipid and sphingolipid metabolism are perturbed by MTDIA (Coorey 2017). Glycerohospholipids and sphingolipids contribute largely to the architecture of the yeast plasma membrane and can also be found at varied levels in organelles (Klug et al 2014).…”

“…The Saccharomyces cerevisiae Y7092 yeast strain derived from the S288c background was kindly provided by Prof. Charlie Boone (University of Toronto) and transformed with the human equilibrative nucleoside transporter (hENT1) (Coorey 2017).…”