5-Methylthioadenosine (MTA) is a product of AdoMet mediated polyamine synthesis. 5-Methylthioadenosine phosphorylase (MTAP) is the sole enzyme to metabolize MTA in humans, producing adenine and methylthioribose-1-phosphate (MTR-1-P). These are salvaged in the methionine and adenine salvage pathways for resynthesis of AdoMet. Previous transition state analysis of MTAP has led to the design and synthesis of powerful transition state analogue inhibitors of MTAP. They show selective anti-cancer effects in human cancer cell lines in culture as well as in mouse human cancer xenografts. A sensitive, continuous assay for characterizing MTAP catalytic activity is needed and will be developed. We propose a new assay with an alternative substrate, 2-amino-5-methylthioadenosine (2AMTA), to yield an intense fluorescent product that can be precisely and continuously quantified. This new technology will provide an accurate method of characterizing kinetic properties of MTAP. It is also applicable to bacterial enzymes using MTA as a substrate. We will further assess MTAP transition state analogues as anti-cancer agents. We will use a mouse model of human Familial Adenomatous Polyposis (FAP), APCMin/+, to determine the efficacy of MTAP transition state analogues as treatments for spontaneously forming cancers in immunocompetent mice. Analysis involves potential delay of disease onset and a test of disease reversal based on early and late treatments. Progression of disease analysis uses microPET live-animal imaging. Finally we will solve the transition state structure of human methionine adenosyl transferase (MAT) enzymes, with the goal of setting the foundation for future transition state analogue design. MAT2A, the cancer-cell specific MAT isoform, is upregulated in FaDu cells made resistant to MTAP transition state analogues. We hypothesize that transition state analogue inhibitors of MAT2A will work synergistically with MTAP transition state analogues and will amplify the anti-cancer effects of both therapeutics.
The goal of this project is to evaluate inhibitors of methionine metabolism for efficacy as anti-cancer therapeutics. This will involve designing a new method for measuring MTAP inhibitory activity when screening potential drug candidates. Additionally we will test the anti-cancer potential of an MTAP inhibitor in an in vivo mouse model of human colorectal cancer. Finally we will solve the transition state structure of another enzyme involved in methionine metabolism, MAT2A, with the goal of designing inhibitors with enhanced anti-cancer effects when used in tandem with MTAP inhibitors.
|Firestone, Ross S; Schramm, Vern L (2017) The Transition-State Structure for Human MAT2A from Isotope Effects. J Am Chem Soc 139:13754-13760|
|Firestone, Ross S; Cameron, Scott A; Karp, Jerome M et al. (2017) Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5'-Methylthioadenosine Phosphorylase. ACS Chem Biol 12:464-473|
|Firestone, Ross S; Cameron, Scott A; Tyler, Peter C et al. (2016) Continuous Fluorescence Assays for Reactions Involving Adenine. Anal Chem 88:11860-11867|