"ABSTRACT Recent studies of the applicants support the role of the endogenous gaseous biological mediator hydrogen sulfide (H2S) in colorectal cancer. In specific, the selective upregulation of cystathionine-?- synthase (CBS) and the subsequent production of H2S in colonic cancer cells serves as a pro- survival factor by stimulating tumor cell bioenergetics, growth, proliferation, migration and invasion. This work identifies CBS as a novel antitumor target. Currently, the most potent inhibitor of CBS known is aminooxyacetic acid (AOAA), which exerts potent anticancer effects in vitro and in vivo. However, the concentrations of AOAA necessary to exert antiproliferative effects in cell-based systems and preclinical animal models are rather high, compared to its high in vitro potency on isolated CBS enzyme. Based on the preliminary data, the cell-based potency of AOAA can be improved by synthesis of cell-permeable prodrugs of AOAA. For instance, YD0171 (aminooxyacetic acid methyl ester) markedly enhances the antiproliferative effect of its parent, AOAA, in HCT116 human colon cancer cells. In order to advance the clinical translation of AOAA, we will pursue the following two Aims: Aim #1. To synthesize diverse prodrug analogs of AOAA. Various classes of ester prodrugs will be synthesized, and tested both in a cell-free CBS enzyme activity assay, and a cell proliferation assay using HCT116 colon cancer cells. Synthetic work and in vitro testing will be pursued in an iterative fashion, in order to identify the most active prodrugs. We will also evaluate prodrug selectivity in a proliferation assay comparing colonic cancer cells to non-transformed colonic epithelial cells, and characterize the release of the active metabolite from the prodrugs upon incubation with cells or plasma. Aim #2. To evaluate a selected subset of novel AOAA prodrugs in preclinical models of colon cancer. First, inhibition of tumor CBS activity will be established using an in vivo/ex vivo approach. Next, in vivo pre- and post-treatment efficacy will be established, in comparison to the parent compound AOAA, in a stringent, well-established preclinical model of human cancer, which utilizes tumor-bearing mice subjected to orthotopic transplantation of patient- derived colon cancer xenografts (PDTX). Successful completion of the current project will be evidenced by the identification of at least one AOAA prodrug, which exerts antiproliferative effects with higher potency than AOAA in vitro and in vivo. Selection of a development candidate will trigger a Phase II project, aimed at IND-enabling studies, followed by clinical development. The applicant team (the PI at CBS Therapeutics, the head of the subcontract at the University of Texas Galveston and all participating co-investigators) has all necessary theoretical and practical expertise, know-how and infrastructure to conduct the proposed work. " |