TITLE: Therapeutic repurposing of benserazide for colon cancer ABSTRACT: We have recently identified cystathionine-beta-synthase (CBS)-derived hydrogen sulfide (H2S) as an endogenous tumor-promoting factor and thus a potential new therapeutic target in colorectal cancer. The importance of CBS-derived H2S in the pathobiology of colorectal cancer is supported by: 1) the significant anti- tumorigenic effects of CBS gene silencing, and 2) induction of an invasive metastatic phenotype when aberrantly overexpressed. Also, treatment with the prototypical CBS inhibitor aminooxyacetic acid (AOAA) reduces colon cancer cell proliferation in vitro and tumor growth and metastasis in vivo. However, the potency, specificity and selectivity of AOAA is suboptimal. Moreover, no clinically used, professional CBS inhibitors exist. To facilitate the translation of CBS inhibitors as anticancer therapeutics, via the repurposing route, we have conducted a virtual screening 260,000 compounds, with special emphasis of (a) commonalities in the emerging structures and (b) potential availability of the identified compounds for rapid repurposing. The virtual screening was conducted using a combination of publicly available software. The results produced several common scaffolds, one of them being a class of compounds which also included benserazide, a clinically used anti- Parkinson compound with its original pharmacological action of being a peripherally acting DOPA decarboxylase inhibitor. We have tested benserazide in vitro on human recombinant CBS, and confirmed it CBS inhibitory activity. In order to begin clinical translation of the compounds into the direction of anticancer effects, we have also tested its effect in HCT116 cells, a human colon cancer line. The compound caused a concentration-dependent inhibition of HCT116 proliferation. We have also conducted Pubmed searches and have compared the antiproliferative concentration range of benserazide with the concentration range of the compound in patients; have done pharmacokinetic/pharmacodynamic modeling using publicly available software (Winnonlin) to estimate the concentrations of benserazide likely achievable in the tumor tissue of patients. Taken together, these data suggest that benserazide, after an appropriately selected set of preclinical studies proposed in the current application, may be a candidate for repurposing for the clinical therapy of colon cancer. Aim 1 will focus on hit validation and repurposing feasibility testing in vitro. Aim 2 will focus on ?hit? validation in vivo. Completion of the proposed project is expected to demonstrate the efficacy of benserazide as an anticancer agent, and is expected to build preclinical rationale for the repurposing of benserazide for the experimental therapy of patients with advanced colorectal cancer. The applicant team has all necessary theoretical and practical expertise to conduct the proposed work.