Recent studies of the applicants support the role of the endogenous gaseous biological mediator hydrogensulfide(H2S)incolorectalcancer.Inspecific,theselectiveupregulationofcystathionine-?- 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 esterprodrugswillbesynthesized,andtestedbothinacell-freeCBSenzymeactivityassay,anda 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 prodrugselectivityinaproliferationassaycomparingcoloniccancercellstonon-transformedcolonic 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 preclinicalmodelsofcoloncancer.First,inhibitionoftumorCBSactivitywillbeestablishedusingan 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 withhigherpotencythanAOAAinvitroandinvivo.Selectionofadevelopmentcandidatewilltrigger a Phase II project, aimed at IND-enabling studies, followed by clinical development. The applicant team(thePIatCBSTherapeutics,theheadofthesubcontractattheUniversityofTexasGalveston andallparticipatingco-investigators)hasallnecessarytheoreticalandpracticalexpertise,know-how andinfrastructuretoconducttheproposedwork.
PROJECTRELEVANCE Recentdatashowthatanimalcellsandhumancellsproducehydrogensulfide(H2S),anduse it for various vital biological functions. The applicants have discovered that tumor cells containaspecificprotein,CBS,whichproduceslargeamountsofH2Sgas.Subsequentwork bytheapplicantsshowedthatH2Ssupportsthegrowthandexpansionofcancercellswithin the cancer patients' body via several mechanisms. First: tumor-produced H2S serves as a 'hormone' that stimulates the growth and expansion of the tumor. Second: tumor-produced H2S serves as a 'fuel' to feed the metabolism of the tumor cells, in order to keep them 'energized'.Third:H2Sstimulatesthegrowthofnewbloodvesselsaroundthetumortissue, sothatthetumorcan'suckaway'freshbloodandnutritientsfromthehost.Fourth:thetumor 'hijacks'thebloodvessels,andforcesittoproduceevenmoreH2S.Insummary,thetumor producesaH2S-richlocalenvironment,inordertothrive.Thetherapeuticimplicationofthese processesis,thatbyblockingthetumorfromoverproducingH2S,thetumorwillloseits'fuel supply',andwillstopgrowing:itwillshrinkandwillbeeliminatedbythehost'simmunological defenses. In the current project the applicants propose to synthesize, test and optimize a diverse range of novel CBS inhibitor compound analogs, that are all prodrug derivatives of the prototypical CBS inhibitor aminooxyacetic acid (AOAA). Based on preliminary data, prodrugs of AOAA have improved cell uptake and anticancer effect compared to AOAA. AppropriatelyselectedAOAAprodrugswouldbeidealcandidatesforfuturedevelopmentfor theexperimentaltherapyofcolorectalcancer.Theprojectplaysacriticalroleinthepractical translationofCBSinhibition,aradicallynewconceptforcancertherapy.
