Abstract 326: Discovery and in vitro and in vivo characterization of a novel, small-molecule Ras inhibitor class

Introduction: Mutations in ras genes that result in constitutive activation of Ras proteins are key drivers of oncogenesis, but no effective drugs have been developed that target these aberrant gene products. Through iterative screening and chemical optimization using a phenotypic assay designed to select for Ras inhibitors, we identified a novel series of compounds that potently and selectively inhibit the growth of tumor cells harboring activated Ras relative to cells lacking activated Ras. Several compounds in the series have favorable drug-like properties with strong antitumor activity in a mouse K-Ras mutant tumor model. Methods: Viable cell number was measured using a luminescent indicator of ATP. Disruption of Ras-Raf binding was determined by pre-incubating GST-Raf beads with cell lysates in the presence of test compounds for 30 min. Ras activation was measured by Ras pull-down and western blotting using an anti-Ras antibody. Cell cycle arrest was measured by DNA content. Antitumor activity was determined in a subcutaneous mouse tumor model involving K-Ras mutant HCT116 colon tumors. Mice were treated with Ras inhibitors administered ip for 14 days bid at a dose of 2.5 or 5 mg/kg. Results: Low nanomolar concentrations of DC070-547 inhibited the growth of multiple tumor cell lines harboring activated K-Ras, N-Ras or H-Ras with a selectivity index greater than 100-fold over cells lacking activated Ras. Ras selectivity was confirmed by transfecting human HT29 colon and H322 lung tumor cells that lack activated Ras with mutant H-Ras, thus inducing sensitivity to DC070-547. The compound also inhibited Ras-Raf binding as evident by Ras-pull down assays of lysates from tumor cells treated with DC070-0547 at concentrations that inhibit tumor cell growth. DC070-547 caused G2 phase cell cycle arrest and induced apoptosis selectively in tumor cells containing activated Ras. Cultured epithelial cells derived from normal colon, mammary, and lung tissues were essentially refractory to treatment.The Ras inhibitors were evaluated for antitumor activity in a subcutaneous mouse model involving K-Ras mutant human HCT116 colon tumors. Treatments were well tolerated and completely suppressed tumor growth with the effect being sustained for at least six weeks after treatment was discontinued. Complete tumor regression was also apparent in some of the treated mice. Conclusion: While Ras is widely considered to be non-druggable, we have identified a novel series of compou