Abstract A140: Novel drug development candidate potently and selectively inhibits growth of tumor cells harboring activated Ras
Authors: Piazza Gary A. , Zhu Bing , Lee Kevin , Canzoneri Joshua , Sigler Sara , Lindsey Ashley , Ramirez-Alcantara Veronica , Madeira da Silva Luciana , Mullins Haddon , Trinh Alisa , Berry Kristy , Valiyaveettil Jacob , Keeton Adam B. , Chen Xi , Boyd Michael R. 
Published: May 2016

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 chemical synthesis and screening using a phenotypic assay designed to select for Ras inhibitors, we identified a small-molecule lead compound that was then optimized for potency and selectivity to inhibit the growth of tumor cells harboring activated Ras relative to cells lacking activated Ras.

Materials and methods: Viable cell number was measured using the CellTiter-Glo® ATP assay (Promega) following 72 hr of treatment. Ras activation status was measured by precipitating GTP-bound Ras from cell lysates with GST-Raf1-RBD/GSH sepharose followed by western blotting using anti-Ras antibody. Ras binding was determined by pre-incubating cell lysates with GST-Raf1-RBD/GSH sepharose, treating with test compounds for 30 min, followed by western blotting using anti-Ras antibody. Cell cycle distribution was measured by DNA content following DyeCycle Green labeling.

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 selectivity indices greater than 100-fold over cells lacking activated Ras. By surveying a large panel of human colon, breast, and lung tumor cell lines, a strong correlation was measured between potency to inhibit tumor cell growth and Ras activation status. Ras selectivity was confirmed by transfecting human H322 lung tumor cells that lack activated Ras with mutant H-Ras and inducing sensitivity to DC070-547. The compound inhibited Ras-Raf binding as shown by Ras-pull down in cell lysates following treatment with DC070-547 at concentrations that inhibit tumor cell growth. DC070-547 also caused cell cycle arrest in the G2 phase selectively in tumor cells containing activated Ras. Interestingly, cultured epithelial cells derived from normal colon, mammary, and lung tissues were essentially refractory to treatment.

Conclusion: While Ras is widely considered to be non-druggable, we have identified a series of compounds that potently and selectively inhibit the growth of tumor cells harboring activated Ras. With promising drug-like properties, DC070-547 has been selected from this series as a prospective drug development candidate that is being evaluated for anti-tumor efficacy and toxicity in preclinical models.

Citation Format: Gary A. Piazza, Bing Zhu, Kevin Lee, Joshua Canzoneri, Sara Sigler, Ashley Lindsey, Veronica Ramirez-Alcantara, Luciana Madeira da Silva, Haddon Mullins, Alisa Trinh, Kristy Berry, Jacob Valiyaveettil, Adam B. Keeton, Xi Chen, Michael R. Boyd. Novel drug development candidate potently and selectively inhibits growth of tumor cells harboring activated Ras. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A140.