Aberrant KRAS signaling plays an important role in the pathogenesis of malignancies including pancreatic (PDAC), colon and lung cancer. Because therapies targeting either downstream or associated factors of KRAS pathway have shown dismal results in clinics, there is a need to identify effective agents targeting active KRAS (GTP-KRAS) protein. In this study, we screened a chemical library for their efficacy to inhibit KRAS activity using in silico methods. Based on their binding energy, we selected a candidate agent (LP1) that exhibited high potential of binding to KRAS protein at a region where KRAS-activator protein “SOS1” binds to, and GDP-GTP exchange takes place. We next investigated if LP1 exhibits the efficacy of KRAS binding in biological solution using recombinant proteins and employing drug development techniques i.e., florescence (FL)-based competition, Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) assays. The FL-based study show that LP1 inhibits the binding of SOS1 protein and GTP molecules to KRAS protein. The ITC and SPR analysis showed that LP1 binds to the KRAS protein (at 10 μM). We next determined the KRAS-inhibitory potential of LP1 in activated-KRAS cells representing premalignant and malignant models of PDAC, colon and lung cancer. Dissociation study involving incubation of KRAS/SOS1 proteins showed the LP1 inhibits the binding of SOS1 to KRAS. Notably, LP1 therapy significantly decreased the (i) KRAS-GTP protein levels (ii) growth and (iii) proliferation of activated-KRAS single cell and 3D Organoid cultures. We next tested KRAS-inhibitory efficacy of LP1 in PDAC patient-derived (PD) tumor explants and found that LP1 therapy (10 days) significantly decreased growth of, and KRAS-GTP levels in PD explants. Based on the in silico, in solution, cell model and PD-explant data, we determined the efficacy of LP1 as a KRAS inhibitor in in vivo conditions. We first performed the pharmacokinetic study of LP1 in mice and observed that LP1 is physiologically available in the blood (peak of 15-20 μM) after oral and intraperitoneal administrations and detectable up to 24h post-administration. We next tested LP1 therapy in tumor xenograft models and show that LP1 therapy significantly reduces the tumorigenicity of KRAS-activated PDAC and colon cell-derived tumors implanted in athymic mice. We next tested efficacy of LP1 administration on the developmental phases of PDAC disease (from normal to high grade neoplasia/early stage carcinoma) and show that LP1 feeding for six months caused a significant inhibition of PanIN development in KPCG12D transgenic mouse model. Notably the analysis of tumors xenograft models and pancreatic tissues of KPCG12D mice receiving LP1 therapy exhibited reduced KRAS-GTP levels. Taken together, these data show that LP1 is a potent KRAS activity inhibitor and a potential candidate for clinical use against KRAS-driven cancers.

Citation Format: Arsheed Ahmad Ganaie, Hifzur R. Siddique, Ishfaq Sheikh, Lei Wang, Aijaz Parray, Jayanth panyam, Peter Villalta, Joshua Liao, Yibin deng, Mohammad saleem. Development of a novel KRAS-targeting agent: systematic validation using in silico, in solution, cell models, PDX and transgenic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1246. doi:10.1158/1538-7445.AM2017-1246