Abstract 1842: PHT-782 an inhibitor of the KRAS signaling nanocluster protein CNKSR1 blocks mutant KRAS signaling and cell growth but does not inhibit wild type KRAS cell growth
Authors: Ihle Nathan T. , Zhang Shuxing , Indarte Martin , Kirkpatrick Lynn , Powis Garth 
Published: August 2012

KRAS is the predominant form of mutated RAS (mut-KRAS) found in 25% of patient tumors across many cancer types. Mut-KRAS plays a critical role in driving tumor growth and resistance to therapy, and its effects are so powerful that it overrides the activity of many molecularly targeted signaling drugs being developed for cancer today such that they cannot be used in patients with mut-KRAS. Using a global siRNA screen and an isogenic MiaPaCa-2 pancreatic cell line with and without oncogenic KRAS, we searched for genes that when inhibited would block the growth of mut-KRAS cancer cells without affecting wild type-KRAS (wt-KRAS) cell growth. A top hit in the screen was CNKSR1 (connector enhancer of kinase suppressor of RAS 1), a protein associated with KRAS in the RAS membrane signaling nanocluster. siRNA knockdown of CNKSR1 inhibited the growth of mut-KRAS non small cell lung cancer (NSCLC) cell lines but not NSCLC cell lines with wt-KRAS. CNKSR1 is a multidomain protein with a pleckstrin homology (PH) domain, a 100 to 120 amino acid, highly conserved but low sequence identity 3D superfold. PH-domains are found in a number of signaling proteins where they are responsible for binding to membrane phosphatidylinositols-3-phosphates (PIP2/3). Over expression of the PH-domain of CNKSR1 in H1373 mut-KRAS NSCLC cells inhibits cell growth suggesting it has a dominant negative effect over native CNKSR1, and that the PH-domain of CNKSR1 is necessary for the effects on mut-KRAS activity. We used in-silico docking of large compound libraries and identified 7 small molecule compounds with predicted low micromolar binding affinity for the PH-domain of CNSKR1. Surface plasmon resonance spectroscopy confirmed the in-silico predictions with binding to the recombinant PH-domain of CNKSR1. The most active compound was PHT-782 (Kd=1.8 μM). PHT-782 inhibited mut-KRAS signaling and the growth of mut-KRAS isogenic and NSCLC cell lines (IC50 ∼ 30 μM) as effectively as siRNA knockdown of KRAS. Importantly, PHT-782 did not block the growth of wt-KRAS cell lines at concentrations up to 100 μM. In mice PHT-782 exhibited a plasma t12 &gt; 4 hr, and had moderate antitumor activity against a mut-KRAS xenograft model without observable toxicity at doses up to 200 mg/kg. To improve potency and selectivity we have undertaken further iterative in-silico lead optimization using a proprietary computational modeling platform PHuDock®. We have identified several candidates with improved binding potency and optimal pharmacodynamic and pharmacokinetic properties. The results of our studies show that the PH domain of CNKSR1 can be targeted by small molecule inhibitors and that the agents selectively block mut-KRAS signaling and cell growth, thus creating a therapeutic potential for patients with oncogenic KRAS for which there is currently no effective therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1842. doi:1538-7445.AM2012-1842