Characterisation of a cyclic peptide that binds to the RAS binding domain of phosphoinositide 3-kinase p110α

P110 alpha is a member of the phosphoinositide 3-kinase (PI3K) enzyme family that functions downstream of RAS. RAS proteins contribute to the activation of p110 alpha by interacting directly with its RAS binding domain (RBD), resulting in the promotion of many cellular functions such as cell growth, proliferation and survival. Previous work from our lab has highlighted the importance of the p110 alpha/RAS interaction in tumour initiation and growth. Here we report the discovery and characterisation of a cyclic peptide inhibitor (cyclo-CRVLIR) that interacts with the p110 alpha-RBD and blocks its interaction with KRAS. cyclo-CRVLIR was discovered by screening a "split-intein cyclisation of peptides and proteins" (SICLOPPS) cyclic peptide library. The primary cyclic peptide hit from the screen initially showed a weak affinity for the p110 alpha-RBD (K-d about 360 mu M). However, two rounds of amino acid substitution led to cyclo-CRVLIR, with an improved affinity for p110 alpha-RBD in the low mu M (K-d 3 mu M). We show that cyclo-CRVLIR binds selectively to the p110 alpha-RBD but not to KRAS or the structurally-related RAF-RBD. Further, using biophysical, biochemical and cellular assays, we show that cyclo-CRVLIR effectively blocks the p110 alpha/KRAS interaction in a dose dependent manner and reduces phospho-AKT levels in several oncogenic KRAS cell lines.