Kinases are intensely pursued as drug targets for cancer treatment. Usually, kinase inhibitors target the active center (ATP binding site). However, the similarity of this site across many kinases often results in non-selective inhibition. Accordingly, an alternative approach is being pursued for the identification of allosteric inhibitors targeting relatively less conserved binding sites, thus higher selectivity can be attained. PI3Kα is the most frequently mutated kinase in human cancers, with 80% of the mutations occurring in two hotspots of the PIK3CA gene, which encodes the catalytic subunit of PI3Kα: 1) the H1047R mutation, which is found at the kinase (membrane binding) domain and 2) the E545K mutation, which is found at the helical domain (amino acid replacement with charge reversal). To assess the activation mechanisms of PIK3CA mutants, extensive microsecond Molecular Dynamics (MD) simulations were performed to examine conformational differences between the wild type (WT) and mutant proteins. The MD results were used to predict alterations in the allosteric networks of the WT upon each of the mutation. Binding site analysis of allosteric action in the kinase domain identified cavities in the vicinity of the H1047R mutation and the membrane-binding region. Virtual screening and subsequent biochemical assays were used to identify several PI3Kα inhibitors acting at a micromolar concentration. The effect of the inhibitors on WT and H1047R mutant PI3Kα-membrane associations was subsequently studied by SPR experiments, which showed that these small molecules alter the protein-membrane affinity, unlike other known PI3Kα inhibitors such as wortmannin. It was also observed that the inhibitory concentration remained unchanged even under conditions of increased ATP concentration, indicating that these inhibitors are non-competitive. The most promising small molecules were further tested in vivo using xenografts and genetically modified mouse models. Micro PET/CT imaging of the tumors showed reduction in volume after treatment. Immunohistochemical analysis of the xenografts showed reduction of proliferation and increase in apoptosis after treatment, in comparison to the controls. Details about the results with the E545K will be presented at the meeting.

Note: This abstract was not presented at the meeting.

Citation Format: Zoe Cournia, Paraskevi Gkeka, Hari Leontiadou, Ioannis Galdadas, Christina Athanasiou, Antriana Papadimitropoulou, Vasiliki Lazani, Maria Pavlaki, Bogos Agianian, Savvas Christoforidis, Argiris Efstratiadis. Νovel small molecule modulators of the hotspot PIK3CA mutants identified by computational and experimental approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3918.