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, another approach is being pursued for the identification of allosteric inhibitors targeting relatively less conserved binding sites, thus higher selectivity can be attained. PIK3CA is the most frequently mutated kinase in human cancers, with 80% of the mutations occuring in two hotspots: 1) an H1047R mutation is found at the kinase (membrane binding) domain and 2) an E545K mutation is found at the helical domain (amino acid replacement with charge reversal).<br/>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 the allosteric networks of the WT and each of the mutated proteins. 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 PIK3CA inhibitors acting at a micromolar concentration. The effect of the inhibitors on WT and H1047R mutant PIK3CA-membrane associations was subsequently studied by SPR experiments, which showed that these small molecules alter the protein-membrane affinity, unlike other known PIK3CA 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 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 with the controls. Details about the results with the E545K will be presented at the meeting.

Νovel small molecule modulators of the hotspot PIK3CA mutants identified by computational and experimental approaches

2019 Spring National Meeting

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