Dissecting isoform selectivity of PI3K inhibitors: the role of non-conserved residues in the catalytic pocket

The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr⁸⁸⁶ and Lys⁸⁹⁰ of the PI3Kγ isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the β isoform (Glu⁸⁵⁸ and Asp⁸⁶²) and those in the α isoform (His⁸⁵⁵ and Gln⁸⁵⁹) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of β-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the α-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kβ activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the α-isoform-selectivity of the compounds studied.