Definition of the binding mode of a new class of phosphoinositide 3-kinase α-selective inhibitors using in vitro mutagenesis of non-conserved amino acids and kinetic analysis

The binding mechanism of a new class of lipid-competitive, ATP non-competitive, p110α isoform-selective PI3K (phosphoinositide 3-kinase) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of nonconserved amino acids in the p110α and p110β isoforms, we have identified three unique binding mechanisms for the p110α-selective inhibitors PIK-75, A-66S and J-32. Each of the inhibitor's p110α-isoform-selective binding was found to be due to interactions with different amino acids within p110. The PIK-75 interaction bound the non-conserved region 2 amino acid p110α Ser ⁷⁷³, A-66S bound the region 1 non-conserved amino acid p110α Gln ⁸⁵⁹, and J-32 binding had an indirect interaction with Lys ⁷⁷⁶ and Ile ⁷⁷¹. The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.