TY - JOUR T1 - Isoform-Selective Inhibition of Phosphoinositide 3-Kinase: Identification of a New Region of Nonconserved Amino Acids Critical for p110α Inhibition JF - Molecular Pharmacology JO - Mol Pharmacol SP - 657 LP - 664 DO - 10.1124/mol.111.072546 VL - 80 IS - 4 AU - Zhaohua Zheng AU - Syazwani I. Amran AU - Philip E. Thompson AU - Ian G. Jennings Y1 - 2011/10/01 UR - http://molpharm.aspetjournals.org/content/80/4/657.abstract N2 - The combination of molecular modeling and X-ray crystallography has failed to yield a consensus model of the mechanism for selective binding of inhibitors to the phosphoinositide 3-kinase (PI3K) p110 α-isoform. Here we have used kinetic analysis to determine that the p110α-selective inhibitor 2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-α]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid (PIK-75) is a competitive inhibitor with respect to a substrate, phosphatidylinositol (PI) in contrast to most other PI3K inhibitors, which bind at or near the ATP site. Using sequence analysis and the existing crystal structures of inhibitor complexes with the p110γ and -δ isoforms, we have identified a new region of nonconserved amino acids (region 2) that was postulated to be involved in PIK-75 p110α selectivity. Analysis of region 2, using in vitro mutation of identified nonconserved amino acids to alanine, showed that Ser773 was a critical amino acid involved in PIK-75 binding, with an 8-fold-increase in the IC50 compared with wild-type. Kinetic analysis showed that, with respect to PI, the PIK-75 Ki for the isoform mutant S773D increased 64-fold compared with wild-type enzyme. In addition, a nonconserved amino acid, His855, from the previously identified region 1 of nonconserved amino acids, was found to be involved in PIK-75 binding. These results show that these two regions of nonconserved amino acids that are close to the substrate binding site could be targeted to produce p110α isoform-selective inhibitors. ER -