Abstract
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.
Footnotes
This work was funded by the National Health and Medical Research Council of Australia [Grant 545943]
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.072546.
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ABBREVIATIONS:
- PI3K
- phosphoinositide 3-kinase
- CAL101
- 5-fluoro-3-phenyl-2-[(1S)-1-(1H-purin-6-ylamino)propyl]-4(3H)-quinazolinone
- PIK-75
- 2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-α]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid
- PI
- phosphatidylinositol
- ZSTK474
- 2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine
- PIK-39
- 5-chloro-3-(2-methoxyphenyl)-2-(7H-purin-6-ylsulfanylmethyl)quinazolin-4-one
- PDB
- Protein Data Bank
- WT
- wild type
- LY294002
- 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
- PIP2
- phosphatidylinositol 4,5-bisphosphate
- IC-87114
- 2-[6-amino-9H-purin-9-yl)methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone
- AS15
- 2-[[3-(2-methoxyphenyl)-4-oxo-5,6,7,8-tetrahydroquinazolin-2-yl]sulfanyl]-N-quinoxalin-6-ylacetamide
- GDC-0941
- 2-(1H-indazol-4-yl)-6-[[4-(methylsulfonyl)-1-piperazinyl]methyl]-4-(4-morpholinyl)thieno[3,2-d]pyrimidine.
- Received March 30, 2011.
- Accepted July 21, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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