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. 2006 May 19;125(4):733-47.
doi: 10.1016/j.cell.2006.03.035. Epub 2006 Apr 27.

A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling

Zachary A Knight  1 , Beatriz Gonzalez, Morri E Feldman, Eli R Zunder, David D Goldenberg, Olusegun Williams, Robbie Loewith, David Stokoe, Andras Balla, Balazs Toth, Tamas Balla, William A Weiss, Roger L Williams, Kevan M Shokat
Affiliations

A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling

Zachary A Knight et al. Cell. .

Abstract

Phosphoinositide 3-kinases (PI3-Ks) are an important emerging class of drug targets, but the unique roles of PI3-K isoforms remain poorly defined. We describe here an approach to pharmacologically interrogate the PI3-K family. A chemically diverse panel of PI3-K inhibitors was synthesized, and their target selectivity was biochemically enumerated, revealing cryptic homologies across targets and chemotypes. Crystal structures of three inhibitors bound to p110gamma identify a conformationally mobile region that is uniquely exploited by selective compounds. This chemical array was then used to define the PI3-K isoforms required for insulin signaling. We find that p110alpha is the primary insulin-responsive PI3-K in cultured cells, whereas p110beta is dispensable but sets a phenotypic threshold for p110alpha activity. Compounds targeting p110alpha block the acute effects of insulin treatment in vivo, whereas a p110beta inhibitor has no effect. These results illustrate systematic target validation using a matrix of inhibitors that span a protein family.

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Figures

Figure 1
Figure 1. Biochemical Analysis of Selected PI3-K Inhibitors
(A) Structures of representative compounds from eleven classes of PI3-K inhibitors. (B) Distribution of IC50 values for selected inhibitors against PI3-K family members. Some IC50 values for AMA-37 and TGX-115 have been reported previously (Knight et al., 2004). (C) Principal component analysis of the relationships between PI3-Ks and inhibitors. (Top) Plot of the similarity between PI3-Ks according to their sequency identity within the kinase domain. (Middle) Plot of the similarity between PI3-Ks according to their IC50 values against small molecule inhibitors. (Bottom) Plot of the similarity between inhibitors based on their IC50 values against PI3-Ks. (D) (Left panel) The ratio of IC50 values for one compound from each chemotype (TGX-115, AMA-37, PIK-39, PIK-75, PIK-90, PIK-93, PI-103, PIK-124) against pairs of class I PI3-Ks. (Right panel) The ratio of IC50 values for one compound from each chemotype with an IC50 < 5 μM against p110α between PIKKs and p110α. Blue dots represent compounds and bars represent the geometric mean of these ratios.
Figure 2
Figure 2. Structures of Isoform-Selective PI3-K Inhibitors
(A) Crystal structure of ATP in the active site of p110γ, highlighting regions designated as the adenine, affinity, and selectivity pockets. Hydrogen bonds are indicated by arrows. (B) Alignment of all reported PI3-K inhibitor cocrystal structures. PIK-39 is colored orange and all other inhibitors are colored blue. Met 804 (red) adopts an up conformation in all structures except PIK-39. (C) Crystal structures and models of isoform-selective PI3-K inhibitors bound to p110γ. (D) Crystal structures and models of multitargeted PI3-K inhibitors bound to p110γ.
Figure 3
Figure 3. Probing the Selectivity and Affinity Pockets
(A) (Top panel) Structure of PIK-39 bound to p110γ and predicted binding mode for IC87114, PIK-293 and PIK-294. Hydrogen bonds are indicated by arrows. (Lower panel) PIK-294 projects a m-phenol (red) into the affinity pocket, and this compound is more potent against the class I PI3-Ks. (B) (Left panel) Ratio of IC50 values for inhibition of mutant and wild-type p110δ. (Center panel) Dose-response curves for inhibition of wild-type, M752I, and M752V p110δ by IC87114 and PIK-39. Error bars represent mean ± SEM. (Right panel) Binding modes of PIK-39 (top) and multitargeted inhibitors (bottom) relative to the Met 752 mutation (red) that differentially perturbs the binding of these inhibitors.
Figure 4
Figure 4. Biochemical Analysis of the PI3-K Pathway in Fat and Muscle Cells
(A) Western blots of lysates from 3T3-L1 adipocytes or L6 myotubes stimulated with insulin (100 nM) in the presence of PI3-K inhibitors. Phosphorylation sites not specified in the figure: ERK1/2 (p202/204), GSK3α/β (p21/9), rpS6 (p235/236), 4E-BP1 (p37/46). (B) Inositol lipid levels following insulin stimulation (100 nM) in the presence of PI3-K inhibitors. (C) Western blots of lysates from L6 myotubes stimulated with LPA (10 μM) in the presence of PI3-K inhibitors.
Figure 5
Figure 5. Effect of Inhibitors on Glucose Transport in Adipocytes and Mice
(A) Insulin-stimulated transport of 3H-2-deoxyglucose into adipocytes. Triangle, insulin plus inhibitor; square, no insulin; diamond, insulin plus cytochalasin B (20 μM); circle, 1% of input 3H-2-deoxyglucose. Error bars represent mean ± SEM. (B) Insulin tolerance tests in mice. Red, insulin plus drug vehicle; blue, insulin plus drug; green, drug plus insulin vehicle; black, insulin vehicle plus drug vehicle. Error bars represent mean ± SEM.
Figure 6
Figure 6. p110α Is the Dominant PI3-K Activity Associated with the IRS-1 Complex
(A) Inhibition of purified class I PI3-Ks by PIK-23 (1.5 μM), TGX-115 (1.5 μM), and PIK-75 (0.05 μM) in vitro in the presence of 10 μM ATP. Error bars represent mean ± SEM. (B) Relative PI3-K activity associated with IRS-1 immunoprecipitates at 1.5, 5, and 30 min after stimulation with insulin (100 nM). Kinase reactions were performed in the presence of DMSO (blue), 1.5 μM PIK-23 (black), 1.5 μM TGX-115 (red), or 0.05 μM PIK-75 (green). Error bars represent mean ± SEM. (C) PI3-K activity associated with immunoprecipitates of the catalytic subunits of class IA enzymes from cells with and without prior insulin stimulation (100 nM, 90 s). Error bars represent mean ± SEM. (D) (Top panel) Western blots for IRS-1 and p85 from immunoprecipitates of each class IA PI3-K. (Bottom panel) Western blot for class IA PI3-Ks from immunoprecipitates of p85. (E) Western blot for IRS-1, p85, and phosphorylated Ser 307, Ser 612, or Ser 636 of IRS-1 from adipocytes treated with insulin (100 nM, 30 min).
Figure 7
Figure 7. p110β Sets a Threshold for p110α Activity in Myotubes, but Not Adipocytes
(A) Dose response for inhibition of Akt Thr 308 phosphorylation in response to TGX-115 (black), PIK-75 (blue), or PIK-75 + 10 μM TGX-115 (red) in adipocytes or myotubes. Representative Western blots are shown below. Error bars represent mean ± SEM. (B) Effect of the p110β inhibitors TGX-115 and TGX-286 on Akt phosphorylation induced by the PTEN inhibitor bpV(pic) (5 μM).
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References

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