Claire Chaussade

Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling

Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110alpha isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signalling. These include a p110alpha-specific inhibitor (PIK-75), a p110alpha-selective inhibitor (PI-103), a p110beta-specific inhibitor (TGX-221) and a p110delta-specific inhibitor (IC87114). Although we find that p110alpha is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110beta or p110delta alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110alpha inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110alpha, p110beta or p110delta. These results provide evidence that p110beta and p110delta can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.

Functional differences between two classes of oncogenic mutation in the PIK3CA gene

PIK3CA codes for the p110alpha isoform of class-IA PI 3-kinase and oncogenic mutations in the helical domain and kinase domain are common in several cancers. We studied the biochemical properties of a common helical domain mutant (E545K) and a common kinase domain mutant (H1047R). Both retain the ability to autophosphorylate Ser608 of p85alpha and are also inhibited by a range of PI 3-kinase inhibitors (Wortmannin, LY294002, PI-103 and PIK-75) to a similar extent as WT p110alpha. Both mutants display an increased V(max) but while a PDGF derived diphosphotyrosylpeptide caused an increase in V(max) for WT p85alpha/p110alpha it did not for the E545K variant and actually decreased V(max) for the H1047R variant. Further, the E545K mutant was activated by H-Ras whereas the H1047R mutant was not. Together these results suggest helical domain mutants are in a state mimicking activation by growth factors whereas kinase domain mutants mimic the state activated by H-Ras.

Novel pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors: Exploring the benzenesulfonohydrazide SAR

Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues.

Phosphoinositide-3-kinase (PI3K) inhibitors: Identification of new scaffolds using virtual screening

In the present work, we used virtual screening (VS) of the ZINC database of 2.5 million compounds to seek new PI3K inhibitory scaffolds. The VS flowchart implemented various filters, including a 3D-database screen, and extensive docking studies, to derive 89 derivatives that were experimentally assayed against the four PI3K isoforms. Seven compounds showed inhibitory activities between 1 and 100 microM, with four being sufficiently potent to constitute potential new scaffolds. The binding conformations of these four were analyzed to provide a rationalization of their activity profile.

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.

Investigating the role of class-IA PI 3-kinase isoforms in adipocyte differentiation.

PI 3-kinases, in particular class-IA, are key signalling molecules controlling many cellular processes including growth, proliferation, migration and differentiation. In this study, we have used a collection of isoform selective PI 3-kinase inhibitors to determine whether attenuation of signalling through class-IA PI 3-kinase isoforms will impact adipocyte differentiation. First, we analysed the expression profiles and found that fibroblastic pre-adipocytes express detectable levels of p110alpha and p110delta and that after differentiation, p110delta levels fall while p110alpha levels rise, together with C/EBPalpha and PPARgamma. When using specific inhibitors during the differentiation process, we observed that neither p110beta nor p110delta inhibition, had any significant effect. In contrast PIK-75, a selective p110alpha inhibitor completely abolished adipocyte differentiation as assessed by morphology, transcript and protein levels of adipocyte markers. These results indicate that long term treatment with p110alpha inhibitors could potentially have a severe impact on fat cell numbers in vivo.

Evidence for a role for the p110-α isoform of PI3K in skeletal function

Signaling through phosphatidylinositol-3 kinases (PI3K) regulates fundamental cellular processes such as survival and growth, and these lipid kinases are currently being investigated as therapeutic targets in several contexts. In skeletal tissue, experiments using pan-specific PI3K inhibitors have suggested that PI3K signaling influences both osteoclast and osteoblast function, but the contributions of specific PI3K isoforms to these effects have not been examined. In the current work, we assessed the effects of pharmacological inhibitors of the class Ia PI3Ks, alpha, beta, and delta, on bone cell growth, differentiation and function in vitro. Each of the class Ia PI3K isoforms is expressed and functionally active in bone cells. No consistent effects of inhibitors of p110-beta or p110-delta on bone cells were observed. Inhibitors of p110-alpha decreased osteoclastogenesis by 60-80% (p<0.001 vs control) by direct actions on osteoclast precursors, and decreased the resorptive activity of mature osteoclasts by 60% (p<0.01 vs control). The p110-alpha inhibitors also decreased the growth of osteoblastic and stromal cells (p<0.001 vs control), and decreased differentiated osteoblast function by 30% (p<0.05 vs control). These data suggest that signaling through the p110-alpha isoform of class Ia PI3Ks positively regulates the development and function of both osteoblasts and osteoclasts. Therapeutic agents that target this enzyme have the potential to significantly affect bone homeostasis, and evaluation of skeletal endpoints in clinical trials of such agents is warranted.

Novel pyrazolo[1,5-a]pyridines as PI3K inhibitors: variation of the central linker group

As part of our investigation into the pyrazolo[1,5-a]pyridines as novel PI3K inhibitors, we report a range of analogues where the central linker portion of the molecule was varied while retaining the pyrazolo[1,5-a]pyridine and arylsulfonyl or arylcarbonyl groups. Isostere generating software BROOD was used to assist with producing ideas. The isoform selectivity of the compounds varied from pan-PI3K for compound 41 to p110α-selective for compound 58 or p110δ-selective for compound 57. The latter two compounds varied only in their sulphur oxidation state.

Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.