The p110 delta structure: mechanisms for selectivity and potency of new PI(3)K inhibitors.

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies.

SHIP prevents lipopolysaccharide from triggering an antiviral response in mice.

Gram-negative bacterial infections, unlike viral infections, do not typically protect against subsequent viral infections. This is puzzling given that lipopolysaccharide (LPS) and double-stranded (ds) RNA both activate the TIR domain-containing adaptor-inducing interferon beta (TRIF) pathway and, thus, are both capable of eliciting an antiviral response by stimulating type I interferon (IFN) production. We demonstrate herein that SH2-containing inositol-5'-phosphatase (SHIP) protein levels are dramatically increased in murine macrophages via the MyD88-dependent pathway, by up-regulating autocrine-acting transforming growth factor-beta (TGFbeta). The increased SHIP then mediates, via inhibition of the phosphatidylinositol-3-kinase (PI3K) pathway, cytosine-phosphate-guanosine (CPG)- and LPS-induced tolerance and cross-tolerance and restrains IFN-beta production induced by a subsequent exposure to LPS or dsRNA. Intriguingly, we found, using isoform-specific PI3K inhibitors, that LPS- or cytosine-phosphate-guanosine-induced interleukin-6 (IL-6) is positively regulated by p110alpha, -gamma, and -delta but negatively regulated by p110beta. This may explain some of the controversy concerning the role of PI3K in Toll-like receptor-induced cytokine production. Consistent with our in vitro findings, SHIP(-/-) mice overproduce IFN-beta in response to LPS, and this leads to antiviral hypothermia. Thus, up-regulation of SHIP in response to Gram-negative bacterial infections probably explains the inability of such infections to protect against subsequent viral infections.

Characterization of inhibitor binding to human kinesin spindle protein by site-directed mutagenesis.

A number of inhibitors of kinesin spindle protein (KSP) have been described, which are known from X-ray crystallography studies to bind to an induced fit pocket defined by the L5 loop. We describe the characterization of eight mutant forms of KSP in which six residues that line this pocket have been altered. Mutants were analyzed by measuring rates of enzyme catalysis, in the presence and absence of six KSP inhibitors of four diverse structural classes and of varied ATP-competition status. Our analysis was in agreement with the model of binding established by the structural studies and suggests that binding energy is well distributed across functional groups in these molecules. The majority of the mutants retained significant enzymatic activity while diminishing inhibitor binding, indicating potential for the development of drug resistance. These data provide detailed information on interactions between inhibitor and binding pocket at the functional group level and enable the development of novel KSP inhibitors.

PLC-γ and PI3K link cytokines to ERK activation in hematopoietic cells with normal and oncogenic Kras.

Oncogenic K-Ras proteins, such as K-Ras(G12D), accumulate in the active, guanosine triphosphate (GTP)-bound conformation and stimulate signaling through effector kinases. The presence of the K-Ras(G12D) oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf-mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. We showed that phospholipase C-γ (PLC-γ), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-Ras(G12D) remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.

Discovery of dual inhibitors of the immune cell PI3Ks p110delta and p110gamma: a prototype for new anti-inflammatory drugs.

PI3Kdelta and PI3Kgamma regulate immune cell signaling, while the related PI3Kalpha and PI3Kbeta regulate cell survival and metabolism. Selective inhibitors of PI3Kdelta/gamma represent a potential class of anti-inflammatory agents lacking the antiproliferative effects associated with PI3Kalpha/beta inhibition. Here we report the discovery of PI3Kdelta/gamma inhibitors that display up to 1000-fold selectivity over PI3Kalpha/beta and evaluate these compounds in a high-content inflammation assay using mixtures of primary human cells. We find selective inhibition of only PI3Kdelta is weakly anti-inflammatory, but PI3Kdelta/gamma inhibitors show superior inflammatory marker suppression through suppression of lipopolysaccharide-induced TNFalpha production and T cell activation. Moreover, PI3Kdelta/gamma inhibition yields an anti-inflammatory signature distinct from pan-PI3K inhibition and known anti-inflammatory drugs, yet bears striking similarities to glucocorticoid receptor agonists. These results highlight the potential of selectively designing drugs that target kinases with shared biological function.

Activity of the p110-alpha subunit of phosphatidylinositol-3-kinase is required for activation of epithelial sodium transport.

The pathways implicated in the control of epithelial Na(+) channel (ENaC)-dependent Na(+) transport in renal collecting duct cells share substantial parallels with those implicated in insulin-regulated glucose metabolism. Notably, both are inhibited by wortmannin and LY294002 and signal through phosphatidylinositol-3-kinase (PI3K)-dependent kinases SGK1 and Akt. The inhibitor pattern is thought to reflect dependence on PI3K activity since wortmannin and LY294002 are both effective inhibitors of this kinase. However, these inhibitors block a variety of kinases from different families and lack specificity within the PI3K family. To begin to dissect more precisely the pathways required for signaling and for control of Na(+) transport in renal collecting duct cells, we have examined the effect of a set of PI3K inhibitors, which selectively block distinct subsets of PI3K catalytic subunit isoforms. We have found that ENaC-dependent Na(+) transport was blocked by inhibitors of the p110-alpha isoform of PI3K, but not by inhibitors of p110-beta, -gamma, or -delta. Inhibitors that block Na(+) current also blocked SGK1 and Akt phosphorylation. In contrast to insulin-stimulated glucose uptake in muscle cells, p110-beta inhibition did not enhance sensitivity to p110-alpha inhibition. These data support the conclusion that ENaC-dependent Na(+) current is controlled exclusively by p110-alpha, the same isoform that is the principal mediator of insulin effects on glucose metabolism, and lacks any dependence on p110-beta. These findings further underscore the extent to which Na(+) and glucose regulation are intertwined and provide additional insight into the interconnections between diabetes and hypertension.

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

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.