Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compound's safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.

BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of the compounds cellular growth arrest phenotypes and microtubule dynamics suggest that the antiproliferative activity of BKM120 is mainly due to microtubule-dependent cytotoxicity rather than through inhibition of PI3K. Crystal structures of BKM120 and derivatives in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results raise concerns over BKM120's generally accepted mode of action, and provide a unique mechanistic basis for next-generation PI3K inhibitors with improved safety profiles and flexibility for use in combination therapies.

Class IB phosphoinositide 3-kinase ␥ (PI3K␥) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3K␥ consists of a catalytic p110␥ and a regulatory p87 PIKAP (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein ␤␥ (G␤␥)-mediated PI3K␥ regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110␥ activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3K␥-complemented bone marrow-derived mast cells (BMMCs) from p110␥ -/-mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein-coupled receptor (GPCR)-induced PI3K␥ activation. Although p101 strongly interacted with G␤␥, thereby mediating PI3K␥ membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing G␤␥-dependent membrane recruitment of p87/p110␥ by direct interaction with p110␥, suggesting the indispensability of Ras for activation of p87/p110␥. Consequently, interference with Ras signaling indeed selectively blocked p87/p110␥, but not p101/ p110␥, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110␥ activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3K␥ that could open up new possibilities for therapeutic intervention.confocal life cell imaging ͉ G protein ͉ receptor signaling ͉ mast cells

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Phosphoinositide 3-kinase gamma (PI3Kgamma) plays a major role in chronic inflammation and allergy. It is a heterodimer of a catalytic p110gamma subunit and an adaptor protein, either p101 or the p101 homolog p84 (p87(PIKAP)). It is unclear whether both PI3Kgamma complexes specifically modulate responses such as chemotaxis and degranulation. In mast cells, the p84:p110gamma complex synergizes with immunoglobulin E (IgE)- and antigen-clustered FcepsilonRI receptor signaling and is required to achieve maximal degranulation. During this process, PI3Kgamma is activated by ligands of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs), in particular adenosine receptors, through autocrine and paracrine pathways. Here, we show that p110gamma needs p84 to relay signals from GPCRs to formation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], phosphorylation of Akt, migration of cells, and synergistic adenosine-enforced degranulation. Furthermore, the absence of adaptor subunits could not be compensated for by increased p110gamma abundance. Differentiated, p110gamma null cells also lost adaptor proteins. Complementation of p110gamma null mast cells with p101 and p110gamma restored the activation of Akt and cell migration, but failed to support degranulation. Lack of degranulation was attributed to a change in the spatiotemporal localization of PI3Kgamma-derived PtdIns(3,4,5)P(3); although both p84:p110gamma and p101:p110gamma complexes initially deposited PtdIns(3,4,5)P(3) at the plasma membrane, p101:p110gamma-derived PtdIns(3,4,5)P(3) was rapidly endocytosed to motile, microtubule-associated vesicles. In addition, p84:p110gamma, but not p101:p110gamma signaling was sensitive to disruption of lipid rafts. Our results demonstrate a nonredundant function for the p101 and p84 PI3Kgamma adaptor proteins and show that distinct pools of PtdIns(3,4,5)P(3) at the plasma membrane can elicit specific cell responses.

Phosphoinositide 3-kinase (PI3K)/protein kinase B/Akt and Ras/mitogen-activated protein kinase pathways are often constitutively activated in melanoma and have thus been considered as promising drug targets. Exposure of melanoma cells to NVP-BAG956, NVP-BBD130, and NVP-BEZ235, a series of novel, potent, and stable dual PI3K/ mammalian target of rapamycin (mTOR) inhibitors, resulted in complete G1 growth arrest, reduction of cyclin D1, and increased levels of p27 KIP1 , but negligible apoptosis. In contrast, treatment of melanoma with the pan-class I PI3K inhibitor ZSTK474 or the mTORC1 inhibitor rapamycin resulted only in minor reduction of cell proliferation. In a syngeneic B16 mouse melanoma tumor model, orally administered NVP-BBD130 and NVP-BEZ235 efficiently attenuated tumor growth at primary and lymph node metastatic sites with no obvious toxicity. Metastatic melanoma in inhibitor-treated mice displayed reduced numbers of proliferating and significantly smaller tumor cells. In addition, neovascularization was blocked and tumoral necrosis increased when compared with vehicle-treated mice. In conclusion, compounds targeting PI3K and mTOR simultaneously were advantageous to attenuate melanoma growth and they develop their potential by targeting tumor growth directly, and indirectly via their interference with angiogenesis. Based on the above results, NVP-BEZ235, which has entered phase I/II clinical trials in patients with advanced solid tumors, has a potential in metastatic melanoma therapy. (Mol Cancer Res 2009;7(4):601-13)

Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. PQR620 (3) is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. PQR620 (3) showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague−Dawley mice, maximum concentration (C max ) in plasma and brain was reached after 30 min, with a half-life (t 1/2 ) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of PQR620 (3) inhibited tumor growth significantly. Moreover, PQR620 (3) attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, PQR620 (3) inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.

During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR−/−) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR−/− macrophages with increasing concentrations of 125I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on 125I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect 125I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR−/− mice treated with LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.