AS-252424

AS-252424 is a potent, selective and novel small-molecule PI3Kγ inhibitor (IC50= 30 nM)

PI3K (phosphatidylinositol-4,5-bisphosphate 3-kinase) is a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. It plays a key role in PI3K/Akt/mTOR pathway.

In THP-1 (human monocytic cell line), AS-252424 inhibited the phosphorylation of PKB/Akt due to MCP1 binding to the GPCR receptor CCR2 at lowest IC50 values of 0.4 μM. In primary monocytes and monocytic wild-type cell line THP-1, AS-252424 inhibited MCP-1-mediated chemotaxis in a concentration-dependent manner (IC50 value= 52 μM and 53 μM, respectively). [1] In Capan 1 and HPAF (pancreatic cancer cell lines), AS52424 specifically inhibited the cell proliferation. [2]

In a mouse model of thioglycollate-induced peritonitis, oral administration of AS-252424 at 10 mg/kg led to moderate reduction of neutrophil recruitment (35% ±14%). [1]

References:
1. Pomel V, Klicic J, Covini D et al. Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase gamma. J Med Chem. 2006 Jun 29;49(13):3857-71.
2.  Edling CE, Selvaggi F, Buus R et al. Key role of phosphoinositide 3-kinase class IB in pancreatic cancer. Clin Cancer Res. 2010 Oct 15;16(20):4928-37.

RXFP1 Receptor Activation by Relaxin-2 Induces Vascular Relaxation in Mice via a Galphai2-Protein/PI3Kss/gamma/Nitric Oxide-Coupled Pathway.[Pubmed:30233409]

Front Physiol. 2018 Sep 4;9:1234.

Background: Relaxins are small peptide hormones, which are novel candidate molecules that play important roles in cardiometablic syndrome. Relaxins are structurally related to the insulin hormone superfamily, which provide vasodilatory effects by activation of G-protein-coupled relaxin receptors (RXFPs) and stimulation of endogenous nitric oxide (NO) generation. Recently, relaxin could be demonstrated to activate Gi proteins and phosphoinositide 3-kinase (PI3K) pathways in cultured endothelial cells in vitro. However, the contribution of the Gi-PI3K pathway and their individual components in relaxin-dependent relaxation of intact arteries remains elusive. Methods: We used Galphai2- (Gnai2(-/-)) and Galphai3-deficient (Gnai3(-/-)) mice, pharmacological tools and wire myography to study G-protein-coupled signaling pathways involved in relaxation of mouse isolated mesenteric arteries by relaxins. Human relaxin-1, relaxin-2, and relaxin-3 were tested. Results: Relaxin-2 ( approximately 50% relaxation at 10(-11) M) was the most potent vasodilatory relaxin in mouse mesenteric arteries, compared to relaxin-1 and relaxin-3. The vasodilatory effects of relaxin-2 were inhibited by removal of the endothelium or treatment of the vessels with N (G)-nitro-L-arginine methyl ester (L-NAME, endothelial nitric oxide synthase (eNOS) inhibitor) or simazine (RXFP1 inhibitor). The vasodilatory effects of relaxin-2 were absent in arteries of mice treated with pertussis toxin (PTX). They were also absent in arteries isolated from Gnai2(-/-) mice, but not from Gnai3(-/-) mice. The effects were not affected by FR900359 (Galphaq protein inhibitor) or PI-103 (PI3Kalpha inhibitor), but inhibited by TGX-221 (PI3Kbeta inhibitor) or AS-252424 (PI3Kgamma inhibitor). Simazine did not influence the anti-contractile effect of perivascular adipose tissue. Conclusion: Our data indicate that relaxin-2 produces endothelium- and NO-dependent relaxation of mouse mesenteric arteries by activation of RXFP1 coupled to Gi2-PI3K-eNOS pathway. Targeting vasodilatory Gi-protein-coupled RXFP1 pathways may provide promising opportunities for drug discovery in endothelial dysfunction and cardiometabolic disease.

The Selective Phosphoinoside-3-Kinase p110delta Inhibitor IPI-3063 Potently Suppresses B Cell Survival, Proliferation, and Differentiation.[Pubmed:28713374]

Front Immunol. 2017 Jun 30;8:747.

The class I phosphoinoside-3-kinases (PI3Ks) are important enzymes that relay signals from cell surface receptors to downstream mediators driving cellular functions. Elevated PI3K signaling is found in B cell malignancies and lymphocytes of patients with autoimmune disease. The p110delta catalytic isoform of PI3K is a rational target since it is critical for B lymphocyte development, survival, activation, and differentiation. In addition, activating mutations in PIK3CD encoding p110delta cause a human immunodeficiency known as activated PI3K delta syndrome. Currently, idelalisib is the only selective p110delta inhibitor that has been FDA approved to treat certain B cell malignancies. p110delta inhibitors can suppress autoantibody production in mouse models, but limited clinical trials in human autoimmunity have been performed with PI3K inhibitors to date. Thus, there is a need for additional tools to understand the effect of pharmacological inhibition of PI3K isoforms in lymphocytes. In this study, we tested the effects of a potent and selective p110delta inhibitor, IPI-3063, in assays of B cell function. We found that IPI-3063 potently reduced mouse B cell proliferation, survival, and plasmablast differentiation while increasing antibody class switching to IgG1, almost to the same degree as a pan-PI3K inhibitor. Similarly, IPI-3063 potently inhibited human B cell proliferation in vitro. The p110gamma isoform has partially overlapping roles with p110delta in B cell development, but little is known about its role in B cell function. We found that the p110gamma inhibitor AS-252424 had no significant impact on B cell responses. A novel dual p110delta/gamma inhibitor, IPI-443, had comparable effects to p110delta inhibition alone. These findings show that p110delta is the dominant isoform mediating B cell responses and establish that IPI-3063 is a highly potent molecule useful for studying p110delta function in immune cells.

Phosphoinositide 3-Kinase Is Involved in Mediating the Anti-inflammation Effects of Vasopressin.[Pubmed:27943011]

Inflammation. 2017 Apr;40(2):435-441.

Vasopressin possesses potent anti-inflammatory capacity. Phosphoinositide 3-kinase (PI3K) and its downstream activator Akt contribute to endogenous anti-inflammation capacity. We sought to elucidate whether PI3K is involved in mediating the anti-inflammation effects of vasopressin. Macrophages (RAW264.7 cells) were randomized to receive endotoxin, endotoxin plus vasopressin, or endotoxin plus vasopressin plus the nonselective PI3K inhibitor (LY294002) or the selective isoform inhibitor of PI3Kalpha (PIK-75), PI3Kbeta (TGX-221), PI3Kdelta (IC-87114), or PI3Kgamma (AS-252424). Compared to macrophages treated with endotoxin, the concentrations of cytokines (tumor necrosis factor-alpha, interleukin-6) and chemokine (macrophage inflammatory protein-2) in macrophages treated with endotoxin plus vasopressin were significantly lower (all P < 0.05). The concentrations of phosphorylated nuclear factor-kappaB p65 (p-NF-kappaB p65) in nuclear extracts and phosphorylated inhibitor-kappaBalpha (p-I-kappaBalpha) in cytosolic extracts as well as NF-kappaB-DNA binding activity were also lower (all P < 0.05). Of note, except for macrophages treated with endotoxin plus vasopressin plus PIK-75, the concentrations of cytokines, chemokine, p-NF-kappaB p65, and p-I-kappaBalpha as well as NF-kappaB-DNA binding activity in macrophages treated with endotoxin plus vasopressin plus LY294002, TGX-221, IC-87114, or AS-252424 were significantly higher than those in macrophages treated with endotoxin plus vasopressin (all P < 0.05). In contrast, the phosphorylated Akt concentration in macrophages treated with endotoxin plus vasopressin was significantly higher than that in macrophages treated with endotoxin or in macrophages treated with endotoxin plus vasopressin plus LY294002, TGX-221, IC-87114, or AS-252424, but not PIK-75. These data confirmed that PI3K, especially the isoforms of PI3Kbeta, PI3Kdelta, and PI3Kgamma, is involved in mediating the anti-inflammatory effects of vasopressin.

Potency and pharmacokinetics of broad spectrum and isoform-specific p110gamma and delta inhibitors in cancers.[Pubmed:26791581]

J Recept Signal Transduct Res. 2016;36(1):26-36.

Emerging data on cancer suggesting that target-based therapy is promising strategy in cancer treatment. PI3K-AKT pathway is extensively studied in many cancers; several inhibitors target this pathway in different levels. Recent finding on this pathway uncovered the therapeutic applications of PI3K-specific inhibitors; PI3K, AKT, and mTORC broad spectrum inhibitors. Noticeably, class I PI3K isoforms, p110gamma and p110delta catalytic subunits have rational therapeutic application than other isoforms. Therefore, three classes of inhibitors: isoform-specific, dual-specific and broad spectrum were selected for molecular docking and dynamics. First, p110delta structure was modelled; active site was analyzed. Then, molecular docking of each class of inhibitors were studied; the docked complexes were further used in 1.2 ns molecular dynamics simulation to report the potency of each class of inhibitor. Remarkably, both the studies retained the similar kind of protein ligand interactions. GDC-0941, XL-147 (broad spectrum); TG100-115 (dual-specific); and AS-252424, PIK-294 (isoform-specific) were found to be potential inhibitors of p110gamma and p110delta, respectively. In addition to that pharmacokinetic properties are within recommended ranges. Finally, molecular phylogeny revealed that p110gamma and p110delta are evolutionarily divergent; they probably need separate strategies for drug development.

Inhibitors of pan-PI3K Signaling Synergize with BRAF or MEK Inhibitors to Prevent BRAF-Mutant Melanoma Cell Growth.[Pubmed:26137449]

Front Oncol. 2015 Jun 16;5:135.

BRAF and MEK inhibitors have improved outcomes for patients with BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistances. Activation of the PI3K pathway can mediate resistance to these agents, providing a strong rationale for combination therapy in melanoma. Here, a panel of nine low-passage human metastatic melanoma cell lines with BRAF mutations was tested in cell proliferation and protein expression assays for sensitivity to inhibitors of MEK (selumetinib) and BRAF (vemurafenib) as single agents and in combination with inhibitors of pan-PI3K (ZSTK474), pan-PI3K/mTOR (BEZ235), individual PI3K isoforms (p110alpha, A66; p110beta, TGX-221; p110gamma, AS-252424; p110delta, idelalisib), or mTORC1/2 (KU-0063794). Selumetinib and vemurafenib potently inhibited cell proliferation in all cell lines, especially in those that expressed low levels of phosphorylated AKT (pAKT). ZSTK474 and BEZ235 also inhibited cell proliferation in all cell lines and enhanced the antitumor activity of selumetinib and vemurafenib in the majority of lines by either interacting synergistically or additively to increase potency or by inducing cytotoxicity by significantly increasing the magnitude of cell growth inhibition. Furthermore, ZSTK474 or BEZ235 combined with selumetinib to produce robust inhibition of pERK, pAKT, and pS6 expression and synergistic inhibition of NZM20 tumor growth. The inhibitors of individual PI3K isoforms or mTORC1/2 were less effective at inhibiting cell proliferation either as single agents or in combination with selumetinib or vemurafenib, although KU-0063794 synergistically interacted with vemurafenib and increased the magnitude of cell growth inhibition with selumetinib or vemurafenib in certain cell lines. Overall, these results suggest that the sensitivity of BRAF-mutant melanoma cells to BRAF or MEK inhibitors is at least partly mediated by activation of the PI3K pathway and can be enhanced by combined inhibition of the BRAF/MEK and PI3K/mTOR signaling pathways.

Phosphoinositide 3-kinase beta, phosphoinositide 3-kinase delta, and phosphoinositide 3-kinase gamma mediate the anti-inflammatory effects of magnesium sulfate.[Pubmed:25972311]

J Surg Res. 2015 Aug;197(2):390-7.

BACKGROUND: We previously demonstrated that inhibiting phosphoinositide 3-kinase (PI3K) or activating L-type calcium channels blocked the anti-inflammatory effects of magnesium sulfate (MgSO(4)). However, the question as which class I PI3K isoform (PI3Kalpha, PI3Kbeta, PI3Kdelta, or PI3Kgamma) is involved in this regard remains unstudied. The question as whether MgSO(4) and L-type calcium channels interact to influence PI3K activation also remains unstudied. We therefore designed this study to test two hypotheses: (1) inhibiting PI3Kalpha, PI3Kbeta, PI3Kdelta, or PI3Kgamma would block the anti-inflammatory effects of MgSO(4) and (2) activating L-type calcium channels would block the effects of MgSO(4) on activating PI3K. MATERIALS AND METHODS: PI3K isoform investigation: macrophages (RAW264.7 cells) were treated with endotoxin, endotoxin plus MgSO(4), or endotoxin plus MgSO(4) plus the selective inhibitor of PI3Kalpha (PIK-75), PI3Kbeta (TGX-221), PI3Kdelta (IC-87114), or PI3Kgamma (AS-252424). Calcium channel investigation: macrophages were treated with endotoxin, endotoxin plus MgSO(4), or endotoxin plus MgSO(4) plus the L-type calcium channel activator BAY-K8644. RESULTS: The endotoxin plus MgSO(4) group presented lower concentrations of inflammatory mediators (macrophage inflammatory protein 2, tumor necrosis factor alpha, and interleukin 6, lower nuclear concentration of phosphorylated nuclear factor kappaB, lower cytosolic concentration of phosphorylated inhibitor kappaBalpha, and higher concentration of phosphorylated Akt (PI3K activation marker) than the endotoxin group (all P < 0.05). These effects of MgSO(4) were significantly reduced by TGX-221, IC-87114, or AS-252424, but not PIK-75. Additionally, BAY-K8644 blocked the effect of MgSO(4) on activating PI3K. CONCLUSIONS: MgSO(4) exerts its anti-inflammatory effects through activating PI3Kbeta, PI3Kdelta, and PI3Kgamma. The underlying mechanism appears to involve inhibition of L-type calcium channels.

Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca(2+)](i), I(Ca), and Ca(2+) transients in HL-1 cardiomyocytes.[Pubmed:22715995]

J Biomed Sci. 2012 Jun 20;19:59.

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca(2+)](i), Ca(2+) transients and membrane Ca(2+) current, I(Ca), in cultured murine HL-1 cardiomyocytes. LY294002 (1-20 muM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca(2+)](i), Ca(2+) transients and I(Ca). We also examined the effect of PI3K isoform specific inhibitors, i.e. alpha (PI3-kinase alpha inhibitor 2; 2-8 nM); beta (TGX-221; 100 nM) and gamma (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca(2+)](i) regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca(2+)](i), and inhibited Ca(2+) transients. Triciribine (1-20 muM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca(2+)](i), and Ca(2+) transients and I(Ca). We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca(2+)](i) in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca(2+)](i) required for excitation-contraction coupling in cardiomyoctyes.

Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase gamma.[Pubmed:16789742]

J Med Chem. 2006 Jun 29;49(13):3857-71.

Class I phosphoinositide 3-kinases (PI3Ks), in particular PI3Kgamma, have become attractive drug targets for inflammatory and autoimmune diseases. Here, we disclose a novel series of furan-2-ylmethylene thiazolidinediones as selective, ATP-competitive PI3Kgamma inhibitors. Structure-based design and X-ray crystallography of complexes formed by inhibitors bound to PI3Kgamma identified key pharmacophore features for potency and selectivity. An acidic NH group on the thiazolidinedione moiety and a hydroxy group on the furan-2-yl-phenyl part of the molecule play crucial roles in binding to PI3K and contribute to class IB PI3K selectivity. Compound 26 (AS-252424), a potent and selective small-molecule PI3Kgamma inhibitor emerging from these efforts, was further profiled in three different cellular PI3K assays and shown to be selective for class IB PI3K-mediated cellular effects. Oral administration of 26 in a mouse model of acute peritonitis led to a significant reduction of leukocyte recruitment.

Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils.[Pubmed:15878979]

Blood. 2005 Aug 15;106(4):1432-40.

It is well established that preexposure of human neutrophils to proinflammatory cytokines markedly augments the production of reactive oxygen species (ROS) to subsequent stimuli. This priming event is thought to be critical for localizing ROS to the vicinity of the inflammation, maximizing their role in the resolution of the inflammation, and minimizing the damage to surrounding tissue. We have used a new generation of isoform-selective phosphoinositide 3-kinase (PI3K) inhibitors to show that ROS production under these circumstances is regulated by temporal control of class I PI3K activity. Stimulation of tumor necrosis factor-alpha (TNF-alpha)-primed human neutrophils with N-formyl-methionyl-leucyl-phenylalanine (fMLP) results in biphasic activation of PI3K; the first phase is largely dependent on PI3Kgamma, and the second phase is largely dependent on PI3Kdelta. The second phase of PI3K activation requires the first phase; it is this second phase that is augmented by TNF-alpha priming and that regulates parallel activation of ROS production. Surprisingly, although TNF-alpha-primed mouse bone marrow-derived neutrophils exhibit superficially similar patterns of PI3K activation and ROS production in response to fMLP, these responses are substantially lower and largely dependent on PI3Kgamma alone. These results start to define which PI3K isoforms are responsible for modulating neutrophil responsiveness to infection and inflammation.