AS-605240

AS-605240 is a selective PI3K γ inhibitor with an IC50 of 8 nM. [1]

PI3Ks are a family of enzymes, which phosphorylate the 3’- OH position of the inositol ring of phosphoinositides.They have been divided into three classes on the basis of structural features and in vitro lipid substrate specificity. The three class-Ia PI3-kinases (p110 α / β / δ ) and the sole class-Ib PI 3-kinase (p110 γ ) couple growth factor receptors and G-protein-coupled receptors, respectively, to a wide range of downstream pathways. Signal transduction via the PI3K/Akt pathway is essential for regulating cellular responses, including proliferation, survival, migration, motility and tumorigenesis, in a variety of cell types. The key selectivity features were identified by co-crystallization with PI3Kγ, which revealed that thiazolidinedione nitrogen makes a salt – bridge interaction with the side chain of Lys-833, and also forms the link to Val-882.[1,2]

AS-605240 is a cell permeable inhibitor for PI3Ks and inhibits p110 α/β/γ/δ in vitro?with IC50 values of 0.06, 0.27, 0.008 0.3 μΜ, respectively. AS-605240 was tested its ability to inhibit C5a-mediated PKB phosphorylation in RAW264 mouse macrophages and AS-605240 showed an IC50 of 0.09 mM. [1]

As in many in?ammatory processes, neutrophils are predominant in the initial in?ux of leukocytes, followed by monocytes-macrophages and lymphocytes. in vivo efficacy in blocking leukocyte migration assays were conducted, AS-605240 was tested in two different mouse models of peritonitis, including RANTES-induced or thioglycollate, the outcome showed ED50 of 9.1 mg/kg and 10 mg/kg respectively. AS-605240 also show that it is an orally active, selective PI3Kγ inhibitor suppresses joint inflammation and damage in a mouse model of collagen-induced arthritis.[1]

References:
[1].  Camps M, Rückle T, Ji H, et al. Blockade of PI3Kγ suppresses joint inflammation and damage in mouse models of rheumatoid arthritis[J]. Nature medicine, 2005, 11(9): 936-943.
[2].  Marone R, Cmiljanovic V, Giese B, et al. Targeting phosphoinositide 3-kinase—moving towards therapy[J]. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2008, 1784(1): 159-185.

Muscarinic m2 receptor-mediated actin polymerization via PI3 kinase gamma and integrin-linked kinase in gastric smooth muscle.[Pubmed:30393912]

Neurogastroenterol Motil. 2019 Feb;31(2):e13495.

BACKGROUND: Actin polymerization plays an important role in smooth muscle contraction. Integrin-linked kinase (ILK) was shown to mediate actin polymerization in airway smooth muscle. The role of ILK in actin polymerization in response to m2 receptor activation was not in gastric smooth muscle. METHODS: Phosphorylation of paxillin, neuronal Wiskott-Aldrich syndrome protein (N-WASp), and association of paxillin with GEF proteins (Cool2/alphaPix [Cool2/PAK-interacting exchange factor alpha], Cool1/betaPix [Cool1/PAK-interacting exchange factor beta], and DOCK 180 [Dedicator of cytokinesis]) and N-WASp with Arp2/3 complex were measured by western blot. Activation of Cdc42 was determined using an antibody for activated Cdc42. Actin polymerization was measured as an increase in F-actin/G-actin ratio. RESULTS: Phosphorylation of paxillin, an association of paxillin with GEF proteins, Cdc42 activity, and actin polymerization were increased in response to m2 receptor activation in gastric smooth muscle cells. The increases in paxillin phosphorylation, Cdc42 activity, and actin polymerization were inhibited by a PI3Kgamma inhibitor (AS-605240), ILK siRNA, and ILK dominant negative mutant (ILK [R211]). Increase in actin polymerization was also inhibited by Cdc42 dominant negative mutant (Cdc42 [T17N]). Increases in the association of paxillin with GEF proteins, phosphorylation of N-WASp and its association with Arp2/3 complex were inhibited by ILK (R211). CONCLUSION: In gastric smooth muscle cells, activation of PI3Kgamma by muscarinic m2 receptors causes ILK-dependent phosphorylation of paxillin, an association of paxillin with Cdc42 GEF proteins and activation of Cdc42, which, in turn, causes phosphorylation of N-WASp and its association with Arp2/3 complex leading to actin polymerization.

Comprehensive analysis of T cell leukemia signals reveals heterogeneity in the PI3 kinase-Akt pathway and limitations of PI3 kinase inhibitors as monotherapy.[Pubmed:29799846]

PLoS One. 2018 May 25;13(5):e0193849.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K gamma-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kgamma-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.

Notch signaling via regulation of RB and p-AKT but not PIK3CG contributes to MIA PaCa-2 cell growth and migration to affect pancreatic carcinogenesis.[Pubmed:29434912]

Oncol Lett. 2018 Feb;15(2):2105-2110.

Pancreatic cancer is one of the leading causes of cancer-associated mortality. The understanding of the expression pattern of key protein factors and their function in pancreatic cancer cells is therefore vital for the diagnosis and treatment of this malignancy. The results of the present study reveal that the levels of neurogenic locus notch homolog protein 2 (Notch2) and phosphorylated (p)-SMAD family member 2 decreased, whereas the expression of Notch3 and phosphoinositide-3 kinase catalytic subunit-gamma protein increased in human pancreatic cancer tissues compared with tumor-adjacent tissues. Using the human pancreatic cancer MIA PaCa-2 cell line, it was observed that retinoblastoma-associated protein (RB) and p-RB expression were inhibited and p-AKT was upregulated when Notch signaling was activated in MIA PaCa-2 cells. Furthermore, inhibition of phosphoinositide-3 kinase catalytic subunit-gamma (PIK3CG) activity by AS-605240 was able to block the growth and migration of MIA PaCa-2 cells. In conclusion, the results of the present study demonstrate that the Notch signal pathway may be involved in pancreatic carcinogenesis by modulating RB and p-AKT. PIK3CG may therefore be a potential target gene for the treatment of pancreatic cancer.

alpha-Tocopheryl Phosphate Induces VEGF Expression via CD36/PI3Kgamma in THP-1 Monocytes.[Pubmed:28059487]

J Cell Biochem. 2017 Jul;118(7):1855-1867.

The CD36 scavenger receptor binds several ligands and mediates ligand uptake and ligand-dependent signal transduction and gene expression, events that may involve CD36 internalization. Here we show that CD36 internalization in THP-1 monocytes is triggered by alpha-tocopherol (alphaT) and more strongly by alpha-tocopheryl phosphate (alphaTP) and EPC-K1, a phosphate diester of alphaTP and L-ascorbic acid. alphaTP-triggered CD36 internalization is prevented by the specific covalent inhibitor of selective lipid transport by CD36, sulfo-N-succinimidyl oleate (SSO). Moreover, SSO inhibited the CD36-mediated uptake of 14C-labelled alphaTP suggesting that alphaTP binding and internalization of CD36 is involved in cellular alphaTP uptake, whereas the uptake of alphaT was less affected. Similar to that, inhibition of selective lipid transport of the SR-BI scavenger receptor resulted mainly in reduction of alphaTP and not alphaT uptake. In contrast, uptake of alphaT was mainly inhibited by Dynasore, an inhibitor of clathrin-mediated endocytosis, suggesting that the differential regulatory effects of alphaTP and alphaT on signaling may be influenced by their different routes of uptake. Interestingly, alphaTP and EPC-K1 also reduced the neutral lipid content of THP-1 cells and the phagocytosis of fluorescent Staphylococcus aureus bioparticles. Moreover, induction of the vascular endothelial growth factor (VEGF) promoter activity by alphaTP occurred via CD36/PI3Kgamma/Akt, as it could be inhibited by specific inhibitors of this pathway (SSO, Wortmannin, AS-605240). These results suggest that alphaTP activates PI3Kgamma/Akt signaling leading to VEGF expression in monocytes after binding to and/or transport by CD36, a receptor known to modulate angiogenesis in response to amyloid beta, oxLDL, and thrombospondin. J. Cell. Biochem. 118: 1855-1867, 2017. (c) 2017 Wiley Periodicals, Inc.

Genetic Deletion and Pharmacological Inhibition of PI3K gamma Reduces Neutrophilic Airway Inflammation and Lung Damage in Mice with Cystic Fibrosis-Like Lung Disease.[Pubmed:26185363]

Mediators Inflamm. 2015;2015:545417.

PURPOSE: Neutrophil-dominated airway inflammation is a key feature of progressive lung damage in cystic fibrosis (CF). Thus, reducing airway inflammation is a major goal to prevent lung damage in CF. However, current anti-inflammatory drugs have shown several limits. PI3Kgamma plays a pivotal role in leukocyte recruitment and activation; in the present study we determined the effects of genetic deletion and pharmacologic inhibition of PI3Kgamma on airway inflammation and structural lung damage in a mouse model of CF lung disease. METHODS: betaENaC overexpressing mice (betaENaC-Tg) were backcrossed with PI3Kgamma-deficient (PI3Kgamma (KO)) mice. Tissue damage was assessed by histology and morphometry and inflammatory cell number was evaluated in bronchoalveolar lavage fluid (BALF). Furthermore, we assessed the effect of a specific PI3Kgamma inhibitor (AS-605240) on inflammatory cell number in BALF. RESULTS: Genetic deletion of PI3Kgamma decreased neutrophil numbers in BALF of PI3Kgamma (KO)/betaENaC-Tg mice, and this was associated with reduced emphysematous changes. Treatment with the PI3Kgamma inhibitor AS-605240 decreased the number of neutrophils in BALF of betaENaC-Tg mice, reproducing the effect observed with genetic deletion of the enzyme. CONCLUSIONS: These results demonstrate the biological efficacy of both genetic deletion and pharmacological inhibition of PI3Kgamma in reducing chronic neutrophilic inflammation in CF-like lung disease in vivo.

Inhibition of phosphoinositide 3-kinase ameliorates dextran sodium sulfate-induced colitis in mice.[Pubmed:19828878]

J Pharmacol Exp Ther. 2010 Jan;332(1):46-56.

The critical role of phosphoinositide 3-kinase gamma (PI3Kgamma) in inflammatory cell activation and recruitment makes it an attractive target for immunomodulatory therapy. 5-Quinoxilin-6-methylene-1,3-thiazolidine-2,4-dione (AS605240), a potent PI3Kgamma inhibitor, has been reported to ameliorate chronic inflammatory disorders including rheumatoid arthritis, systemic lupus erythematosus, and atherosclerosis. However, its in vivo effect on intestinal inflammation remains unknown. Here we evaluated the protective and therapeutic potentials of AS605240 in mice with dextran sodium sulfate (DSS)-induced acute and chronic colitis. Our results showed that AS605240 improved survival rate, disease activity index, and histological damage score in mice administered DSS in both preventive and therapeutic studies. AS605240 treatment also significantly inhibited the increase in myeloperoxidase levels, macrophage infiltration, and CD4(+) T-cell number in the colon of DSS-fed mice. The DSS-induced overproduction of colonic proinflammatory cytokines including interleukin (IL)-1beta, tumor necrosis factor-alpha, and interferon-gamma was significantly suppressed in mice undergoing AS605240 therapy, whereas colonic anti-inflammatory cytokines such as IL-4 were up-regulated. The down-regulation of the phospho-Akt level in immunological cells from the inflamed colon tissue and spleen of AS605240-treated mice was detected both by immunohistochemical analysis and Western blotting. These findings demonstrate that AS605240 may represent a promising novel agent for the treatment of inflammatory bowel disease by suppressing leukocyte infiltration as well as by immunoregulating the imbalance between proinflammatory and anti-inflammatory cytokines.