as-605240 and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Our previous study showed that PI3Kγ inhibition with AS605240 plus a standard rat-dose tPA (10 mg/kg) combination attenuates delayed tPA-induced brain hemorrhage and ameliorates acute stroke injury 3 days after ischemic stroke in rats. The purpose of this study was to investigate whether combining AS605240 with tPA can enhance thrombolytic efficacy, so that lower doses of tPA can be applied to improve long-term outcome after ischemic stroke. The results showed that AS605240 plus low-dose tPA (5 mg/kg) combination therapy at 4 h after stroke onset significantly reduced infarct volume and neurological deficits at 24 h after stroke compared with saline, AS605240 or low-dose tPA alone group. Importantly, the combination therapy significantly reduced the delayed tPA-associated brain hemorrhage. Moreover, the combination therapy significantly decreased the size of the residual embolus within the middle cerebral artery, which was associated with a decrease in plasma plasminogen activator inhibitor-1 (PAI-1) activity compared with saline and tPA alone. Finally, AS605240 plus low-dose tPA combination improved long-term outcome for at least 35 days after stroke compared with the saline-treated group. Taken together, these findings suggest that PI3Kγ inhibition with AS605240 might act as an adjunct approach for enhancing tPA thrombolytic efficacy in acute ischemic stroke.

Topics: Animals; Disease Models, Animal; Drug Therapy, Combination; Embolic Stroke; Fibrinolytic Agents; Male; Phosphoinositide-3 Kinase Inhibitors; Quinoxalines; Rats; Rats, Sprague-Dawley; Thiazolidinediones; Thrombolytic Therapy; Tissue Plasminogen Activator; Treatment Outcome

A series of (1,2,4)triazole[4,3-a]pyridine (TZP) derivatives have been designed and synthesized. Compound 8d was identified as having the most potent inhibitory activity on NO release in response to lipopolysaccharide (LPS) stimulation and inhibition of the migration induced by MCP-1 protein on RAW264.7 macrophages. Based on the screening data, an immunofluorescence assay and a real-time qPCR assay were conducted, indicating that compound 8d suppressed NF-κB p65 translocation and expression of inflammatory genes by concanavalin A (Con A)-induced RAW264.7 macrophages. More importantly, 8d also exhibited potent efficacy, alleviating Con A-induced hepatitis by downregulating the levels of plasma alanine transaminase (ALT), aspartate transaminase (AST) and inflammatory infiltration in a mouse autoimmune hepatitis (AIH) model. In addition, the flow cytometry (FCM) data showed that compound 8d inhibited the accumulation of MDSCs in the liver of Con A-induced mice. These findings raise the possibility that compound 8d might serve as a potential agent for the treatment of AIH.

Topics: Animals; Cell Survival; Cells, Cultured; Concanavalin A; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Design; Female; Hepatitis; Humans; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Molecular Structure; Nitric Oxide; Pyridines; RAW 264.7 Cells; Structure-Activity Relationship; Triazoles

There is a major clinical need for new therapies for the treatment of chronic itch. Many of the molecular components involved in itch neurotransmission are known, including the neuropeptide NPPB, a transmitter required for normal itch responses to multiple pruritogens in mice. Here, we investigated the potential for a novel strategy for the treatment of itch that involves the inhibition of the NPPB receptor NPR1 (natriuretic peptide receptor 1). Because there are no available effective human NPR1 (hNPR1) antagonists, we performed a high-throughput cell-based screen and identified 15 small-molecule hNPR1 inhibitors. Using in vitro assays, we demonstrated that these compounds specifically inhibit hNPR1 and murine NPR1 (mNPR1). In vivo, NPR1 antagonism attenuated behavioral responses to both acute itch- and chronic itch-challenged mice. Together, our results suggest that inhibiting NPR1 might be an effective strategy for treating acute and chronic itch.

Topics: Animals; Behavior, Animal; Cell-Free System; Dermatitis, Contact; Disease Models, Animal; Ganglia, Spinal; Humans; Mice, Inbred C57BL; Mice, Knockout; Neurons; Pruritus; Receptors, Atrial Natriuretic Factor; Reproducibility of Results; Signal Transduction; Small Molecule Libraries

The intense and prolonged inflammatory response after ischemic stroke significantly contributes to the secondary neural injury. PI3Kγ, which is involved in the regulation of vascular permeability, chemotactic leukocyte migration and microglia activation, is a key target for intervention in the inflammatory response. In this study, we identified the protective effect of the PI3Kγ inhibitor AS605240 against stroke-related injury in the mouse model of transient intraluminal middle cerebral artery occlusion (tMCAO). The results showed that administration of AS605240 could improve the neurological function score, reduce the infarct size and decrease astrocyte activation in the tMCAO mice after injury. The inhibitory effect of AS605240 on microglia activation is relatively clear. Therefore, in this study, the effects of AS605240 on astrocytes were studied in cell cultures. IL-6 and its soluble receptor were used to construct the astrocyte activation model. AS605240 treatment significantly reduced the astrocyte activation markers and the morphological changes of cells. We also identified 13 inflammatory factors whose expression was significantly upregulated by IL-6/sIL-6R and significantly inhibited by AS605240 at the protein level, and seven of those factors were verified at the mRNA level. These results indicated that specific inhibition of PI3Kγ could reduce astrocyte activation induced by inflammation, which might aid the repair and remodeling of neurons in the later stage after ischemic stroke.

Topics: Animals; Astrocytes; Brain Ischemia; Class Ib Phosphatidylinositol 3-Kinase; Cytokines; Disease Models, Animal; Infarction, Middle Cerebral Artery; Inflammation; Male; Mice; Mice, Inbred C57BL; NF-kappaB-Inducing Kinase; Protective Agents; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Quinoxalines; Rats; Rats, Sprague-Dawley; Signal Transduction; STAT3 Transcription Factor; Stroke; Thiazolidinediones

Anthracyclines, such as doxorubicin (DOX), are potent anticancer agents for the treatment of solid tumors and hematologic malignancies. However, their clinical use is hampered by cardiotoxicity. This study sought to investigate the role of phosphoinositide 3-kinase γ (PI3Kγ) in DOX-induced cardiotoxicity and the potential cardioprotective and anticancer effects of PI3Kγ inhibition.. Mice expressing a kinase-inactive PI3Kγ or receiving PI3Kγ-selective inhibitors were subjected to chronic DOX treatment. Cardiac function was analyzed by echocardiography, and DOX-mediated signaling was assessed in whole hearts or isolated cardiomyocytes. The dual cardioprotective and antitumor action of PI3Kγ inhibition was assessed in mouse mammary tumor models.. PI3Kγ kinase-dead mice showed preserved cardiac function after chronic low-dose DOX treatment and were protected against DOX-induced cardiotoxicity. The beneficial effects of PI3Kγ inhibition were causally linked to enhanced autophagic disposal of DOX-damaged mitochondria. Consistently, either pharmacological or genetic blockade of autophagy in vivo abrogated the resistance of PI3Kγ kinase-dead mice to DOX cardiotoxicity. Mechanistically, PI3Kγ was triggered in DOX-treated hearts, downstream of Toll-like receptor 9, by the mitochondrial DNA released by injured organelles and contained in autolysosomes. This autolysosomal PI3Kγ/Akt/mTOR/Ulk1 signaling provided maladaptive feedback inhibition of autophagy. PI3Kγ blockade in models of mammary gland tumors prevented DOX-induced cardiac dysfunction and concomitantly synergized with the antitumor action of DOX by unleashing anticancer immunity.. Blockade of PI3Kγ may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

Topics: Animals; Antibiotics, Antineoplastic; Autophagy; Autophagy-Related Proteins; Breast Neoplasms; Cardiotoxicity; Class Ib Phosphatidylinositol 3-Kinase; Cytoprotection; Disease Models, Animal; Doxorubicin; Female; Genes, erbB-2; Heart Diseases; Mice, Inbred BALB C; Mice, Transgenic; Mutation; Myocytes, Cardiac; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinoxalines; Thiazolidinediones; Toll-Like Receptor 9; Tumor Burden

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi (T. cruzi) and is an important cause of severe inflammatory heart disease. However, the mechanisms driving Chagas disease cardiomyopathy have not been completely elucidated. Here, we show that the canonical PI3Kγ pathway is upregulated in both human chagasic hearts and hearts of acutely infected mice. PI3Kγ-deficient mice and mutant mice carrying catalytically inactive PI3Kγ are more susceptible to T. cruzi infection. The canonical PI3Kγ signaling in myeloid cells is essential to restrict T. cruzi heart parasitism and ultimately to avoid myocarditis, heart damage, and death of mice. Furthermore, high PIK3CG expression correlates with low parasitism in human Chagas' hearts. In conclusion, these results indicate an essential role of the canonical PI3Kγ signaling pathway in the control of T. cruzi infection, providing further insight into the molecular mechanisms involved in the pathophysiology of chagasic heart disease.

Topics: Adult; Animals; Biopsy; Cell Line; Chagas Cardiomyopathy; Class Ib Phosphatidylinositol 3-Kinase; Disease Models, Animal; Female; Heart; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Myeloid Cells; Myocardium; Phosphoinositide-3 Kinase Inhibitors; Quinoxalines; Signal Transduction; Thiazolidinediones; Trypanosoma cruzi; Up-Regulation

Chronic kidney disease (CKD) poses a formidable challenge for public healthcare worldwide as vast majority of patients with CKD are also at risk of accelerated cardiovascular disease and death. Renal fibrosis is the common manifestation of CKD that usually leads to end-stage renal disease although the molecular events leading to chronic renal fibrosis and eventually chronic renal failure remain to be fully understood. Nonetheless, emerging evidence suggests that an aberrant activation of PI3Kγ signaling may play an important role in regulating profibrotic phenotypes. Here, we investigate whether a blockade of PI3Kγ signaling exerts any beneficial effect on alleviating kidney injury and renal fibrosis. Using a mouse model of angiotensin II (Ang II)-induced renal damage, we demonstrate that PI3Kγ inhibitor AS605240 effectively mitigates Ang II-induced increases in serum creatinine and blood urea nitrogen, renal interstitial collagen deposition, the accumulation of ECM proteins and the expression of α-Sma and fibrosis-related genes in vivo. Mechanistically, we reveal that AS605240 effectively inhibits Ang II-induced cell proliferation and phosphorylation of Akt in fibroblast cells. Furthermore, we demonstrate that Ang II-upregulated expression of IL-6, Tnf-α, IL-1β and Tgf-β1 is significantly attenuated in the mice treated with AS605240. Taken together, our results demonstrate that PI3Kγ may function as a critical mediator of Ang II-induced renal injury and fibrosis. It is thus conceivable that targeted inhibition of PI3Kγ signaling may constitute a novel therapeutic approach to the clinical management of renal fibrosis, renal hypertension and/or CKD.

Topics: Angiotensin II; Animals; Cell Proliferation; Class Ib Phosphatidylinositol 3-Kinase; Cytokines; Disease Models, Animal; Fibroblasts; Fibrosis; Kidney; Mice; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Quinoxalines; Renal Insufficiency, Chronic; Thiazolidinediones

A key component during sepsis is the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, of which the PI3K-γ isoform is a major regulator in many inflammatory responses. However, the role of PI3K-γ in the development of sepsis-induced myocardial dysfunction (SIMD) is unknown. In this study, we established a model of SIMD induced by lipopolysaccharide (LPS), subsequently used the selective inhibitor LY294002 and AS605240 to block the effect of PI3K and PI3K-γ, respectively. Cardiac function was evaluated by echocardiography, hearts were obtained for histological and protein expression examinations. ELISA was used to measure the serum levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), cardiac troponin I (cTnI) and heart-type fatty acid binding protein (H-FABP). LPS-treated mice showed an increase to cardiac inflammation, myocardial damage and production of TNF-α, IL-6, NF-κB, cTnI and H-FABP. Administration of AS605240 to LPS-treated mice reduced some patho-physiological characteristics of SIMD and reduced TNF-α, IL-6, cTnI and H-FABP production. However, administration of LY294002 did not improve those same conditions. The results showed that PI3K-γ is likely a crucial element in SIMD by regulating the PI3K/Akt pathway, and become a new marker of myocardial injury. Inhibition of PI3K-γ might be a potential therapeutic target in SIMD.

Topics: Animals; Cardiomyopathies; Chromones; Class II Phosphatidylinositol 3-Kinases; Cytokines; Disease Models, Animal; Down-Regulation; Interleukin-6; Lipopolysaccharides; Mice; Morpholines; Myocardium; Quinoxalines; Sepsis; Signal Transduction; Thiazolidinediones; Tumor Necrosis Factor-alpha

With a steady increase in its incidence and lack of curative treatment, type 1 diabetes (T1D) has emerged as a major health problem worldwide. To design novel effective therapies, there is a pressing need to identify regulatory targets controlling the balance of autoreactive to regulatory-T-cells (Tregs). We previously showed that the inhibition of the γ-subunit of the Phosphoinositide-3-kinase (PI3K), significantly suppress autoimmune-diabetes. To further delineate the mechanisms and the selectivity of specific immune modulation by PI3Kγ-inhibition, we developed a new NOD mouse model of T1D lacking the γ-subunit of PI3K. Strikingly, the loss of PI3Kγ protected 92% of the NOD-mice from developing spontaneous diabetes. The NOD.PI3Kγ-/- mice are protected from insulitis secondary to a defect in CD4 and CD8 autoreactive-T-cells activation and survival. In addition, PI3Kγ-deficiency promoted Treg generation in-vitro and in-vivo. Furthermore, PI3Kγ-inhibitor (AS605240) inhibited proliferation and cytokine production of a human CD4+ T-cell clone specific for GAD555-567 peptide that was isolated from a patient with T1D. These studies demonstrate the key role of the PI3Kγ pathway in regulating autoimmune-diabetes and provide rationales for future devise of anti- PI3Kγ therapy in T1D.

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Proliferation; Cell Survival; Class Ib Phosphatidylinositol 3-Kinase; Cytokines; Diabetes Mellitus, Type 1; Disease Models, Animal; Female; Humans; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Pancreas; Peptides; Phosphoinositide-3 Kinase Inhibitors; Quinoxalines; T-Lymphocytes, Regulatory; Thiazolidinediones

Phosphatidylinositol 3-kinase (PI3K) is an enzyme involved in different pathophysiological processes, including neurological disorders. However, its role in seizures and postictal outcomes is still not fully understood. We investigated the role of PI3Kγ on seizures, production of neurotrophic and inflammatory mediators, expression of a marker for microglia, neuronal death and hippocampal neurogenesis in mice (WT and PI3Kγ(-/-)) subjected to intrahippocampal microinjection of pilocarpine. PI3Kγ(-/-) mice presented a more severe status epilepticus (SE) than WT mice. In hippocampal synaptosomes, genetic or pharmacological blockade of PI3Kγ enhanced the release of glutamate and the cytosolic calcium concentration induced by KCl. There was an enhanced neuronal death and a decrease in the doublecortin positive cells in the dentate gyrus of PI3Kγ(-/-) animals after the induction of SE. Levels of BDNF were significantly increased in the hippocampus of WT and PI3Kγ(-/-) mice, although in the prefrontal cortex, only PI3Kγ(-/-) animals showed significant increase in the levels of this neurotrophic factor. Pilocarpine increased hippocampal microglial immunolabeling in both groups, albeit in the prelimbic, medial and motor regions of the prefrontal cortex this increase was observed only in PI3Kγ(-/-) mice. Regarding the levels of inflammatory mediators, pilocarpine injection increased interleukin (IL) 6 in the hippocampus of WT and PI3Kγ(-/-) animals and in the prefrontal cortex of PI3Kγ(-/-) animals 24h after the stimulus. Levels of TNFα were enhanced in the hippocampus and prefrontal cortex of only PI3Kγ(-/-) mice at this time point. On the other hand, PI3Kγ deletion impaired the increase in IL-10 in the hippocampus induced by pilocarpine. In conclusion, the lack of PI3Kγ revealed a deleterious effect in an animal model of convulsions induced by pilocarpine, suggesting that this enzyme may play a protective role in seizures and pathological outcomes associated with this condition.

Topics: Animals; Calcium; Calcium-Binding Proteins; Class Ib Phosphatidylinositol 3-Kinase; Cytokines; Disease Models, Animal; Doublecortin Domain Proteins; Enzyme Inhibitors; Glutamic Acid; Hippocampus; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Microtubule-Associated Proteins; Muscarinic Agonists; Neuropeptides; Pilocarpine; Prefrontal Cortex; Quinoxalines; Reaction Time; Seizures; Synaptosomes; Thiazolidinediones; Time Factors

Experimental cerebral malaria (ECM) is characterized by a strong immune response, with leukocyte recruitment, blood-brain barrier breakdown and hemorrhage in the central nervous system. Phosphatidylinositol 3-kinase γ (PI3Kγ) is central in signaling diverse cellular functions. Using PI3Kγ-deficient mice (PI3Kγ-/-) and a specific PI3Kγ inhibitor, we investigated the relevance of PI3Kγ for the outcome and the neuroinflammatory process triggered by Plasmodium berghei ANKA (PbA) infection. Infected PI3Kγ-/- mice had greater survival despite similar parasitemia levels in comparison with infected wild type mice. Histopathological analysis demonstrated reduced hemorrhage, leukocyte accumulation and vascular obstruction in the brain of infected PI3Kγ-/- mice. PI3Kγ deficiency also presented lower microglial activation (Iba-1+ reactive microglia) and T cell cytotoxicity (Granzyme B expression) in the brain. Additionally, on day 6 post-infection, CD3+CD8+ T cells were significantly reduced in the brain of infected PI3Kγ-/- mice when compared to infected wild type mice. Furthermore, expression of CD44 in CD8+ T cell population in the brain tissue and levels of phospho-IkB-α in the whole brain were also markedly lower in infected PI3Kγ-/- mice when compared with infected wild type mice. Finally, AS605240, a specific PI3Kγ inhibitor, significantly delayed lethality in infected wild type mice. In brief, our results indicate a pivotal role for PI3Kγ in the pathogenesis of ECM.

Topics: Animals; Brain; Class Ib Phosphatidylinositol 3-Kinase; Disease Models, Animal; Extracellular Matrix; Female; Humans; Lung; Malaria, Cerebral; Mice; Phosphoinositide-3 Kinase Inhibitors; Plasmodium berghei; Quinoxalines; Survival Analysis; Thiazolidinediones

The gastrin-releasing peptide (GRP) and its receptor (GRPR) are important components of itch transmission. Upstream, but not downstream, aspects of GRPR signaling have been investigated extensively. We hypothesize that GRPR signals in part through the PI3Kγ/Akt pathway. We used pharmacological, electrophysiological, and behavioral approaches to further evaluate GRPR downstream signaling pathways. Our data show that GRP directly activates small-size capsaicin-sensitive DRG neurons, an effect that translates into transient calcium flux and membrane depolarization (∼ 20 mV). GRPR activation also induces Akt phosphorylation, a proxy for PI3Kγ activity, in ex vivo naive mouse spinal cords and in GRPR transiently expressing HEK293 cells. The intrathecal injection of GRP led to intense scratching, an effect largely reduced by either GRPR antagonists or PI3Kγ inhibitor. Scratching behavior was also induced by the intrathecal injection of an Akt activator. In a dry skin model of itch, we show that GRPR blockade or PI3Kγ inhibition reversed the scratching behavior. Altogether, these findings are highly suggestive that GRPR is expressed by the central terminals of DRG nociceptive afferents, which transmit itch via the PI3Kγ/Akt pathway.. Itch is the most common symptom of the skin and is related to noncutaneous diseases. It severely impairs patients' quality of life when it becomes chronic and there is no specific or effective available therapy, mainly because itch pathophysiology is not completely elucidated. Our findings indicate that the enzyme PI3Kγ is a key central mediator of itch transmission. Therefore, we suggest PI3Kγ as an attractive target for the development of new anti-pruritic drugs. With this study, we take a step forward in our understanding of the mechanisms underlying the central transmission of itch sensation.

Topics: Action Potentials; Animals; Anticarcinogenic Agents; Bombesin; Capsaicin; Central Nervous System; Disease Models, Animal; Enzyme Inhibitors; Ganglia, Spinal; Gastrin-Releasing Peptide; Indoles; Male; Mice; Neurons; p-Methoxy-N-methylphenethylamine; Pain Threshold; Peptide Fragments; Phosphatidylinositol 3-Kinase; Pruritus; Quinoxalines; Reaction Time; Receptors, Bombesin; Synaptic Transmission; Thiazolidinediones

A large amount of evidence suggests that monocytes/macrophages infiltration is implicated in a variety of inflammatory diseases including acute liver injury. Monocyte chemoattractant protein 1 (MCP-1) plays a crucial role in the process of macrophages recruitment. We herein presented a small-molecule library and a feasible quick screening method of evaluating potency of inhibition of chemotaxis of RAW264.7 cells stimulated by MCP-1. Fifty-three small molecules were synthesized and screened, and four compounds (2g, 2h, 4f, and 6h) showed inhibitory effects with IC(50) values range from 0.72 to 20.47 microM, with compound 4f being the most efficient. Further in vivo studies demonstrated that oral administration of 2g, 2h, 4f, or 6h decreases, most significantly for 4f, the serum levels of alanine aminotransaminase (ALT) and asparate aminotransaminase (AST) in ConA-induced acute livery injury BALB/c mice. Histopathological evaluation liver sections confirmed 4f as a potent, orally active compound for hepatoprotective effects against ConA-induced acute liver injury in BALB/c mice.

Topics: Animals; Cell Movement; Cells, Cultured; Chemical and Drug Induced Liver Injury; Concanavalin A; Disease Models, Animal; Drug Discovery; Drug Evaluation, Preclinical; Female; Liver Failure, Acute; Liver Function Tests; Mice; Mice, Inbred BALB C; Small Molecule Libraries; Stereoisomerism; Structure-Activity Relationship; Thiazolidinediones

Phosphoinositide 3-kinase gamma(PI3Kgamma) is a critical mediator of directional cell movement. Here, we sought to characterise the role of PI3Kgamma in mediating the different steps of polymorphonuclear leukocyte (PMN) trafficking in the lung. In a murine model of lipopolysaccharide (LPS)-induced lung injury, PMN migration into the different lung compartments was determined in PI3Kgamma gene-deficient (PI3Kgamma(-/-)) and wild-type mice. Bone marrow chimeras were created to characterise the role of PI3Kgamma on haematopoietic versus nonhaematopoietic cells. A small-molecule PI3Kgamma inhibitor was tested in vitro and in vivo. PMN adhesion to the pulmonary endothelium and transendothelial migration into the lung interstitium was enhanced in PI3Kgamma(-/-) mice. However, transepithelial migration into the alveolar space was reduced in these mice. When irradiated PI3Kgamma(-/-) mice were reconstituted with bone marrow from wild-type mice, migratory activity into the alveolar space was restored partially. A small-molecule PI3Kgamma inhibitor reduced chemokine-induced PMN migration in vitro when PMNs or epithelial cells, but not when endothelial cells, were treated. The inhibitor also reduced LPS-induced PMN migration in vivo. We conclude that PI3Kgamma is required for transepithelial but not for transendothelial migration in LPS-induced lung injury. Inhibition of PI3Kgamma activity may be effective at curbing excessive PMN infiltration in lung injury.

Topics: Acute Lung Injury; Analysis of Variance; Animals; Bronchoalveolar Lavage Fluid; Cell Movement; Chimera; Class Ib Phosphatidylinositol 3-Kinase; Disease Models, Animal; Flow Cytometry; Humans; Intracellular Signaling Peptides and Proteins; Isoenzymes; Lipopolysaccharides; Lung; Male; Membrane Proteins; Mice; Neutrophils; Phosphatidylinositol 3-Kinases; Quinoxalines; Thiazolidinediones

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.

Topics: Acute Disease; Animals; Blotting, Western; CD4 Lymphocyte Count; CD4-Positive T-Lymphocytes; Chronic Disease; Colitis; Colon; Cytokines; Dextran Sulfate; Disease Models, Animal; Enzyme Inhibitors; Flow Cytometry; Fluorescent Antibody Technique; Immunohistochemistry; Intestinal Mucosa; Male; Mice; Mice, Inbred C57BL; Organ Size; Phosphoinositide-3 Kinase Inhibitors; Quinoxalines; Severity of Illness Index; Spleen; Thiazolidinediones

Phosphoinositide 3-kinase (PI3K)gamma is expressed in hematopoietic cells, endothelial cells (ECs), and cardiomyocytes and regulates different cellular functions relevant to inflammation, tissue remodeling and cicatrization. Recently, PI3Kgamma inhibitors have been indicated for the treatment of chronic inflammatory/autoimmune diseases and atherosclerosis.. We aimed to determine PI3Kgamma contribution to the angiogenic capacity of ECs and the effect of PI3Kgamma inhibition on healing of myocardial infarction (MI).. Human umbilical ECs were treated with a selective PI3Kgamma inhibitor, AS605240, or a pan-phosphoinositide 3-kinases inhibitor, LY294002. Both inhibitory treatments and small interfering RNA-mediated PI3Kgamma knockdown strongly impaired ECs angiogenic capacity, because of suppression of the PI3K/Akt and mitogen-activated protein kinase pathways. Constitutive activation of Akt rescued the angiogenic defect. Reparative angiogenesis was studied in vivo in a model of MI. AS605240 did not affect MI-induced PI3Kgamma upregulation, whereas it suppressed Akt activation and downstream signaling. AS605240 strongly reduced inflammation, enhanced cardiomyocyte apoptosis, and impaired survival and proliferation of ECs in peri-infarct zone, which resulted in defective reparative neovascularization. As a consequence, AS605240-treated MI hearts showed increased infarct size and impaired recovery of left ventricular function. Similarly, PI3Kgamma-deficient mice showed impaired reparative neovascularization, enhanced cardiomyocyte apoptosis and marked deterioration of cardiac function following MI. Mice expressing catalytically inactive PI3Kgamma also failed to mount a proper neovascularization, although cardiac dysfunction was similar to wild-type controls.. PI3Kgamma expression and catalytic activity are involved at different levels in reparative neovascularization and healing of MI.

Topics: Animals; Apoptosis; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Chromones; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Humans; Inflammation; Leukocytes; Male; Mice; Morpholines; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinoxalines; Recovery of Function; Regeneration; RNA Interference; Signal Transduction; Thiazolidinediones; Time Factors; Transfection; Ventricular Function, Left

Topics: Animals; Cardiovascular Agents; Disease Models, Animal; Genotype; Mice; Mice, Knockout; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Phenotype; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinoxalines; Thiazolidinediones; Ventricular Function, Left; Ventricular Remodeling

The role of inflammation at all stages of the atherosclerotic process has become an active area of investigation, and there is a notable quest for novel and innovative drugs for the treatment of atherosclerosis. The lipid kinase phosphoinositide 3-kinase-gamma (PI3Kgamma) is thought to be a key player in various inflammatory, autoimmune, and allergic processes. These properties and the expression of PI3Kgamma in the cardiovascular system suggest that PI3Kgamma plays a role in atherosclerosis.. Here, we demonstrate that a specific PI3Kgamma inhibitor (AS605240) is effective in murine models of established atherosclerosis. Intraperitoneal administration of AS605240 (10 mg/kg daily) significantly decreased early atherosclerotic lesions in apolipoprotein E-deficient mice and attenuated advanced atherosclerosis in low-density lipoprotein receptor-deficient mice. Furthermore, PI3Kgamma levels were elevated in both human and murine atherosclerotic lesions. Comparison of low-density lipoprotein receptor-deficient mice transplanted with wild-type or PI3Kgamma-deficient bone marrow demonstrated that functional PI3Kgamma in the hematopoietic lineage is required for atherosclerotic progression. Alleviation of atherosclerosis by targeting of PI3Kgamma activity was accompanied by decreased macrophage and T-cell infiltration, as well as increased plaque stabilization.. These data identify PI3Kgamma as a new target in atherosclerosis with the potential to modulate multiple stages of atherosclerotic lesion formation, such as fatty streak constitution, cellular composition, and final fibrous cap establishment.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Class Ib Phosphatidylinositol 3-Kinase; Disease Models, Animal; Humans; Inflammation; Intramolecular Oxidoreductases; Isoenzymes; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphoinositide-3 Kinase Inhibitors; Prostaglandin-E Synthases; Quinoxalines; Receptors, LDL; Thiazolidinediones

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.

Topics: Animals; Arthritis, Rheumatoid; Binding Sites; Chemotaxis, Leukocyte; Dioxoles; Disease Models, Animal; Enzyme Inhibitors; Isoenzymes; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred DBA; Mice, Knockout; Molecular Sequence Data; Molecular Structure; Peritonitis; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinoxalines; Signal Transduction; Structure-Activity Relationship; Thiazolidinediones

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease generated by deregulation of T cell-mediated B-cell activation, which results in glomerulonephritis and renal failure. Disease is treated with immunosuppressants and cytostatic agents that have numerous side effects. Here we examine the use of inhibitors of phosphoinositide 3-kinase (PI3K) gamma, a lipid kinase that regulates inflammation, in the MRL-lpr mouse model of SLE. Treatment reduced glomerulonephritis and prolonged lifespan, suggesting that P13Kgamma may be a useful target in the treatment of chronic inflammation.

Topics: Animals; Disease Models, Animal; Enzyme Inhibitors; Female; Lupus Erythematosus, Systemic; Lupus Nephritis; Male; Mice; Mice, Mutant Strains; Phosphoinositide-3 Kinase Inhibitors; Quinoxalines; Thiazolidinediones