Pierre-Alain Vitte

Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis

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 PI3Kγ (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 PI3Kγ as a therapeutic target. We also describe that oral treatment with a PI3Kγ 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 PI3Kγ inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.

Inhibition of c-Jun N-terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats

Myocardial ischemia/reperfusion is associated with inflammation, apoptosis and necrosis. During this process, c‐jun N‐terminal kinase is activated in cardiac myocytes resulting in apoptosis. This study investigates the effects of AS601245, a nonpeptide ATP competitive JNK inhibitor, on infarct size caused by myocardial ischemia/reperfusion in anaesthetized rats. The left descending coronary artery of anaesthetized rats was occluded for 30 min and then reperfused for 3 h. AS601245 was administered 5 min before the end of the ischemia period as an i.v. bolus (1.5, 4.5 or 15 mg kg−1 i.v.) followed by continuous i.v. infusion (18, 55 and 183 μg kg−1 min−1, respectively) during reperfusion. Controls received saline only. 3‐Aminobenzamide, a poly(ADP‐ribose) polymerase inhibitor, was used as reference compound at 10 mg kg−1 i.v. bolus plus 0.17 mg kg−1 min−1 continuous infusion. AS601245 significantly reduced infarct size at 4.5 mg kg−1 (−44%; P<0.001) and 15 mg kg−1 i.v. (−40.3%; P<0.001) similarly to 3‐aminobenzamide (−44.2%; P<0.001). This protective effect was obtained without affecting hemodinamics or reducing ST‐segment displacement. The beneficial effects on infarct size correlated well with the reduction of c‐jun phosphorylation (−85%; P<0.001 versus control) and of TUNEL‐positive cells (−82.1%; P<0.001) in post‐ischemic cardiomyocytes. No change in the phosphorylation state of p38 MAPK and ERK in post‐ischemic heart was observed in the presence of AS601245 in comparison to the vehicle‐treated group. These results demonstrate that blocking the JNK pathway may represent a novel therapeutic approach for treating myocardial ischemia/reperfusion‐induced cardiomyocyte death.

Regulation of the severity of neuroinflammation and demyelination by TLR-ASK1-p38 pathway

Apoptosis signal‐regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen‐activated protein kinase (MAPK) kinase kinase which plays important roles in stress and immune responses. Here, we show that ASK1 deficiency attenuates neuroinflammation in experimental autoimmune encephalomyelitis (EAE), without affecting the proliferation capability of T cells. Moreover, we found that EAE upregulates expression of Toll‐like receptors (TLRs) in activated astrocytes and microglia, and that TLRs can synergize with ASK1‐p38 MAPK signalling in the release of key chemokines from astrocytes. Consequently, oral treatment with a specific small molecular weight inhibitor of ASK1 suppressed EAE‐induced autoimmune inflammation in both spinal cords and optic nerves. These results suggest that the TLR‐ASK1‐p38 pathway in glial cells may serve as a valid therapeutic target for autoimmune demyelinating disorders including multiple sclerosis.

An engineered monomer of CCL2 has anti-inflammatory properties emphasizing the importance of oligomerization for chemokine activity in vivo

We demonstrated recently that P8A‐CCL2, a monomeric variant of the chemokine CCL2/MCP‐1, is unable to induce cellular recruitment in vivo, despite full activity in vitro. Here, we show that this variant is able to inhibit CCL2 and thioglycollate‐mediated recruitment of leukocytes into the peritoneal cavity and recruitment of cells into lungs of OVA‐sensitized mice. This anti‐inflammatory activity translated into a reduction of clinical score in the more complex inflammatory model of murine experimental autoimmune encephalomyelitis. Several hypotheses for the mechanism of action of P8A‐CCL2 were tested. Plasma exposure following s.c. injection is similar for P8A‐CCL2 and wild‐type (WT) CCL2, ruling out the hypothesis that P8A‐CCL2 disrupts the chemokine gradient through systemic exposure. P8A‐CCL2 and WT induce CCR2 internalization in vitro and in vivo; CCR2 then recycles to the cell surface, but the cells remain refractory to chemotaxis in vitro for several hours. Although the response to P8A‐CCL2 is similar to WT, this finding is novel and suggests that despite the presence of the receptor on the cell surface, coupling to the signaling machinery is retarded. In contrast to CCL2, P8A‐CCL2 does not oligomerize on glycosaminoglycans (GAGs). However, it retains the ability to bind GAGs and displaces endogenous JE (murine MCP‐1) from endothelial surfaces. Intravital microscopy studies indicate that P8A‐CCL2 prevents leukocyte adhesion, while CCL2 has no effect, and this phenomenon may be related to the mechanism. These results suggest that oligomerization‐deficient chemokines can exhibit anti‐inflammatory properties in vivo and may represent new therapeutic modalities.

Control of death receptor and mitochondrial-dependent apoptosis by c-Jun N-terminal kinase in hippocampal CA1 neurones following global transient ischaemia.

c‐Jun N‐terminal kinase (JNK), a member of the mitogen‐activated protein kinase family, is activated in response to a number of extracellular stimuli, including inflammatory cytokines, UV irradiation and ischaemia. A large body of evidence supports a role for JNK signalling in stress‐induced apoptosis. It has been hypothesized that JNK may contribute to the apoptotic response by regulating the intrinsic cell death pathway involving the mitochondria. Here, we examined the role of the JNK signalling pathway in hippocampal CA1 apoptotic neurones following transient ischaemia in gerbils. We showed early activation of death receptor‐dependent apoptosis (caspase‐8 activation 2 days after ischaemia) and a biphasic activation of caspase‐3 and caspase‐9 after ischaemia. Activation of the mitochondrial pathway, as measured by cytochrome c release, appeared as a late event (5–7 days after ischaemia). AS601245, a novel JNK inhibitor, antagonized activation of both pathways and significantly protected CA1 neurones from cell death. Our results suggest a key role of JNK in the control of death receptor and mitochondrial‐dependent apoptosis after transient ischaemia.

Interleukin-6 attenuates the development of experimental diabetes-related neuropathy.

Neuropathy is the most severe and the least understood complication of diabetes. We investigated the potential neuroprotective effect of IL‐6 therapy in an experimental model of diabetic neuropathy. A single i.v. injection of streptozotocin (STZ, 55 mg/kg) was used to induce experimental diabetes in adult males. IL‐6 (1, 10 or 30 µg/kg) was administrated either intraperitoneally on a daily basis or subcutaneously (s.c.) on a daily, on a three times or one time per week basis, starting at day 10 post‐STZ. A decrease in sensory nerve conduction velocity (SNCV), indicative of neuropathy, is seen in STZ rats as early as day 10 post‐STZ, a time at which blood glycaemia is already maximal. At later time points, this electrophysiological impairment became severe and clinically apparent by affecting tail flick latency. Motor dysfunction defined by a significant increase in compound muscle action potential (CMAP) latency was also recorded. At the completion of the study (day 40 post‐STZ), histological examination revealed significant axonopathy and myelin loss, along with an increase in the proportion of fibers with abnormal appearance in sciatic nerves of STZ rats. These changes were not observed in non‐diabetic rats and were significantly prevented by IL‐6 treatment. The optimal dose appeared to be 10 µg/kg s.c. three injections per week, which showed a better effect in most of the parameters studied than 4‐methylcatechol, a NGF‐like neuroprotective compound. Once weekly and three times weekly administrations of IL‐6 were as effective as daily treatment. Taken together, these results support the potential neuroprotective actions of IL‐6. The fact that the half‐life of IL‐6 is only approximately 5 h while weekly dosing was neuroprotective strongly suggests activation by IL‐6 of effector molecule(s) with longer duration of action.

An Adiponectin-Like Molecule with Antidiabetic Properties

Adiponectin increases glucose transport, reduces inflammation, and controls vascular functions. Hence, we propose that treatment with a recombinant globular domain of adiponectin (rgAd110-244) has significant therapeutic potential to treat insulin resistance. Mice were fed for 3 months on a high-fat diet (HFD) to induce insulin resistance, diabetes, and moderate weight gain. The mice were first infused iv with different doses of rgAd110-244 (0.12, 0.4, and 1.2 microg/kg x min) for 5 h. Basal and insulin-sensitive glucose use rates were assessed by the use of a submaximal rate of insulin in the awake free-moving mouse. rgAd110-244 reduced, with dose dependence, epinephrine-induced hyperglycemia and HFD-induced insulin resistance by increasing whole-body glucose use (35% at the highest dose) and glycolysis rates. Similarly, the reduction of plasma free fatty acid concentrations by insulin was dramatically improved. Basal hepatic glucose production was unchanged by rgAd110-244 infusion. This acute rgAd110-244 treatment improved glucose homeostasis and was associated with an increased content of muscle phospho-Akt, glycogen synthase kinase-3beta, and AMP-activated kinase. Second, HFD mice were chronically treated with sc rgAd110-244 injections (10, 30, and 100 microg/kg). Fasting glycemia and insulin-sensitive glucose use were improved by rgAd110-244 at the highest dose at completion of the treatment, with concomitant reduction in body weight gain. We here show for the first time that a recombinant adiponectin fragment (110-244 amino acids called rgAd110-244) is able to treat insulin-resistant diabetes. Our results strongly suggest further pharmacological investigation of rgAd110-244 with the objective of developing a new treatment of insulin-resistant diabetes.

Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells.

Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild—yet chronic—neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-β. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1—but not pertussis toxin, which affects Gi protein-dependent responses—abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis.

GLEPP1/Protein-tyrosine Phosphatase ϕ Inhibitors Block Chemotaxis in Vitro and in Vivo and Improve Murine Ulcerative Colitis

We describe novel, cell-permeable, and bioavailable salicylic acid derivatives that are potent and selective inhibitors of GLEPP1/protein-tyrosine phosphatase ϕ. Two previously described GLEPP1 substrates, paxillin and Syk, are both required for cytoskeletal rearrangement and cellular motility of leukocytes in chemotaxis. We show here that GLEPP1 inhibitors prevent dephosphorylation of Syk1 and paxillin in resting cells and block primary human monocyte and mouse bone marrow-derived macrophage chemotaxis in a gradient of monocyte chemotactic protein-1. In mice, the GLEPP1 inhibitors also reduce thioglycolate-induced peritoneal chemotaxis of neutrophils, lymphocytes, and macrophages. In murine disease models, the GLEPP1 inhibitors significantly reduce severity of contact hypersensitivity, a model for allergic dermatitis, and dextran sulfate sodium-induced ulcerative colitis, a model for inflammatory bowel disease. Taken together, our data provide confirmation that GLEPP1 plays an important role in controlling chemotaxis of multiple types of leukocytes and that pharmacological inhibition of this phosphatase may have therapeutic use.

Discovery of a novel series of potent S1P1 agonists.

The discovery of a novel series of S1P1 agonists is described. Starting from a micromolar HTS positive, iterative optimization gave rise to several single-digit nanomolar S1P1 agonists. The compounds were able to induce internalization of the S1P1 receptor, and a selected compound was shown to be able to induce lymphopenia in mice after oral dosing.