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.

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.

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.

Several lines of evidence support the hypothesis that c-Jun N-terminal kinase (JNKs) plays a critical role in a wide range of diseases including cell death (apoptosis)-related disorders (neurodegenerative diseases, brain, heart, and renal ischemia, epilepsy) and inflammatory disorders (multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases). Screening of our internal compound collection for inhibitors of JNK3 led to the identification of (benzothiazol-2-yl)acetonitrile derivatives as potent and selective JNK1, -2, -3 inhibitors. Starting from initial hit 1 (AS007149), the chemistry and initial structure-activity relationship (SAR) of this novel and unique kinase inhibitor template were explored. Investigation of the SAR rapidly revealed that the benzothiazol-2-ylacetonitrile pyrimidine core was crucial to retain a good level of potency on rat JNK3. Therefore, compound 6 was further optimized by exploring a number of distal combinations in place of the chlorine atom. This led to the observation that the presence of an aromatic group, two carbons away from the aminopyrimidine moiety and bearing substituents conferring hydrogen bond acceptor (HBA) properties, could improve the potency. Further improvements to the biological and biopharmaceutical profile of the most promising compounds were performed, resulting in the discovery of compound 59 (AS601245). The in vitro and in vivo anti-inflammatory potential of this new JNK inhibitor was investigated and found to demonstrate efficacy per oral route in an experimental model of rheumatoid arthritis (RA).

1 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. 2 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 À1 i.v.) followed by continuous i.v. infusion (18, 55 and 183 mg kg À1 min À1, respectively) during reperfusion. Controls received saline only. 3-Aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, was used as reference compound at 10 mg kg À1 i.v. bolus plus 0.17 mg kg À1 min À1 continuous infusion. 3 AS601245 significantly reduced infarct size at 4.5 mg kg À1 (À44%; Po0.001) and 15 mg kg À1 i.v. (À40.3%; Po0.001) similarly to 3-aminobenzamide (À44.2%; Po0.001). This protective effect was obtained without affecting hemodinamics or reducing ST-segment displacement. 4 The beneficial effects on infarct size correlated well with the reduction of c-jun phosphorylation (À85%; Po0.001 versus control) and of TUNEL-positive cells (À82.1%; Po0.001) 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. 5 These results demonstrate that blocking the JNK pathway may represent a novel therapeutic approach for treating myocardial ischemia/reperfusion-induced cardiomyocyte death.