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.

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).

Leukocyte trafficking to inflammatory sites is a gradual process, which is dominated in its early phases by chemokine-and cytokine-mediated neutrophil recruitment. The chemokine regulated on activation normal T cell expressed and secreted (RANTES) has been shown to be highly expressed in the joints of patient with rheumatoid arthritis and to promote leukocyte trafficking into the synovial tissue. In this study, we investigated the effect of RANTES in a murine model of peritoneal chemotaxis, and we found that RANTES dose-dependently induces neutrophil recruitment. Then, through morphological and histological analyses, we observed that activated neutrophils represent the major infiltrating population in response to RANTES chemotactic stimulus. Furthermore, we demonstrated that oral administration of either nonisoform-specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (morpholin-4-yl-8-phenylchromen-4-one) or selective PI3K␥ inhibitor AS041164 (5-benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione) blocks RANTES-induced chemotaxis and reduces the level of AKT phosphorylation. Because the two compounds showed a similar pharmacokinetic profile in terms of bioavailability and half-life after oral route administration, the selective inhibition of the PI3K␥-isoform pathway through AS041164 was three times more potent in reducing neutrophil recruitment. Finally, to confirm the blockade of neutrophil infiltration that occurs in the early phase of the inflammatory response, AS041164 was also tested in a model of carrageenan-induced paw edema in rats. Therefore, the PI3K␥ pathway plays an important role in controlling neutrophil chemotaxis during early steps of inflammation.During inflammation, neutrophils are rapidly recruited at sites of acute infection and dominate the initial influx of leukocytes (Issekutz and Movat, 1980). Later, during the progression of inflammation, monocytes and macrophages replace neutrophils, suggesting a bimodal recruitment pattern involving a switch from neutrophils to monocytes (Doherty et al., 1988;Henderson et al., 2003). The first step in approaching a site of insult requires neutrophils to transmigrate across endothelial barriers, a process that depends on chemokines (Yoshie et al., 2001). In response to a chemotactic gradient, CXC and CC chemokines activate leukocytes by binding to seven transmembrane receptors coupled to G proteins (Proudfoot, 2002) linked to heterotrimeric G protein complexes (Proudfoot, 2002). Upon stimulation, the G protein complex dissociates and subsequently recruits various signaling components, such as nucleotide exchange factors, phospholipid lipases, and lipid kinase phosphoinositide-3ЈOH-kinase isoforms, such as phosphoinositide 3-kinase (PI3K) (Akasaki et al., 1999). Neutrophils have been shown to express different chemokine receptors, including CXCR2 and CCR1 (Lee et al., 1995;Zhang et al., 1999). Furthermore, the blockade of the chemokine receptor CXCR2 or its ligands IL-8 and macrophage inflammatory protein (MIP)-2, or alternatively of t...

We describe novel, cell-permeable, and bioavailable salicylic acid derivatives that are potent and selective inhibitors of GLEPP1/protein-tyrosine phosphatase . 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.

A novel high-affinity inhibitor of tumor necrosis factor (TNF) is described, which is created by the fusion of the extracellular domains of TNF-binding protein 1 (TBP-1) to both the ␣ and ␤ chains of an inactive version of the heterodimeric protein hormone, human chorionic gonadotropin. The resulting molecule, termed TNF-soluble high-affinity receptor complex (SHARC), self-assembles into a heterodimeric protein containing two functional TBP-1 moieties. The TNF-SHARC is a potent inhibitor of TNF-␣ bioactivity in vitro and has a prolonged pharmacokinetic profile compared with monomeric TBP-1 in vivo. Consistent with the long half-life, the duration of action in an lipopolysaccharide-mediated proinflammatory mouse model is prolonged similarly. In a collagen-induced arthritis mouse model, this molecule demonstrates improved efficacy over monomeric TBP-1. Based on these results, we demonstrated that inactivated heterodimeric protein hormones are flexible and efficient scaffolds for the creation of soluble high-affinity receptor complexes.