The JAKs receive continued interest as therapeutic targets for autoimmune, inflammatory, and oncological diseases. JAKs play critical roles in the development and biology of the hematopoietic system, as evidenced by mouse and human genetics. JAK1 is critical for the signal transduction of many type I and type II inflammatory cytokine receptors. In a search for JAK small molecule inhibitors, GLPG0634 was identified as a lead compound belonging to a novel class of JAK inhibitors. It displayed a JAK1/JAK2 inhibitor profile in biochemical assays, but subsequent studies in cellular and whole blood assays revealed a selectivity of ∼30-fold for JAK1- over JAK2-dependent signaling. GLPG0634 dose-dependently inhibited Th1 and Th2 differentiation and to a lesser extent the differentiation of Th17 cells in vitro. GLPG0634 was well exposed in rodents upon oral dosing, and exposure levels correlated with repression of Mx2 expression in leukocytes. Oral dosing of GLPG0634 in a therapeutic set-up in a collagen-induced arthritis model in rodents resulted in a significant dose-dependent reduction of the disease progression. Paw swelling, bone and cartilage degradation, and levels of inflammatory cytokines were reduced by GLPG0634 treatment. Efficacy of GLPG0634 in the collagen-induced arthritis models was comparable to the results obtained with etanercept. In conclusion, the JAK1 selective inhibitor GLPG0634 is a promising novel therapeutic with potential for oral treatment of rheumatoid arthritis and possibly other immune-inflammatory diseases.

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The structures of ternary complexes of human inositol monophosphatase with inhibitory Gd3+ and either D- or L-myo-inositol 1-phosphate have been determined to 2.2-2.3 A resolution using X-ray crystallography. Substrate and metal are bound identically in each active site of the phosphatase dimer. The substrate is present at full occupancy, while the metal is present at only 35% occupancy, suggesting that Li+ from the crystallization solvent partially replaces Gd3+ upon substrate binding. The phosphate groups of both substrates interact with the phosphatase in the same manner with one phosphate oxygen bound to the octahedrally coordinated active site metal and another oxygen forming hydrogen bonds with the amide groups of residues 94 and 95. The active site orientations of the inositol rings of D- and L-myo-inositol 1-phosphate differ by rotation of nearly 60 degrees about the phosphate ester bond. Each substrate utilizes the same key residues (Asp 93, Ala 196, Glu 213, and Asp 220) to form the same number of hydrogen bonds with the enzyme. Mutagenesis experiments confirm the interaction of Glu 213 with the inositol ring and suggest that interactions with Ser 165 may develop during the transition state. The structural data suggest that the active site nucleophile is a metal-bound water that is activated by interaction with Glu 70 and Thr 95. Expulsion of the ester oxygen appears to be promoted by three aspartate residues acting together (90, 93, and 220), either to donate a proton to the leaving group or to form another metal binding site from which a second Mg2+ coordinates the leaving group during the transition state.

myo-Inositol monophosphatase (myo-inositol-1-phosphate phosphohydrolase, EC 3.1.3.25) is an attractive target for mechanistic Investigation due to its critical role in the phosphatidylinositol signaling pathway and the possible relevance of Its Ihibition by Li+ to manic depression therapy. The x-ray crystallographic structure of human inositol monophosphatase in the presence of the inhibitory metal Gd3+ showed only one metal bound per active site, whereas in the presence ofMn2+, three ions were present with one being displaced upon phosphate binding. We report here modeling, kinetic, and mutagenesis studies on the enzyme, which reveal the requirement for two metal ions in the catalytic mechanism. Activity titration curves with Zn2+ or Mn2+ in the presence or absence of Mg2+ are consistent with a two-metal mechanism. Modeling studies based on the various x-ray crystallographic structures (including those with Gd3+ and substrate bound) further support a two-metal mechanism and define the positions of the two metal ions relative to substrate. While the first metal ion may activate water for nucleophilic attack, a second metal ion, coordinated by three aspartate residues, appears to act as a Lewis acid, stabiing the leaving inositol oxyanion. In this model, the 6-OH group of substrate acts as a ligand for this second metal ion, consistent with the reduced catalytic activity observed with substrate analogues lacking the 6-OH. Evidence from Tb3+ fluorescence quenching and the two-metal kinetic titration curves suggests that Li+ binds at the site of this second metal ion.In the phosphatidylinositol second-messenger pathway, receptor-activated phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate, to form two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol, leading to Ca2+ and protein kinase C-mediated signal transduction, respectively (1-3). Inositol 1,4,5-trisphosphate is subsequently metabolized in a series of pathways, regenerating inositol for reincorporation into inositol phospholipids, thereby maintaining this signaling cycle. myo-Inositol monophosphatase (IMPase; myo-l-phosphate phosphohydrolase, EC 3.1.3.25) catalyzes the last hydrolytic step in these pathways, regenerating myo-D-inositol from myo-D-inositol 1-phosphate [Ins(l)P], Ins(3)P, or Ins(4)P (4).IMPase, a homodimer of two 30-kDa subunits, requires Li+ parameters gave contacts in agreement with those seen in the Cambridge Crystallographic Database (13), but to achieve similarly satisfactory results the van der Waals radius parameter of Gd3+ was reduced to 2.1 A. Hydrogens were added to the protein by using the Sybyl BIOPOLY ADDH command. The standard Sybyl TIP3P water model was used for crystallographically observed water molecules. Charges were taken from the standard Kollman parameter set for water molecules and protein residues. Starting coordinates for dianionic Ins(l)P were obtained from the crystal structure of the IMPase/Gd/Ins(l)P complex, supplemented with hydrogens at idealized sites on the inositol moiet...

GPR84 is a medium chain free fatty acid-binding G-protein-coupled receptor associated with inflammatory and fibrotic diseases. As the only reported antagonist of GPR84 (PBI-4050) that displays relatively low potency and selectivity, a clear need exists for an improved modulator. Structural optimization of GPR84 antagonist hit 1, identified through high-throughput screening, led to the identification of potent and selective GPR84 inhibitor GLPG1205 (36). Compared with the initial hit, 36 showed improved potency in a guanosine 5′-O-[γ-thio]­triphosphate assay, exhibited metabolic stability, and lacked activity against phosphodiesterase-4. This novel pharmacological tool allowed investigation of the therapeutic potential of GPR84 inhibition. At once-daily doses of 3 and 10 mg/kg, GLPG1205 reduced disease activity index score and neutrophil infiltration in a mouse dextran sodium sulfate-induced chronic inflammatory bowel disease model, with efficacy similar to positive-control compound sulfasalazine. The drug discovery steps leading to GLPG1205 identification, currently under phase II clinical investigation, are described herein.