Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune, inflammatory, and oncology disease indications. The most advanced Syk inhibitor, R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed, at least in part, to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications.

Phosphorylated prolactin has been identified and isolated from bovine pituitaries. The biological activity of this phosphoprotein is severely reduced in comparison with nonphosphorylated prolactin. The sites of phosphorylation are serines 26, 34, and 90, and the stoichiometry is 1:1:10, respectively. In this report, the phosphoserine residues have been individually replaced with glutamic acid in recombinant methionyl bovine prolactins in order to mimic phosphorylation at each site. Substitution of glutamic acid for serine at positions 26, 34, and 90 reduced protein helical contents by 10, 6, and 14%, respectively. UV absorbances for S26E and S34E bovine prolactins were blue-shifted, similar to the biological isolates of phosphorylated bovine prolactin, but the biological activities of the S26E and S34E mutants (ED 50 values of 16.3 and 18.8 pM, respectively) were similar to that of wild-type prolactin (ED 50 value of 18.6 pM) in the Nb2 rat lymphoma assay. S90E bovine prolactin had the greatest reduction in helical content but showed similar UV and fluorescent spectra to the wildtype bovine prolactin. The biological activity of S90E bovine prolactin (ED 50 value of 672 pM) was reduced to an activity similar to that of phosphorylated bovine prolactin. The data indicate that the phosphorylation of serine 90 is responsible for the reduction in biological activity.Prolactin binds and activates receptors on the plasma membrane of target cells (1). The PRL 1 receptor is a member of the cytokine receptor superfamily (2), receptors with extracellular and intracellular domains connected by a single transmembrane domain. Several receptors in this superfamily activate cells by formation of receptor dimers brought about by the binding of two receptors binding to unique surfaces of the ligand (sites 1 and 2) (3). Receptor dimerization promotes the association and/or activation of the Janus family of tyrosine kinases (4) and phosphorylation of specific tyrosines of the receptor and substrate proteins that regulate transcription in the nucleus or the activity of other kinase systems. Substrate proteins appear to be presented to the Janus kinases by binding to the phosphorylated loci of the intracellular domain of the receptor (5).The three-dimensional structure of PRL has not been solved, but it is likely to be a four-helix bundle motif (6) similar to the up-up-down-down structure of hGH, a protein related by sequence able to bind prolactin receptors and induce lactogenic actions. The sequence of several variants of the PRL receptor have been reported; most variation between receptor subtypes is observed in the intracellular domain (7). The three-dimensional structure of hGH bound to the extracellular domain of the hPRL receptor in a 1:1 complex has been reported (8).These data have defined the site 1 surface area and have shown that the four-helix bundle of hGH has undergone little structural change when bound to either the hPRL or hGH receptor. However, there is a major shift of the first ancillary helix of the loop connecti...

Agents that target B-cell receptor (BCR) signaling in lymphoid malignancies including idelalisib (GS-1101) and fostamatinib which inhibit the delta isoform of PI3 kinase (PI3Kd) and spleen tyrosine kinase (Syk) respectively have shown significant clinical activity. By disrupting B-cell signaling pathways, idelalisib treatment has been associated with a dramatic lymph node response, but eradication of disease and relapse in high risk disease remain challenges. Targeting the BCR signaling pathway with simultaneous inhibition of PI3Kd and Syk has not yet been reported. We evaluated the pre-clinical activity of idelalisib combined with the novel and selective Syk inhibitor GS-9973 in primary peripheral blood and bone marrow Chronic Lymphocytic Leukemia (CLL) samples. Both PI3Kd and Syk inhibition reduced CLL survival and in combination induced synergistic growth inhibition and further disrupted chemokine signaling at nanomolar concentrations including in bone marrow derived and poor risk samples. Simultaneous targeting of these kinases may significantly increase clinical activity.

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