R406, an Orally Available Spleen Tyrosine Kinase Inhibitor Blocks Fc Receptor Signaling and Reduces Immune Complex-Mediated Inflammation

Abstract

Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to selfantigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)-and IgG-mediated activation of Fc receptor signaling (EC 50 for degranulation ϭ 56 -64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K i ϭ 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Sykindependent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.

Spleen tyrosine kinase (Syk) is a potent target for GvHD prevention at different cellular levels

“…18 However, R406 is a 5 --100-fold less potent inhibitor of other kinases, such as Flt-3, c-Kit, Lck and Janus kinase-1 and 3, which are involved in the IL-2-induced primary Tc activation pathway. 18 We had studied the effects of R406 at concentrations that were below those described for the inhibition of Janus kinasedependent anti CD3/CD28 TCR-induced IL-2 production at 448 nM 18 and found no inhibitory effect. At the higher doses a certain degree of inhibition of other kinases although being less Figure 6.…”

“…17 Although initial studies were performed with broader protein tyrosine kinase inhibitors such as Curcumin or Piceatannol, recently more specific Syk kinase inhibitors, such as Syk Inhibitor I to IV or R406, of which the orally bioavailable prodrug is R788 (Fostamatinib) have been developed and safely tested in humans. 18 Recent phase II clinical trials demonstrated that Fostamatinib treatment effectively improved response rates in patients with rheumatoid arthritis. 19,20 Therefore, by using pharmacological inhibitors we aimed to delineate the relevance of Syk with respect to protection from GvHD, graft-versus-leukemia effects and anti-viral immune responses.…”

Spleen tyrosine kinase inhibition in the treatment of autoimmune, allergic and autoinflammatory diseases

“…R406, an orally available active metabolite of the prodrug R788 (fostamatinib), is a competitive Syk inhibitor [37,38]. The selectivity for R406 in inhibiting Syk is limited as it may inhibit additional kinases and non-kinase targets.…”

“…Administration of R406 reduced clinical arthritis in two antibody-induced arthritis models (K/BXN serum and collagen antibody). In addition, R406 suppressed bone erosions detected by radiography, pannus formation, and synovitis in these animal models [37]. It was also observed that the expression of Syk in synovial tissues correlated with the levels of inflammatory cell infiltrates in the joints and was virtually undetectable in R406-treated mice subjected to collagen-induced arthritis in rats [38].…”

“…In addition, Syk inhibition reduced synovial fluid cytokine levels and cartilage oligomeric matrix protein in serum in these animals [38]. R406 was also found to limit an Arthus reaction in mice [37] and rats [38] and reverse passive Arthus reaction in murine chimeras with Syk-deficient hematopoietic cells [43,44]. This effect is probably due to the suppression of immune-complex-mediated inflammation by inhibiting the Fc receptor signaling.…”