Selective inhibition of spleen tyrosine kinase (SYK) with a novel orally bioavailable small molecule inhibitor, RO9021, impinges on various innate and adaptive immune responses: implications for SYK inhibitors in autoimmune disease therapy

Abstract

Introduction: Spleen tyrosine kinase (SYK) is a key integrator of intracellular signals triggered by activated immunoreceptors, including Bcell receptors (BCR) and Fc receptors, which are important for the development and function of lymphoid cells. Given the clinical efficacy of Bcell depletion in the treatment of rheumatoid arthritis and multiple sclerosis, pharmacological modulation of Bcells using orally active small molecules that selectively target SYK presents an attractive alternative therapeutic strategy.

Methods: A SYK inhibitor was developed and assayed in various in vitro systems and in the mouse model of collagen-induced arthritis (mCIA).

Results: A novel ATP-competitive inhibitor of SYK, 6-[(1R,2S)-2-Amino-cyclohexylamino]-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylic acid amide, designated RO9021, with an adequate kinase selectivity profile and oral bioavailability, was developed. In addition to suppression of BCR signaling in human peripheral blood mononuclear cells (PBMC) and whole blood, FcγR signaling in human monocytes, and FcϵR signaling in human mast cells, RO9021 blocked osteoclastogenesis from mouse bone marrow macrophages in vitro. Interestingly, Toll-like Receptor (TLR) 9 signaling in human Bcells was inhibited by RO9021, resulting in decreased levels of plasmablasts, immunoglobulin (Ig) M and IgG upon B-cell differentiation. RO9021 also potently inhibited type I interferon production by human plasmacytoid dendritic cells (pDC) upon TLR9 activation. This effect is specific to TLR9 as RO9021 did not inhibit TLR4- or JAK-STAT-mediated signaling. Finally, oral administration of RO9021 inhibited arthritis progression in the mCIA model, with observable pharmacokinetics (PK)-pharmacodynamic (PD) correlation.

Conclusions: Inhibition of SYK kinase activity impinges on various innate and adaptive immune responses. RO9021 could serve as a starting point for the development of selective SYK inhibitors for the treatment of inflammation-related and autoimmune-related disorders.

Figure 1

Structure, potency and selectivity of a novel spleen tyrosine kinase inhibitor, RO9021. (A) Compound structure of RO9021, 6-((1R,2S)-2-amino-cyclohexylamino)-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylic acid amide. (B) Inhibition of spleen tyrosine kinase (SYK) enzymatic activity, measured by incorporation of ³³P-ATP into SYK substrate peptide. The half-maximal inhibitory concentration (IC₅₀) is reported as the average value of three independent assays. (C) Kinome selectivity of RO9021. RO9021 was profiled against 392 nonmutant kinases by KinomeScan and presented as a kinome dendrogram. Circle size is proportional to percentage inhibition at the test concentration (1 μM): largest circle, 99% inhibition; medium circle, 90 to 99% inhibition; smallest circles, 51 to 90% inhibition. Arrow, SYK kinase (blue circle). (D) Selectivity score of RO9021. The selectivity score is a quantitative measure of compound selectivity, calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants. (E) Structural basis of RO9021 selectivity. Crystal structure of RO9021 bound to SYK. Orange dotted lines, possible hydrophobic interactions between RO9021 and the Pro455/Gly454 region (surface shaded red).

Figure 2

Inhibition of B-cell receptor and Fc Receptor pathways by RO9021. (A) RO9021 inhibited phosphorylation of PLCγ2(Y1217), BTK(Y223), AKT(S476) and ERK(p42/44) (T202/Y204) in anti-IgM stimulated Ramos cells. The levels of total Bruton’s tyrosine kinase (BTK) and β-tubulin were used as loading controls. (B) RO9021 suppressed anti-IgM mediated calcium flux in Ramos cells (Ramos-BCR, squares), but not anti-CD3-mediated calcium flux in Jurkat cells (Jurkat-TCR, triangles). Intracellular calcium concentrations were measured using a calcium-sensitive fluorescent dye and presented as calculated percentage inhibition. (C), (D) Inhibition of anti-IgM-induced CD69 expression in human peripheral blood mononuclear cells (PBMCs) (C) and whole blood (D). Data were in duplicate and are shown as mean ± standard deviation (SD). (E) RO9021 inhibited FcγR signaling (human IgG-coated beads, dots), but not toll-like receptor-4 (TLR4)-induced TNFα production (lipopolysaccharide (LPS), triangles) in human monocytes. Percentage of inhibition by compound presented. Data were in quadruplicate and are shown as mean ± SD. (F) Inhibition of histamine release in human mast cells induced by anti-4-hydroxy-3-nitrophenylacetyl hapten (NP) IgE and NP. Anti-NP IgE and NP cross-linking-induced a robust histamine release (cells alone, triangle; IgEx, inverted triangle). RO9021 attenuated IgE-NP-induced histamine release in a concentration-dependent manner (circle). Data were in triplicate and are shown as mean ± SD.

Figure 3

Marginal effect of RO9021 in the JAK–STAT pathway. Human peripheral blood mononuclear cells were pretreated with RO9021 or the janus kinase (JAK) inhibitor tofacitinib and then stimulated with IL-2 to induce STAT5 phosphorylation in CD3⁺ T cells (A) or IFNγ to induce STAT1 phosphorylation in CD14⁺ monocytes/macrophages (B). Left panels, representative overlaid histograms of flow cytometric analysis. Filled gray, unstimulated; filled red, stimulated with IL-2 or IFNγ. Blue line, treatment with 1 μM RO9021; green line, treatment with 1 μM JAK inhibitor (tofacitinib). Right panels: concentration-dependent inhibition curves of RO9021 (dots) and tofacitinib (squares).

Figure 4

Dose-dependent inhibitory effects of RO9021 on osteoclastogenesis. (A) Representative images of tartrate-resistant acid phosphate (TRAP) staining showing receptor activator of nuclear factor kappa-B ligand (RANK-L) and monocyte colony-stimulating factor (M-CSF) mediated osteoclastogenesis from mouse bone marrow macrophages (BMM). Osteoclasts were shown as TRAP⁺ multinuclear cells. Negative and positive controls labeled as (–) RANK-L (upper left) and (+) RANK-L (upper right), respectively. The concentration of RO9021 was labeled on the upper right corner of each image. (B) TRAP⁺ multinuclear osteoclasts were counted under a microscope and percentage of inhibition on osteoclastogenesis was presented. Results were in triplicate and are plotted as mean ± standard deviation. IC₅₀, half-maximal inhibitory concentration.

Figure 5

Inhibition of Toll-like receptor-9 pathway in human B cells and plasmacytoid dendritic cells by RO9021. (A), (B) Human B cells were stimulated with CpG-B (ODN2006) and IFNα for 2 days. Production of IL-6 (A) and B-cell proliferation (B) was blocked by RO9021. The proliferation was presented by total alive cells, which were quantitated by Celltiter Glow as relative luminescent units. (C) RO9021 inhibited human plasmablast differentiation. Human B cells were differentiated with ODN2006 and IL-2 for 6 days. The plasmablasts were identified as CD19⁺CD38⁺CD20^– cells and presented as the percentage in total CD19⁺ B cells. The levels of IgM, IgG and IL-6 in the supernatants were also blocked by RO9021. (D), (E) RO9021 blocked Toll-like receptor 9 (TLR9)-mediated cytokine production in human plasmacytoid dendritic cells (pDCs). Purified pDC cells were stimulated with CpG-A (ODN2216) for 2 days. The production of IFNα (D) and TNFα (E) in supernatant were determined. Data were in duplicate and are shown as mean ± standard deviation. Statistical analysis of each treatment compared with the positive group was performed by Student’s t test. *P <0.05; **P <0.01; ***P <0.001; ns, not statistically significant. Plots shown are representative of four independent experiments tested in different donors. IC₅₀, half-maximal inhibitory concentration.

Figure 6

Oral administration of RO9021 abrogated collagen-induced arthritis in mice. (A) Dose-dependent attenuation of clinical scores by RO9021 (n = 14/group). ***P <0.001 compared with vehicle-treated group. po, per orally. (B) Representative toluidine blue-stained paraffin section of hind paws of vehicle (left upper), 5 mg/kg RO9021 (left bottom) and 45 mg/kg RO9021 (right bottom) treated mice. Photomicrograph of naïve mice also shown (right upper). Intense blue staining within joint space (arrows) indicated representative affected joints with inflammation and pannus formation. (C) Histopathological quantitation of inflammation, pannus formation, cartilage damage, and bone resorption in the hind paws of a subgroup of mice (n = 15/group). *P <0.05.(D) Serum level of IL-6 and KC(CXCL1) after 14 days of treatment with vehicle or RO9021. ***P <0.001 compared with vehicle-treated group. (E) Inhibition of anti-IgD induced CD69 expression in ex vivo whole blood assay at 2 and 5 hours post dose, but not at 24 hours post dose. CD69 expression in B220⁺ cells was induced ex vivo with anti-IgD in terminal whole blood from subgroups of mice. CD69 expression in unstimulated blood (inverted triangle) was also examined and used as baseline control (n = 5/group).