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Research Area
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Description
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Cancer |
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Biological Activity
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Description
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R406 is a potent spleen tyrosine kinase (Syk) inhibitor with EC50 and Ki of 56 nM and 30 nM, respectively. |
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Targets
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Syk |
Syk |
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IC50 |
56 nM (EC50) |
30 nM (Ki) [1] |
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In Vitro
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R406 is a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling. R406 inhibits the anti-IgE-induced production and release of LTC4 and cytokines and chemokines, including TNFα, IL-8, and GM-CSF. R406 inhibits phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 binds to the ATP binding pocket of Syk and inhibits its kinase activity as an ATP-competitive inhibitor with Ki of 30 nM. R406 blocks Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and Bcr-mediated activation of B lymphocytes. [1] R406 significantly induces chronic lymphocytic leukemia (CLL) cell apoptosis in nurselike cells cocultures and blocks CCL3 and CCL4 secretion by CLL cells in response to B-cell antigen receptor (Bcr) triggering. [2] R406 is a potent inhibitor of platelet signaling and functions initiated by FcγRIIA cross-linking by specific antibodies or by sera from HIT patients. [3] |
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In Vivo
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R406 reduces cutaneous reverse passive Arthus reaction by approximately 86% at 5 mg/kg in prophylactic treated mice. R406 also shows efficacy in inhibiting paw inflammation in antibody-induced arthritis mouse models. [1] R406 does not adversely affect macrophage or neutrophil function in innate immune responses and has minimal functional immunotoxicity notwithstanding its lymphocytopenic effect. [4] |
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Clinical Trials
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R406’s prodrug, Fostamatinib, is completed in Phase I clinical trials in subjects with rheumatoid arthritis or renal impairment. |
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Features
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Lead drug candidate for rheumatoid arthritis |
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Protocol
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Kinase Assay
[1]
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| In Vitro Fluorescence Polarization Kinase Assay |
R406 is serially diluted in DMSO and then diluted to 1% DMSO in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated BGG). ATP and substrate in kinase buffer are added at room temperature, resulting in a final DMSO concentration on 0.2%. The kinase reactions are performed in a final volume of 20?mL containing 5?mM HS1 peptide substrate, 4?mM ATP and started by addition of 0.125 ng of Syk in kinase buffer. The reaction is allowed to proceed for 40 minutes at room temperature. The reaction is stopped by the addition of 20?mL of PTK quench mix containing EDTA/anti-phosphotyrosine antibody (1X final)/fluorescent phosphopeptide tracer (0.5X final) diluted in FP Dilution Buffer. The plate is incubated for 30 minutes in the dark at room temperature and then read on a Polarion fluorescence polarization plate reader. Data are converted to amount of phosphopeptide present using a calibration curve generated by competition with the phosphopeptide competitor provided in the Tyrosine Kinase Assay Kit. For IC50 determination, R406 is tested at eleven concentrations and curve-fitting is performed by non-linear regression analysis. |
| Ki Determination |
For Ki determination, duplicate 200-μL reactions are set up at eight different ATP concentrations from 200 μM (2-fold serial dilutions) in the presence of either DMSO or R406 at 125, 62.5, 31.25, 15.5, or 7.8 nM. At different time points, 20 μL of each reaction is removed and quenched to stop the reaction. For each concentration of R406, the rate of reaction at each concentration of ATP is determined and plotted against the ATP concentration to determine the apparent Km and Vmax (maximal rate). Finally the apparent Km (or apparent Km/Vmax) is plotted against the inhibitor concentration to determine the Ki. |
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Animal Study
[1]
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| Animal Models |
Arthritis is induced in C57BL/6 mice by intraperitoneal injection of 150 μL of pooled sera from adult K/BxN mice. |
| Formulation |
35% TPGS, 60% PEG 400, and 5% propylene glycol |
| Doses |
1 or 5 mg/kg |
| Administration |
Administered orally |
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References |
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[1] Braselmann S, et al. J Pharmacol Exp Ther, 2006, 319(3), 998-1008.
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[2] Quiroga MP, et al. Blood, 2009, 114(5), 1029-1037.
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[3] Lhermusier T, et al. J Thromb Haemost, 2011, 9(10), 2067-2076.
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[4] Zhu Y, et al. Toxicol Appl Pharmacol, 2007, 221(3), 268-277.
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