RN486 is an effective and specific BTK inhibitor (IC50: 4 nM).

BTK:4 nM

In the AIA model, RN486, either alone or in combination with methotrexate, effectively suppresses joint and systemic inflammation, reducing both paw swelling and the levels of inflammatory markers in the blood. Its functional activity in rodent models parallels these effects, efficiently preventing type I and III hypersensitivity reactions. Furthermore, RN486 demonstrates significant bone protection and anti-inflammatory properties in mouse CIA and rat adjuvant-induced arthritis models.

RN486 selectively inhibits Bruton’s tyrosine kinase and demonstrates functional activity across multiple human cell types in vitro. It blocks mast cell degranulation induced by Fcε receptor cross-linking (IC50: 2.9 nM), inhibits the production of tumor necrosis factor α in monocytes triggered by the engagement of Fcγ receptors (IC50: 7.0 nM), and reduces the expression of the activation marker CD69 in B cells following B cell antigen receptor engagement in whole blood (IC50: 21.0 nM).

Radiometric kinase assay: The enzymatic kinase activity is assessed by measuring the phosphorylation of a synthetic substrate by the purified GST-fusion FGFR3-K650E kinase domain, in the presence of radiolabeled ATP. Enzyme activities are measured by mixing 10 μL of a 3-fold concentrated BGJ398 solution or control with 10 μL of the corresponding substrate mixture (peptidic substrate, ATP and [γ33P]ATP). The reactions are initiated by addition of 10 μL of a 3-fold concentrated solution of the enzyme in assay buffer. The final concentrations of the assay components are as following: 10 ng of GST-FGFR3-K650E, 20 mM Tris-HCl, pH 7.5, 3 mM MnCl2, 3 mM MgCl2, 1 mM DTT, 250 μg/mL PEG 20000, 2 μg/mL poly(EY) 4:1, 1% DMSO and 0.5 μM ATP (γ-[33P]-ATP 0.1 μCi). The assay is carried out according to the filter binding (FB) method in 96-well plates at room temperature for 10 minutes in a final volume of 30 μL including BGJ398. The enzymatic reactions are stopped by the addition of 20 μL of 125 mM EDTA, and the incorporation of 33P into the polypeptidic substrates is quantified as following: 30 μL of the stopped reaction mixture are transferred onto Immobilon-PVDF membranes previously soaked for 5 minutes with methanol, rinsed with water, soaked for 5 min with 0.5% H3PO4, and mounted on vacuum manifold with disconnected vacuum source. After spotting, vacuum is connected, and each well rinsed with 0.5% H3PO4 (200 μL). Free membranes are removed and ished four times on a shaker with 1% H3PO4 and once with ethanol. Membranes are dried and overlaid with addition of 10 μL/well of a scintillation fluid. The plates are eventually sealed and counted in a microplate scintillation counter. IC50 values are calculated by linear regression analysis of the percentage inhibition of the BGJ398.