TPCA-1

2 years -80 in solvent

Administered via i.p. or b.i.d.

U87, MT330, SJ-G2, and GBM6 human glioma lines

3, 10, or 20 mg/kg

17.9 nM [1], 17.9 nM [1], 17.9 nM [1], 17.9 nM [1], 17.9 nM [1], 17.9 nM [1]

Prophylactic administration of TPCA-1 at 3, 10, or 20 mg/kg, i.p., b.i.d., results in a dose-dependent reduction in the severity of murine collagen-induced arthritis (CIA). The significantly reduced disease severity and delay of disease onset resulting from administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. are comparable to the effects of the antirheumatic drug, etanercept, when administered prophylactically at 4 mg/kg, i.p., every other day. Nuclear localization of p65, as well as levels of IL-1beta, IL-6, TNF-alpha, and interferon-gamma, is significantly reduced in the paw tissue of TPCA-1- and etanercept-treated mice. In addition, administration of TPCA-1 in vivo results in significantly decreased collagen-induced T cell proliferation ex vivo. Therapeutic administration of TPCA-1 at 20 mg/kg, but not at 3 or 10 mg/kg, i.p., b.i.d., significantly reduces the severity of CIA, as does etanercept administration at 12.5 mg/kg, i.p., every other day. [1]

IKK-2 Assay, Recombinant human IKK-2 (residues 1-756) is expressed in baculovirus as an N-terminal GST-tagged fusion protein, and its activity is assessed using a time-resolved fluorescence resonance energy transfer assay. In brief, IKK-2 (5 nM final) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM CHAPS, pH 7.4, with 1 mM DTT and 0.01% w/v BSA) is added to wells containing various concentrations of compound or dimethyl sulfoxide (DMSO) vehicle (3% final). The reaction is initiated by the addition of GST-IkappaBalpha substrate (25 nM final)/ATP (1 uM final), in a total volume of 30 uL. The reaction is incubated for 30 min at room temperature, then terminated by the addition of 15 uL of 50 mM EDTA. Detection reagent (15 uL) in buffer (100 mM HEPES, pH 7.4, 150 mM NaCl, and 0.1% w/v BSA) containing antiphosphoserine- IkappaBalpha-32/36 monoclonal antibody 12C2, labeled with W-1024 europium chelate, and an allophycocyanin-labeled anti-GST antibody is added, and the reaction is further incubated for 60 min at room temperature. The degree of phosphorylation of GST- IkappaBalphais measured as a ratio of specific 665-nm energy transfer signal to reference europium 620-nm signal, using a Packard Discovery plate reader.

DMSO 56 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL

3-Thiophenecarboxamide, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-