In vitro activity:1400W is either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS. Inhibition of human iNOS by 1400W is time-dependent. 1400W is competitive with L-arginine.

Kinase Assay: 1400W is a slow, tight binding, potent, and highly selective inhibitor of inducible nitric-oxide synthase (iNOS) with Ki values of 7 nM. The slow onset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s-1 and a binding constant of 2.0 microM. Inhibition was dependent on the cofactor NADPH. L-Arginine was a competitive inhibitor of 1400W binding with a Ks value of 3.0 microM. Inhibited enzyme did not recover activity after 2 h. Thus, 1400W was either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS with a Kd value

Cell Assay: RAW264.7 cells are treated with LPS/IFNγ for 16 h and GAPDH-p300 binding is abolished by the iNOS inhibitor 1400W (100 μM). Cell lysates are immunoprecipitated with an anti-p300 antibody and the immunoprecipitates are analysed by western blotting with an anti-GAPDH antibody.