The inhibitory potency and selectivity of arginine substrate site nitric-oxide synthase inhibitors is solely determined by their affinity toward the different isoenzymes

We have investigated various nitric oxide (NO) synthase inhibitors for thei r affinity and selectivity toward the three human isoenzymes in radioligand binding experiments. Therefore, we developed the new radioligand [H-3]2-am ino-4-picoline to measure binding of these compounds to the three human NO synthase (NOS) isoenzymes. Aminopicoline is a potent and nonselective inhib itor of all three isoforms. [H-3]2-amino-4-picoline bound saturably and wit h high affinity to human NOSs. Affinity constants (K-D values) of 59, 111, and 136 nM were obtained for the inducible, neuronal, and endothelial NOS i soforms (iNOS, nNOS, eNOS). Binding of [H-3]2-amino-4-picoline was competit ive with the substrate arginine. From all the inhibitors tested, AMT (2-ami no-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride) showed the highest a ffinity and no selectivity. L-NIL [L-N-6-(1-Iminoethyl) lysine hydrochlorid e] and aminoguanidine were moderately iNOS-selective while L-NA (N-G-nitro- L-arginine) and L-NAME (N-G-nitro-L-arginine methyl ester hydrochloride) sh owed selectivity toward the constitutive isoforms. High iNOS versus eNOS se lectivity was found for 1400W, whereas several isothiourea derivatives and 1400W displayed moderate n- versus eNOS selectivity. To relate the affinity of these compounds to their inhibitory potency, we measured the inhibitory potency under almost identical conditions using a new microtiter plate ass ay. The inhibitory potency of selective and nonselective NOS inhibitors was almost exactly mirrored by their affinity toward the different isoenzymes. Highly significant correlations were obtained between the potency of enzym e inhibition and the inhibition of [H-3]2-amino-4-picoline binding for all three isoenzymes. These data show that the potency and selectivity of NOS i nhibitors are solely determined by their affinity toward the different isof orms. Furthermore, these data identify the new radioligand [3H]2-amino-4-pi coline as a very useful radiolabel for the investigation of the substrate b inding site of all three isoforms.