RT Journal Article SR Electronic T1 The Inhibitory Potency and Selectivity of Arginine Substrate Site Nitric-Oxide Synthase Inhibitors Is Solely Determined by Their Affinity toward the Different Isoenzymes JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 1026 OP 1034 DO 10.1124/mol.58.5.1026 VO 58 IS 5 A1 Rainer Boer A1 Wolf-RĂ¼diger Ulrich A1 Thomas Klein A1 Berit Mirau A1 Sabine Haas A1 Ilka Baur YR 2000 UL http://molpharm.aspetjournals.org/content/58/5/1026.abstract AB We have investigated various nitric oxide (NO) synthase inhibitors for their affinity and selectivity toward the three human isoenzymes in radioligand binding experiments. Therefore, we developed the new radioligand [3H]2-amino-4-picoline to measure binding of these compounds to the three human NO synthase (NOS) isoenzymes. Aminopicoline is a potent and nonselective inhibitor of all three isoforms. [3H]2-amino-4-picoline bound saturably and with 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 isoforms (iNOS, nNOS, eNOS). Binding of [3H]2-amino-4-picoline was competitive with the substrate arginine. From all the inhibitors tested, AMT (2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochloride) showed the highest affinity and no selectivity.l-NIL [l-N 6-(1-Iminoethyl)lysine hydrochloride] and aminoguanidine were moderately iNOS-selective whilel-NA ( N G-nitro-l-arginine) and l-NAME(N G-nitro-l-arginine methyl ester hydrochloride) showed selectivity toward the constitutive isoforms. High iNOS versus eNOS selectivity 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 assay. 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 enzyme inhibition and the inhibition of [3H]2-amino-4-picoline binding for all three isoenzymes. These data show that the potency and selectivity of NOS inhibitors are solely determined by their affinity toward the different isoforms. Furthermore, these data identify the new radioligand [3H]2-amino-4-picoline as a very useful radiolabel for the investigation of the substrate binding site of all three isoforms.