Boer R, Ulrich W R, Klein T, Mirau B, Haas S, Baur I
Byk Gulden Pharmaceuticals, Konstanz, Germany.
Mol Pharmacol. 2000 Nov;58(5):1026-34.
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 [(3)H]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. [(3)H]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 [(3)H]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 while L-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 [(3)H]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 [(3)H]2-amino-4-picoline as a very useful radiolabel for the investigation of the substrate binding site of all three isoforms.
在放射性配体结合实验中，我们研究了多种一氧化氮（NO）合酶抑制剂对三种人类同工酶的亲和力和选择性。因此，我们开发了新的放射性配体[³H]2-氨基-4-甲基吡啶，以测定这些化合物与三种人类NO合酶（NOS）同工酶的结合情况。氨基甲基吡啶是所有三种同工型的强效非选择性抑制剂。[³H]2-氨基-4-甲基吡啶与人NOSs的结合具有饱和性且亲和力高。对于诱导型、神经元型和内皮型NOS同工酶（iNOS、nNOS、eNOS），获得的亲和常数（KD值）分别为59、111和136 nM。[³H]2-氨基-4-甲基吡啶的结合与底物精氨酸具有竞争性。在所有测试的抑制剂中，AMT（2-氨基-5,6-二氢-6-甲基-4H-1,3-噻嗪盐酸盐）表现出最高的亲和力且无选择性。L-NIL [L-N⁶-(1-亚氨基乙基)赖氨酸盐酸盐]和氨基胍对iNOS具有中等选择性，而L-NA（Nᴳ-硝基-L-精氨酸）和L-NAME（Nᴳ-硝基-L-精氨酸甲酯盐酸盐）对组成型同工型具有选择性。发现1400W对iNOS与eNOS具有高选择性，而几种异硫脲衍生物和1400W对nNOS与eNOS表现出中等选择性。为了将这些化合物的亲和力与其抑制效力相关联，我们使用一种新的微量滴定板测定法在几乎相同的条件下测量了抑制效力。选择性和非选择性NOS抑制剂的抑制效力几乎完全与其对不同同工酶的亲和力相对应。对于所有三种同工酶，酶抑制效力与[³H]2-氨基-4-甲基吡啶结合的抑制之间均获得了高度显著的相关性。这些数据表明，NOS抑制剂的效力和选择性完全由它们对不同同工型的亲和力决定。此外，这些数据表明新的放射性配体[³H]2-氨基-4-甲基吡啶是研究所有三种同工型底物结合位点的非常有用的放射性标记物。
