MOLECULAR MECHANISMS OF INHIBITION OF PORCINE BRAIN NITRIC-OXIDE SYNTHASE BY THE ANTINOCICEPTIVE DRUG 7-NITRO-INDAZOLE
MOLECULAR MECHANISMS OF INHIBITION OF PORCINE BRAIN NITRIC-OXIDE SYNTHASE BY THE ANTINOCICEPTIVE DRUG 7-NITRO-INDAZOLE
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DOI：
10.1016/0028-3908(94)90024-8
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发表时间：
1994-11-01
影响因子：
4.7
通讯作者：
SCHMIDT, K
中科院分区：
文献类型：
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作者：
MAYER, B;KLATT, P;SCHMIDT, K
7-硝基-吲唑(7-NI)是一种新型的一氧化氮合酶(NOS)抑制剂，在体内对神经元同工酶具有选择性[Moore等人]。Br.J·法马克。110、219-224(1993)]。在本研究中，我们用纯化的猪脑一氧化氮合酶来研究7-NI抑制酶的分子机制。该药与L精氨酸具有竞争性，动力学K~(-1)为2.8mU~(-1)，V-max略有降低。作为一种细胞色素P-450，在没有L-精氨酸的情况下，一氧化氮合酶催化血红素介导的分子氧还原，导致过氧化氢的形成。7-NI对H_2O_2的形成有很强的抑制作用(IC_(50)=0.28+/-0.096µM)，但不影响黄素介导的电子传递。因此，7-NT类似于咪唑，一种已知的一氧化氮合酶的血红素位点抑制剂。我们发现咪唑是L-瓜氨酸形成的纯竞争性抑制剂(K-1=263mU M)，并在相似浓度下抑制H_2O_2的形成(IC_(50)=280+/-38mU M)。根据它们在瓜氨酸分析中的L-精氨酸竞争效应，这两种药物都拮抗放射性标记的N-G-硝基-L-精氨酸(L-NNA)的结合，后者是一种用于可逆标记一氧化氮合酶底物位置的高亲和力探针[Kiatt等，J.Biol]。化学。269、14781-14787(1994年)]。7-NI和咪唑的K-1分别为0.09+/-0.024亩M和200+/-63亩M。最后，功能未知的核素标记四氢生物蝶呤的结合也被7-NI拮抗，K-1为0.12+/-0.023 mU，但咪唑是唯一弱的蝶啶结合拮抗剂(K-1类似于50 mM)。这些数据表明，7-NI和咪唑都以L-精氨酸竞争的方式与一氧化氮合酶的人工血红素基团结合，并且7-NI的结合还影响酶的蝶啶位置。
7-Nitro-indazole (7-NI) has been described as novel nitric oxide synthase (NOS) inhibitor with in vivo selectivity for the neuronal isozyme [Moore et al. Br. J. Pharmac. 110, 219-224 (1993)]. In the present study we have used purified porcine brain NOS to invesigate the molecular mechanisms of enzyme inhibition by 7-NI. The drug was competitive with L-arginine, exhibited a kinetic K-1 of 2.8 mu M, and additionally induced a slight reduction in V-max. As a cytochrome P-450, NOS catalyzes a heme-mediated reduction of molecular oxygen, resulting in the formation of H2O2 in the absence of L-arginine. 7-NI turned out as a potent inhibitor of H2O2 formation (IC50 = 0.28 +/- 0.096 mu M) but did not affect flavin-mediated electron transfer. Thus, 7-NT resembled imidazole, a known heme-site inhibitor of NOS. We found that imidazole was a purely competitive inhibitor of L-citrulline formation (K-1 = 263 mu M) and blocked H2O2 formation at similar concentrations (IC50 = 280 +/- 38 mu M). In accordance with their L-arginine-competitive effects in the citrulline assay, both drugs antagonized binding of radiolabeled N-G-nitro-L-arginine (L-NNA), a high affinity probe for reversible labelling of the substrate site of NOS [Kiatt et al., J. Biol. Chem. 269, 14781-14787 (1994)]. The calculated K-1 values for 7-NI and imidazole were 0.09 +/- 0.024 mu M and 200 +/- 63 mu M, respectively. Finally, binding of radiolabelled tetrahydrobiopterin, a NOS cofactor with unknown function, was also antagonized by 7-NI with a K-1 of 0.12 +/- 0.023 mu M. Imidazole, however, was an only weak antagonist of pteridine binding (K-1 similar to 50 mM). These data indicate that both 7-NI and imidazole bind in an L-arginine-competitive manner to the prosthetic heme group of NOS, and that binding of 7-NI additionally affects the pteridine site of the enzyme.