Vasu Dhananjayan, Do Ha T, Li Huiying, Hardy Christine D, Poulos Thomas L, Silverman Richard B
Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Developmental Therapeutics, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208-3113, United States.
Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine, CA 92697-3900, United States.
Bioorg Med Chem. 2025 Jul 1;124:118193. doi: 10.1016/j.bmc.2025.118193. Epub 2025 Apr 10.
Neuronal nitric oxide synthase (nNOS) is a promising target for addressing various neurological disorders and melanoma. Our discovery of a series of truncated pyridinylbenzylamines has yielded potent, selective, and membrane permeable inhibitors of human neuronal nitric oxide synthase. By implementing an efficient synthetic procedure using the Suzuki-Miyaura cross-coupling reaction, we were able to rapidly identify a potent inhibitor. This new inhibitor (18, 6-(2,3-difluoro-5-((methylamino)methyl)phenyl)-4-methylpyridin-2-amine dihydrochloride) exhibits excellent potency, with K values of 30 nM for human nNOS and 40 nM for rat nNOS. It also demonstrates high isoform selectivity, showing an 821-fold preference for human nNOS over human endothelial NOS (eNOS) and a 75-fold selectivity over human inducible NOS (iNOS). Additionally, inhibitor 18 displays high permeability (P = 10.7 × 10 cm s) in an artificial membrane permeability assay. The crystal structures of several NOS-inhibitor complexes provide valuable structural insights into the potency and selectivity of this series of novel inhibitors. A particularly notable finding is the unexpected role of a Cl anion bound to heNOS, which contributes to the high isoform selectivity of these inhibitors and explains why heNOS binds Cl, while hnNOS does not. This unique Cl binding site could be important in future inhibitor design, opening new avenues for the development of more selective NOS inhibitors. Additionally, the presented crystal structures reveal the key factors required to maintain both high potency and selectivity in the simplified inhibitors discussed in this study. Abbreviations: NO, nitric oxide; nNOS, neuronal nitric oxide synthase; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase; rnNOS, rat neuronal nitric oxide synthase; hnNOS, human neuronal nitric oxide synthase; hiNOS, human inducible nitric oxide synthase; heNOS, human endothelial nitric oxide synthase; l-Arg, l-arginine; NADPH, reduced nicotinamide adenine dinucleotide phosphate; CaM, calmodulin; HB, (6R)-5,6,7,8-tetrahydrobiopterin; FAD, flavin adenine dinucleotide; FMN, Flavin mononucleotide, BBB, blood-brain barrier; CNS, central nervous system; PAMPA, parallel artificial membrane permeability assay; P-gp, P-glycoprotein; ER, efflux ratio; P, effective permeability; P, apparent permeability; Caco-2, cancer coli-2; TLC, thin layer chromatography; TBAF, tetra-n-butylammonium fluoride; TFA, trifluoroacetic acid.
神经元型一氧化氮合酶（nNOS）是治疗各种神经系统疾病和黑色素瘤的一个有前景的靶点。我们发现的一系列截短的吡啶基苄胺产生了对人神经元型一氧化氮合酶具有强效、选择性和膜通透性的抑制剂。通过使用铃木-宫浦交叉偶联反应实施高效合成方法，我们能够快速鉴定出一种强效抑制剂。这种新抑制剂（18，6-(2,3-二氟-5-((甲氨基)甲基)phenyl)-4-甲基吡啶-2-胺二盐酸盐）表现出优异的效力，对人nNOS的K值为30 nM，对大鼠nNOS的K值为40 nM。它还表现出高同工型选择性，对人nNOS的偏好性比对人内皮型一氧化氮合酶（eNOS）高821倍，对人诱导型一氧化氮合酶（iNOS）的选择性高75倍。此外，抑制剂18在人工膜通透性测定中显示出高通透性（P = 10.7×10 cm s）。几种NOS-抑制剂复合物的晶体结构为这一系列新型抑制剂的效力和选择性提供了有价值的结构见解。一个特别值得注意的发现是与heNOS结合的Cl阴离子的意外作用，它有助于这些抑制剂的高同工型选择性，并解释了为什么heNOS结合Cl，而hnNOS不结合。这个独特的Cl结合位点在未来的抑制剂设计中可能很重要，为开发更具选择性的NOS抑制剂开辟了新途径。此外，所呈现的晶体结构揭示了在本研究中讨论的简化抑制剂中保持高效力和选择性所需的关键因素。缩写：NO，一氧化氮；nNOS，神经元型一氧化氮合酶；iNOS，诱导型一氧化氮合酶；eNOS，内皮型一氧化氮合酶；rnNOS，大鼠神经元型一氧化氮合酶；hnNOS，人神经元型一氧化氮合酶；hiNOS，人诱导型一氧化氮合酶；heNOS，人内皮型一氧化氮合酶；l-Arg，L-精氨酸；NADPH，还原型烟酰胺腺嘌呤二核苷酸磷酸；CaM，钙调蛋白；HB，(6R)-5,6,7,8-四氢生物蝶呤；FAD，黄素腺嘌呤二核苷酸；FMN，黄素单核苷酸；BBB，血脑屏障；CNS，中枢神经系统；PAMPA，并行人造膜通透性测定；P-gp，P-糖蛋白；ER，外排率；P，有效通透性；P，表观通透性；Caco-2，癌细胞系-2；TLC，薄层色谱法；TBAF，四正丁基氟化铵；TFA，三氟乙酸。
