Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Centre National de la Recherche Scientifique UMR8601, Université Paris Descartes, 75270 Paris, France.
Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12526-31. doi: 10.1073/pnas.1205645109. Epub 2012 Jul 16.
We report the structure-based design and synthesis of a unique NOS inhibitor, called nanoshutter NS1, with two-photon absorption properties. NS1 targets the NADPH site of NOS by a nucleotide moiety mimicking NADPH linked to a conjugated push-pull chromophore with nonlinear absorption properties. Because NS1 could not provide reducing equivalents to the protein and competed with NADPH binding, it efficiently inhibited NOS catalysis. NS1 became fluorescent once bound to NOS with an excellent signal-to-noise ratio because of two-photon excitation avoiding interference from the flavin-autofluorescence and because free NS1 was not fluorescent in aqueous solutions. NS1 fluorescence enhancement was selective for constitutive NOS in vitro, in particular for endothelial NOS (eNOS). Molecular dynamics simulations suggested that two variable residues among NOS isoforms induced differences in binding of NS1 and in local solvation around NS1 nitro group, consistent with changes of NS1 fluorescence yield. NS1 colocalized with eNOS in living human umbilical vein endothelial cells. Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800-950-nm range, with great perspectives for eNOS imaging in living tissues.
我们报告了一种基于结构设计和合成的独特的 NOS 抑制剂，称为纳米快门 NS1，它具有双光子吸收特性。NS1 通过模拟 NADPH 的核苷酸部分靶向 NOS 的 NADPH 结合部位，并与具有非线性吸收特性的共轭推挽发色团相连。由于 NS1 不能向蛋白质提供还原当量，并与 NADPH 结合竞争，因此它有效地抑制了 NOS 催化作用。NS1 与 NOS 结合后会发出荧光，因为它具有良好的信噪比，是通过双光子激发产生的，从而避免了黄素自发荧光的干扰，并且游离的 NS1 在水溶液中没有荧光。NS1 在体外对组成型 NOS，特别是内皮型 NOS (eNOS) 的荧光增强具有选择性。分子动力学模拟表明，NOS 同工型中的两个可变残基诱导 NS1 的结合和 NS1 硝基基团周围局部溶剂化的差异，这与 NS1 荧光产率的变化一致。NS1 在人脐静脉内皮细胞中与 eNOS 共定位。因此，NS1 构成了一类独特的 eNOS 探针，具有 800-950nm 范围内的双光子激发，为活体组织中的 eNOS 成像提供了广阔的前景。
