Department of Chemistry, University of Massachusetts , 104 LGRT, 710 N. Pleasant St., Amherst, Massachussetts 01003, United States.
ACS Chem Biol. 2013 Dec 20;8(12):2744-52. doi: 10.1021/cb400612h. Epub 2013 Oct 28.
Dengue virus is the flavivirus that causes dengue fever, dengue hemorrhagic disease, and dengue shock syndrome, which are currently increasing in incidence worldwide. Dengue virus protease (NS2B-NS3pro) is essential for dengue virus infection and is thus a target of therapeutic interest. To date, attention has focused on developing active-site inhibitors of NS2B-NS3pro. The flat and charged nature of the NS2B-NS3pro active site may contribute to difficulties in developing inhibitors and suggests that a strategy of identifying allosteric sites may be useful. We report an approach that allowed us to scan the NS2B-NS3pro surface by cysteine mutagenesis and use cysteine reactive probes to identify regions of the protein that are susceptible to allosteric inhibition. This method identified a new allosteric site utilizing a circumscribed panel of just eight cysteine variants and only five cysteine reactive probes. The allosterically sensitive site is centered at Ala125, between the 120s loop and the 150s loop. The crystal structures of WT and modified NS2B-NS3pro demonstrate that the 120s loop is flexible. Our work suggests that binding at this site prevents a conformational rearrangement of the NS2B region of the protein, which is required for activation. Preventing this movement locks the protein into the open, inactive conformation, suggesting that this site may be useful in the future development of therapeutic allosteric inhibitors.
登革热病毒是引起登革热、登革出血热和登革休克综合征的黄病毒，目前在全球的发病率正在上升。登革热病毒蛋白酶（NS2B-NS3pro）是登革热病毒感染所必需的，因此是治疗的靶点。迄今为止，人们一直关注开发 NS2B-NS3pro 的活性位点抑制剂。NS2B-NS3pro 活性位点的平坦和带电性质可能导致开发抑制剂的困难，并表明识别变构位点的策略可能是有用的。我们报告了一种方法，该方法允许我们通过半胱氨酸诱变扫描 NS2B-NS3pro 表面，并使用半胱氨酸反应性探针来鉴定该蛋白质易受变构抑制的区域。该方法仅使用了 8 种半胱氨酸变体和 5 种半胱氨酸反应性探针就鉴定出了一个新的变构位点。变构敏感位点位于 Ala125 处，在 120s 环和 150s 环之间。WT 和修饰的 NS2B-NS3pro 的晶体结构表明，120s 环是灵活的。我们的工作表明，该位点的结合阻止了蛋白质 NS2B 区域的构象重排，这是激活所必需的。阻止这种运动将蛋白质锁定在开放的非活性构象中，这表明该位点可能在未来开发治疗性变构抑制剂方面具有用途。
