West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3^pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3^pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3^pro, we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3^pro. Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC₅₀ ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3^pro. To identify the matching protein binding site, chemical shift perturbation studies employing ¹H,¹⁵N-TROSY-HSQC experiments with uniformly ²H,¹⁵N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3^pro inhibitors.

西尼罗河病毒（WNV）属于属黄病毒家族的黄病毒科。在过去的二十年中，这种对人类具有高度致病性的蚊媒病毒已演变成全球性威胁。尽管付出了许多努力，但是抗病毒药和疫苗都没有。病毒蛋白酶NS2B-NS3 ^pro对于病毒复制至关重要，因此被视为主要药物靶标。但是，到目前为止，开发特定的NS2B-NS3 ^pro抑制剂的成功中等。在寻找对NS2B-NS3 ^pro具有结合亲和力的新结构基序，我们使用饱和转移差异（STD）NMR实验作为读数筛选了片段库Maybridge Ro5库。429个片段中约有30％显示出与NS2B-NS3 ^pro的结合。随后的使用已知的活性位点片段A作为报道分子配体的STD-NMR竞争实验产生14个竞争结合片段，以及22个未与A竞争的片段。在基于荧光团的蛋白酶测定中，所有这些片段均显示出在微摩尔范围内的抑制作用。有趣的是，在化合物A的存在下，这22个片段中的10个显示STD强度显着增加，表明协同结合。最有前途的非竞争性抑制剂1和2（IC ₅₀〜500μM）的份额，其可以引导新颖第二现场发展的结构基序NS2B-NS3的（潜在变构）抑制剂^亲。为了鉴定匹配的蛋白质结合位点，在1存在下以及在不存在A的情况下，采用¹ H，¹⁵ N-TROSY-HSQC实验和均一的² H，¹⁵ N标记的蛋白酶进行化学位移扰动研究。数据表明1与NS2B的Met 52 *相互作用，鉴定出与A结合位点相邻的第二位点。因此，我们的研究为新型双齿NS2B-NS3 ^pro抑制剂的合成铺平了道路。