Mishra Arjun K, Singh Nidhi, Agnihotri Pragati, Mishra Shikha, Singh Saurabh P, Kolli Bala K, Chang Kwang Poo, Sahasrabuddhe Amogh A, Siddiqi M I, Pratap J Venkatesh
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, B.S. 10/1, sector 10, Jankipuram Extension, Sitapur Road, Lucknow, Uttar Pradesh, 226031, India.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO, 63110, USA.
J Comput Aided Mol Des. 2017 Jun;31(6):547-562. doi: 10.1007/s10822-017-0022-9. Epub 2017 May 27.
Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl. It belongs to the hexagonal space group P622 with unit cell parameters a = b = 115.18, c = 62.18 Å and α = β = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and R values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation-based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed ~45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.
核苷二磷酸激酶（NDK）是一种普遍存在的酶，可催化γ-磷酸基团从NTP供体转移至NDP受体，这对于维持细胞内三磷酸核苷（NTP）水平至关重要。锥虫无法从头合成嘌呤且依赖补救途径，这使得NDK成为开发针对其所致疾病药物的一个有吸引力的靶点。在此，我们基于对来自亚马逊利什曼原虫的纯化重组NDK的结构和功能表征，报告了新型利什曼原虫NDK抑制剂的发现。重组LaNDK具有自磷酸化、磷酸转移酶和激酶活性，其中组氨酸117发挥着关键作用。LaNDK晶体通过悬滴气相扩散法在含有18%聚乙二醇单甲醚500、100 mM双三羟甲基氨基甲烷丙烷pH 6.0和50 mM氯化镁的溶液中生长。它属于六方空间群P622，晶胞参数为a = b = 115.18，c = 62.18 Å，α = β = 90°，γ = 120°。通过分子置换法解析的结构分别精修至晶体学R因子和R值为22.54%和26.52%。基于分子对接和动力学模拟的虚拟筛选确定了推定的结合化合物。对选定命中化合物的蛋白质抑制研究确定了五种在微摩尔浓度下有效的抑制剂。其中一种化合物对利什曼原虫前鞭毛体增殖显示出约45%的抑制作用。抑制剂-NDK复合物的分析揭示了它们的结合模式，有助于设计新化合物以优化作为抗利什曼病药物的活性。
