Neisseria Research Group, Molecular Microbiology, School of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, England, SO16 6YD.
Medical Biotechnology Laboratory, Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, North Campus, Delhi, 110007, India.
Biol Res. 2024 Sep 5;57(1):62. doi: 10.1186/s40659-024-00543-9.
Neisseria gonorrhoeae (Ng) causes the sexually transmitted disease gonorrhoea. There are no vaccines and infections are treated principally with antibiotics. However, gonococci rapidly develop resistance to every antibiotic class used and there is a need for developing new antimicrobial treatments. In this study we focused on two gonococcal enzymes as potential antimicrobial targets, namely the serine protease L,D-carboxypeptidase LdcA (NgO1274/NEIS1546) and the lytic transglycosylase LtgD (NgO0626/NEIS1212). To identify compounds that could interact with these enzymes as potential antimicrobials, we used the AtomNet virtual high-throughput screening technology. We then did a computational modelling study to examine the interactions of the most bioactive compounds with their target enzymes. The identified compounds were tested against gonococci to determine minimum inhibitory and bactericidal concentrations (MIC/MBC), specificity, and compound toxicity in vitro.
AtomNet identified 74 compounds that could potentially interact with Ng-LdcA and 84 compounds that could potentially interact with Ng-LtgD. Through MIC and MBC assays, we selected the three best performing compounds for both enzymes. Compound 16 was the most active against Ng-LdcA, with a MIC50 value < 1.56 µM and MBC50/90 values between 0.195 and 0.39 µM. In general, the Ng-LdcA compounds showed higher activity than the compounds directed against Ng-LtgD, of which compound 45 had MIC50 values of 1.56-3.125 µM and MBC50/90 values between 3.125 and 6.25 µM. The compounds were specific for gonococci and did not kill other bacteria. They were also non-toxic for human conjunctival epithelial cells as judged by a resazurin assay. To support our biological data, in-depth computational modelling study detailed the interactions of the compounds with their target enzymes. Protein models were generated in silico and validated, the active binding sites and amino acids involved elucidated, and the interactions of the compounds interacting with the enzymes visualised through molecular docking and Molecular Dynamics Simulations for 50 ns and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA).
We have identified bioactive compounds that appear to target the N. gonorrhoeae LdcA and LtgD enzymes. By using a reductionist approach involving biological and computational data, we propose that compound Ng-LdcA-16 and Ng-LtgD-45 are promising anti-gonococcal compounds for further development.
淋病奈瑟菌（Ng）引起性传播疾病淋病。目前尚无疫苗，感染主要用抗生素治疗。然而，淋病奈瑟菌对抗生素的每一类药物都迅速产生耐药性，因此需要开发新的抗菌治疗方法。在这项研究中，我们将两种淋病奈瑟菌酶作为潜在的抗菌靶点，即丝氨酸蛋白酶 L,D-羧肽酶 LdcA（NgO1274/NEIS1546）和溶菌转糖基酶 LtgD（NgO0626/NEIS1212）。为了鉴定可能与这些酶相互作用的化合物作为潜在的抗菌剂，我们使用了 AtomNet 虚拟高通量筛选技术。然后，我们进行了计算建模研究，以研究最具生物活性的化合物与靶酶的相互作用。鉴定的化合物在体外针对淋病奈瑟菌进行了最低抑菌浓度（MIC/MBC）、特异性和化合物毒性试验。
AtomNet 鉴定出 74 种可能与 Ng-LdcA 相互作用的化合物和 84 种可能与 Ng-LtgD 相互作用的化合物。通过 MIC 和 MBC 测定，我们选择了两种酶中表现最好的三种化合物。化合物 16 对 Ng-LdcA 的活性最强，MIC50 值<1.56 µM，MBC50/90 值在 0.195 和 0.39 µM 之间。一般来说，Ng-LdcA 化合物的活性高于针对 Ng-LtgD 的化合物，其中化合物 45 的 MIC50 值为 1.56-3.125 µM，MBC50/90 值在 3.125 和 6.25 µM 之间。这些化合物对淋病奈瑟菌具有特异性，不会杀死其他细菌。它们对人结膜上皮细胞也没有毒性，这可以通过 Resazurin 测定来判断。为了支持我们的生物学数据，深入的计算建模研究详细描述了化合物与靶酶的相互作用。通过从头计算生成了蛋白质模型，并对其进行了验证，阐明了参与的活性结合位点和氨基酸，并通过分子对接和分子动力学模拟（50 ns）和分子力学泊松-玻尔兹曼表面面积（MM-PBSA）可视化了化合物与酶的相互作用。
我们已经鉴定出了一些生物活性化合物，这些化合物似乎靶向淋病奈瑟菌的 LdcA 和 LtgD 酶。通过使用涉及生物学和计算数据的简化方法，我们提出化合物 Ng-LdcA-16 和 Ng-LtgD-45 是有前途的抗淋病奈瑟菌化合物，值得进一步开发。
