Kythreoti Georgia, Thireou Trias, Karoussiotis Christos, Georgoussi Zafiroula, Liggri Panagiota G V, Papachristos Dimitrios P, Michaelakis Antonios, Karras Vasileios, Zographos Spyros E, Schulz Stefan, Iatrou Kostas
National Centre for Scientific Research "Demokritos", Institute of Biosciences and Applications, Athens, Greece.
Department of Biotechnology, Agricultural University of Athens, Athens, Greece.
J Biol Chem. 2024 Dec;300(12):107939. doi: 10.1016/j.jbc.2024.107939. Epub 2024 Oct 29.
Insect olfactory receptors are heteromeric ligand-gated cation channels composed of an obligatory receptor subunit, ORco, and one of many variable subunits, ORx, in as yet undefined molar ratios. When expressed alone ex vivo, ORco forms homotetrameric channels gated by ORco-specific ligands acting as channel agonists. Using an insect cell-based system as a functional platform for expressing mosquito odorant receptors ex vivo, we identified small molecules of natural origin acting as specific ORco channel antagonists, orthosteric or allosteric relative to a postulated ORco agonist binding site, which cause severe inhibition of olfactory function in mosquitoes. In the present communication, we have compiled common structural features of such orthosteric antagonists and developed a ligand-based pharmacophore whose properties are deemed necessary for binding to the agonist binding site and causing inhibition of ORco's biological function. In silico screening of an available collection of natural volatile compounds with the pharmacophore resulted in identification of several ORco antagonist hits. Cell-based functional screening of the same compound collection resulted in the identification of several compounds acting as orthosteric and allosteric antagonists of ORco channel function ex vivo and inducing anosmic behaviors to Aedes albopictus mosquitoes in vivo. Comparison of the in silico screening results with those of the functional assays revealed that the pharmacophore predicted correctly seven out of the eight confirmed orthosteric antagonists and none of the allosteric ones. Because the pharmacophore screen produced additional hits that did not cause inhibition of the ORco channel function, we also generated a support vector machine (SVM) model based on two descriptors of all pharmacophore hits. Training of the SVM on the ex vivo validated compound collection resulted in the selection of the confirmed orthosteric antagonists with a very low cross-validation out-of-sample misclassification rate. Employment of the combined pharmacophore-SVM platform for in silico screening of a larger collection of olfaction-relevant volatiles produced several new hits. Functional validation of randomly selected hits and rejected compounds from this screen confirmed the power of this virtual screening platform as a convenient tool for accelerating the pace of discovery of novel vector control agents. To the best of our knowledge, this study is the first one that combines a pharmacophore with a SVM model for identification of AgamORco antagonists and specifically orthosteric ones.
昆虫嗅觉受体是异源寡聚配体门控阳离子通道，由一个必需的受体亚基ORco和许多可变亚基之一ORx以尚未确定的摩尔比组成。当在体外单独表达时，ORco形成同四聚体通道，由作为通道激动剂的ORco特异性配体门控。利用基于昆虫细胞的系统作为在体外表达蚊虫气味受体的功能平台，我们鉴定出了天然来源的小分子，它们作为特定的ORco通道拮抗剂，相对于假定的ORco激动剂结合位点为正构或别构拮抗剂，可导致蚊虫嗅觉功能严重抑制。在本通讯中，我们汇总了此类正构拮抗剂的共同结构特征，并开发了一种基于配体的药效团，其性质被认为是与激动剂结合位点结合并导致ORco生物学功能抑制所必需的。用该药效团对现有的天然挥发性化合物集合进行虚拟筛选，鉴定出了几个ORco拮抗剂命中物。对同一化合物集合进行基于细胞的功能筛选，鉴定出了几种在体外作为ORco通道功能的正构和别构拮抗剂并在体内诱导白纹伊蚊出现嗅觉缺失行为的化合物。将虚拟筛选结果与功能测定结果进行比较发现，该药效团正确预测了8种已确认的正构拮抗剂中的7种，而别构拮抗剂无一被正确预测。由于药效团筛选产生了未导致ORco通道功能抑制的额外命中物，我们还基于所有药效团命中物的两个描述符生成了一个支持向量机（SVM）模型。用体外验证的化合物集合对SVM进行训练，结果筛选出了已确认的正构拮抗剂，其交叉验证的样本外错误分类率非常低。利用组合的药效团-SVM平台对更多与嗅觉相关的挥发性化合物进行虚拟筛选，产生了几个新的命中物。对该筛选中随机选择的命中物和被拒化合物进行功能验证，证实了这个虚拟筛选平台作为加速新型病媒控制剂发现步伐的便捷工具的强大功能。据我们所知，本研究是首次将药效团与SVM模型相结合来鉴定AgamORco拮抗剂，特别是正构拮抗剂。
