Screening privileged alkylguanidinium motifs under host-mimicking conditions reveals a novel antibiotic with an unconventional mode of action

Screening large molecule libraries against pathogenic bacteria is often challenged by a low hit rate due to limited uptake, underrepresentation of antibiotic structural motifs and assays which do not resemble the infection conditions. To address these limitations, we here screen a library focused on alkylguanidinium compounds, a structural motif associated with antibiotic activity and enhanced uptake, under host-mimicking infection conditions against a panel of disease-associated bacteria. Several hit molecules were obtained with activities against Gram-positive as well as Gram-negative bacteria highlighting the fidelity of the general concept. Based on the best antibiotic activity, we selected one compound (L15) for in-depth mode of action studies. L15 exhibited bactericidal activity against Staphylococcus aureus USA300 Lac (JE2) with a minimum inhibitory concentration of 1.5 µM whereby a structure-activity relationship study with 18 derivatives proved the necessity of the guanidinium motif for antibiotic activity. Electron microscopy studies and depolarization assays at high L15 concentrations showed an impact on the cell membrane and integrity which was absent at the MIC concentration. Sequencing of L15-resistant strains revealed a mutation in an efflux pump but did not provide hints for direct targets. We thus performed affinity-based protein profiling with a L15 probe and identified signal peptidase IB (SpsB) as the most promising hit. Validation by activity assays, binding site identification and molecular docking demonstrated SpsB overactivation by L15, similar to a previously reported activator molecule that dysregulates protein secretion of this essential enzyme. Overall, this study highlights the need for unconventional screening strategies to identify novel antibiotics.

针对致病菌开展大型分子库筛选时，常因细菌摄取效率低下、抗生素结构基序（structural motif）占比不足，以及实验体系未模拟感染微环境等问题，面临活性分子命中率偏低的挑战。为克服上述局限，本研究采用模拟宿主感染的实验体系，针对一组致病细菌，筛选了一类以烷基胍类（alkylguanidinium）化合物为核心的分子库——该类化合物的结构基序与抗生素活性及增强的细胞摄取能力密切相关。本研究成功获得数株活性分子，可同时对抗革兰氏阳性菌与革兰氏阴性菌，这一结果验证了本研究思路的可靠性。基于最优的抗生素活性，我们选取化合物L15开展深入的作用机制研究。L15对金黄色葡萄球菌（Staphylococcus aureus）USA300 Lac（JE2）表现出杀菌活性，最低抑菌浓度（minimum inhibitory concentration, MIC）为1.5 µM；通过对18种衍生物开展构效关系（structure-activity relationship）研究，证实了胍基基序（guanidinium motif）对抗生素活性的必要性。高浓度L15下的电子显微镜观察与细胞膜去极化实验显示，该化合物会破坏细菌细胞膜的结构与完整性，但在MIC浓度下未观察到此现象。对L15耐药菌株的基因组测序结果显示，其外排泵（efflux pump）基因发生了突变，但未提示存在直接作用靶点。因此，我们使用L15探针开展了亲和蛋白质组分析（affinity-based protein profiling），鉴定出信号肽酶IB（signal peptidase IB, SpsB）为最具潜力的候选靶点。通过活性实验、结合位点鉴定与分子对接（molecular docking）验证，证实L15可过度激活SpsB——这与此前报道的一种可扰乱该必需酶蛋白分泌过程的激活剂分子作用机制高度相似。综上，本研究凸显了采用非常规筛选策略开发新型抗生素的必要性。