Biophysical characterization of E-coli TolC interaction with the known blocker hexaamminecobalt
Biophysical characterization of E-coli TolC interaction with the known blocker hexaamminecobalt
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DOI：
10.1016/j.bbagen.2017.07.014
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发表时间：
2017-11-01
影响因子：
3
通讯作者：
Windshuegel, B.
中科院分区：
文献类型：
--
作者：
Gilardi, A.;Bhamidimarri, S. P.;Windshuegel, B.
背景：大肠杆菌AcrAB-TolC三联外排泵。大肠杆菌通过将抗生素运输出细胞而产生抗药性。外膜蛋白TolC可以被各种阳离子，包括hexaamminecobalt，从而TolC代表一个潜在的目标，用于减少抗微生物药物的耐药性，因为它的堵塞可能会提高efficacy of antibiotics.Methods：我们利用单通道电生理测量研究TolC电导在不存在和存在的已知TolC阻滞剂hexaamminecobalt。利用表面等离子体共振技术测定了六氨合钴的缔合和解离常数。最低抑菌浓度（MIC）测定在存在和不存在抗生素进行调查的抗菌效果hexaaminecobalt和其潜在的降低MICs.Results：TolC门控在没有任何配体的情况下是电压依赖性和不对称的高电压。六氨合钴以高亲和力与TolC结合，动力学数据显示快速缔合和解离速率。尽管六氨合钴与TolC有很强的结合力，但它对大肠杆菌没有内在的抗菌活性。结论：小分子药物能有效阻断TolC开放。为了研究TolC作为药物靶点的资格，需要更有效的通道阻断剂。一般意义：TolC是一个潜在的有趣的药物靶点，可以通过小分子来阻断通道。结合过程的生物物理表征将支持未来更有效的TolC阻断剂的鉴定和优化，以验证TolC作为药物靶标。
Background: The tripartite efflux pump AcrAB-TolC in E. coli is involved in drug resistance by transporting antibiotics out of the cell. The outer membrane protein TolC can be blocked by various cations, including hexaamminecobalt, thereby TolC represents a potential target for reducing antimicrobial resistance as its blockage may improve efficacy of antibiotics.Methods: We utilized single channel electrophysiology measurements for studying TolC conductance in the absence and presence of the known TolC blocker hexaamminecobalt. Association and dissociation constants of hexaamminecobalt were determined using surface plasmon resonance measurements. Minimum inhibitory concentration (MIC) assays in the absence and presence of antibiotics were carried out for investigating the antibacterial effect of hexaamminecobalt and its potential to reduce MICs.Results: TolC gating in the absence of any ligand is voltage dependent and asymmetric at high applied voltages. Hexaamminecobalt binds to TolC with high affinity and kinetic data revealed fast association and dissociation rates. Despite potent binding to TolC, hexaamminecobalt does not possess an intrinsic antimicrobial activity against E. colt nor does it reduce MIC values of antibiotics erythromycin and fusidic acid.Conclusions: TolC opening can be effectively blocked by small molecules. More potent channel blockers are needed in order to investigate the eligibility of TolC as drug target.General significance: TolC, a potentially interesting pharmaceutical target can be addressed by small molecules, blocking the channel. Biophysical characterization of the binding processes will support future identification and optimisation of more potent TolC blockers in order to validate TolC as a pharmaceutical target.