Modeling atomic structure of the EmrE multidrug pump to design inhibitor peptides

描述（由申请人提供）：对强效抗生素具有耐药性的病原体的出现是发展中国家治疗传染病（如结核病和疟疾）的最严重问题之一。耐碳青霉烯类肠杆菌科（CRE）如克雷伯氏菌在医院环境中的迅速增加对美国的公共卫生构成日益严重的威胁。细菌中发生多药耐药性的一种常见机制涉及多药耐药性（MDR）蛋白转运蛋白的作用，它结合各种细胞毒性化合物并将它们主动挤出细胞。多药耐药转运蛋白（MDR transporters）是一个广泛存在于细菌中的多药耐药转运蛋白家族，包括M.肺结核，B。Pertussis，百日咳新属N.脑膜炎，B。Anthracis和S.奥瑞斯因此，成功的SMR抑制剂在治疗广泛的细菌感染中将是重要的，特别是与抗生素药物联合使用。本研究的目的是设计能够干扰SMR家族EmrE转运蛋白功能的小肽。具体地，肽类似物将被设计为抑制细菌膜中活性EmrE二聚体的形成。在该项目的第一阶段，EmrE的低分辨率X射线晶体结构和冷冻电子显微镜（EM）图将与分子动力学（MD）和自由能计算机模拟方法一起使用，以构建和验证脂质膜内EmrE的原子分辨率二聚体结构。为了建立结构的生理相关性，将通过自由能模拟计算配体解离常数并与实验进行比较。在第二阶段，原子结构将用于创建EmrE-肽抑制复合物的模型。将通过使用MD和蒙特-卡罗模拟的直接计算机诱变优化模型，生成对EmrE具有最高亲和力的肽抑制剂。优化的肽将被稳定化， 通过全烃交联抵抗蛋白水解。交联肽是本问题的特别有吸引力的候选物，因为它们无毒，抗蛋白水解，并且使用与EmrE结合所需的高膜渗透性相容的疏水交联。将对计算机模拟设计的最终肽抑制剂进行实验测试。

DESCRIPTION (provided by applicant): The emergence of pathogens resistant to powerful antibiotics is among the most serious problems for the treatment of infectious diseases in the developing world, such as tuberculosis and malaria. The rapid rise of carbapenem-resistant Enterobacteriacea (CRE) such as Klebsiella in the hospital setting is a growing threat to public health in the US. A common mechanism by which multidrug resistance occurs in bacteria involves the action of multidrug resistance (MDR) protein transporters, which bind various cytotoxic compounds and actively extrude them out of the cell. MDR transporters from the small multidrug resistance (SMR) family are widely distributed in bacteria, including the pathogens M. Tuberculosis, B. Pertussis, N. Meningitis, B. Anthracis and S. Aureus. Therefore, successful inhibitors of SMR will be important in the treatment of a broad range of bacterial infections, especially in combination with antibiotic drugs. The objective of the present research is to design small peptides that will interfere with the function of the EmrE transporter of the SMR family. Specifically, peptide analogs will be designed to inhibit the formation of the active EmrE dimer in the bacterial membrane. In the first stage of the project, a low resolution X-ray crystal structure and cryoelectron microscopy (EM) maps of EmrE will be used together with molecular dynamics (MD) and free energy computer simulation methods to construct and validate an atomic-resolution dimer structure of EmrE inside the lipid membrane. To establish the physiological relevance of the structure, ligand dissociation constants will be computed by free energy simulations and compared with experiments. In the second stage, the atomic structure will be used to create models of EmrE-peptide inhibition complexes. The models will be optimized by directed in silico mutagenesis using MD and Monte-Carlo simulations generate peptide inhibitors with the highest affinity for EmrE. The optimized peptides will be stabilized to resist proteolysis by all-hydrocarbon cross-links. Cross-linked peptides are especially attractive candidates for the present problem because they are nontoxic, resistant to proteolysis, and use hydrophobic cross-links compatible with a high membrane permeability required for EmrE binding. The final peptide inhibitors designed in silico will be tested experimentally.