Establishing a Gram-Negative Permeation Rule Set Leveraging a Unique Small Molecule Library

摘要 自首次描述分子间的相互关系以来，已有30多年的历史。 抗生素的性质及其在革兰氏阴性杆菌中的蓄积能力。1，2相当大的进展 了解介导药物摄取和RND外排泵的孔蛋白通道的结构和功能 3然而，尽管取得了这些进展，但对规则的简要了解 革兰氏阴性细胞内小分子积累的定义仍然难以捉摸。因此，抗菌剂 药物发现过程已经陷入僵局。迫切需要解决这一发现瓶颈的办法 有效应对多重耐药革兰氏阴性病原体构成的威胁 革兰氏阴性菌被两层膜包裹，外膜不对称 (OM)对小分子，包括抗菌药物，起到强大的渗透屏障的作用。 亲水性抗生素主要通过穿孔蛋白进入革兰氏阴性细胞的机制 据信需要增加药物极性和低分子量才能有利于通过的通道。6-9目前 一些抗菌药物不遵守这些规则。10因此，这些药物一定有一定程度的可塑性 最终需要学习和利用的规则。 为周质粒细胞建立简明的规则将大大有助于抗菌药物的发现 通过改善外膜穿透而积累。尽管药物进行了详尽的筛查活动 对于探索公司来说，实现这一目标的进展受到一些重要因素的限制：i)很少 在革兰氏负片中具有可测量的渗透性以从中获得信息的化学类别，ii)最小 这些化学类别之间的化学多样性和III)普遍缺乏广泛适用的、生物上的- 多药耐药革兰氏阴性病原菌小分子蓄积的相关检测。 这项建议概述了一种多方面的方法来研究小分子渗透的决定因素。 来自VenatoRx制药公司的独特化合物集合中的革兰氏阴性细菌 β-内酰胺酶抑制剂，青霉素结合蛋白的药物发现计划。外膜 渗透性使能参数将从这个小分子训练集得出，通过QSAR和 用主成分分析制定规则来指导提高周质积累的努力 重要的病原体。建立的外膜穿透规则集将通过直接验证 肠杆菌科青霉素结合蛋白活性药物发现计划的应用 并旨在通过改善铜绿假单胞菌和鲍曼不动杆菌的积累来扩大光谱。最后， 拟议的工作将集中在揭开外膜通透性的神秘面纱，以缓解 革兰氏负片的分子不渗透性和允许新的革兰氏阴性偏置化学物质的基本设计 文库显著提高将分子筛查命中转化为治疗的成功率 活性抗生素。

ABSTRACT It has been more than 30 years since the first descriptions of the relationship between molecular properties of antibiotics and their ability to accumulate within gram-negative bacteria.1,2 Considerable progress towards understanding the structure and function of porin channels mediating drug uptake and RND efflux pumps involved in drug elimination has followed.3 Yet, despite these advances, concise knowledge of the rules that define small molecule accumulation within the gram-negative cell remains elusive. As such, the antibacterial drug discovery process has reached an impasse. A solution to this discovery bottleneck is desperately needed to effectively confront the threat posed by multidrug resistant gram-negative pathogens.4 Gram-negative bacteria are encapsulated by two membranes, with the asymmetric outer membrane (OM) acting as a formidable permeability barrier to small molecules, including antibacterial drugs.5 Leaving aside mechanisms of self-promoted uptake, hydrophilic antibiotics enter gram-negative cells largely through porin channels that are believed to require increased drug polarity and low molecular weight to favor passage.6–9 Yet some antibacterial drugs do not abide by these rule.10 Therefore, there must be some level of plasticity in these rules that ultimately need to be learned and exploited. Antibacterial drug discovery would greatly benefit from establishing concise rules for periplasmic accumulation through improved outer membrane penetration. Despite exhaustive screening campaigns by drug discovery companies, progress towards this goal has been limited by a number of important factors: i) few chemical classes having measurable permeability in Gram-negatives from which to derive information, ii) minimal chemical diversity among these chemical classes and iii) a general lack of broadly applicable, biologically- relevant assays to measure small molecule accumulation in MDR gram-negative pathogens. This proposal outlines a multifaceted approach to investigate determinants of small molecule permeation in gram-negative bacteria from a unique compound collection assembled at VenatoRx Pharmaceuticals as part of drug discovery programs for β-lactamase inhibitors, Penicillin Binding Proteins. Outer membrane permeability-enabling parameters will be derived from this small molecule training set, analyzed by QSAR and Principle Component Analysis to formulate rules to guide efforts to improve periplasmic accumulation in these important pathogens. The established rule set for outer membrane penetration will be validated through direct application to an active drug discovery program for Penicillin Binding Proteins focused on Enterobacteriaceae and aiming to expand the spectrum through improved accumulation in P. aeruginosa and A. baumannii. Finally, the proposed work will focus on demystifying outer membrane permeability to relieve the bottleneck of small molecule impermeability in gram negatives and allow the rationale design of new gram-negative-biased chemical libraries to significantly improve the success rates of translating molecular screening hits into therapeutically active antibiotics.