Characterization of MsbA inhibitors as potential antibiotic leads to treat carbapenem-resistant Enterobacteriaceae (CRE)

2013年，疾控中心将耐碳青霉烯类肠杆菌科(CRE)列为紧急威胁，2017年， 世界卫生组织将其列为优先事项1“关键超级细菌”。由于治疗慢性粒细胞白血病的疗法寥寥无几，“泛-泛型”的风险 也存在目前任何可用的抗生素都无法治疗的“耐药”CRE。全新的特工和小说 对SOC制剂没有交叉耐药性的作用机制(MOA)逐渐衰弱。开发出这样的一流产品 该制剂还提供了亟需的口服CRE治疗的可能性，从而提供了一种新的 阶梯式治疗，以减少住院时间和不断增长的医疗成本。我们的建议旨在开发一种 治疗由多药耐药(MDR)引起的危及生命的细菌感染的全新疗法 肠杆菌科，包括Cre。使用创新的基于过度表达的协同文化屏幕 在大肠杆菌(EC)中，我们鉴定了四个结构不同的针对MSBA的小分子抑制剂系列， 一种广泛保守的革兰氏阴性(GN)ABC转运蛋白，负责内毒素 革兰氏阴性外膜(OM)的生物发生和构建。建立在坚实的基础上 在初步数据的基础上，我们的目标是： 目的(A)1.扩展的MOA研究。目标1的研究将在我们初步的MOA和微生物学的基础上扩展 MSBA抑制剂(MsbAi)包括其他GN细菌，特别是克雷伯氏菌的HITS特征 肺炎(Kp)。具体地说，我们将在(1)体外生化试验中演示KP MSBA靶标抑制 和(2)通过测定我们的MsbAI对KpΔTolC株的MIC以及随后 在这种KP背景下筛选MsbAisR突变体。 目的2.证明MsbAis的化学可操作性。目标2的目标是执行Hit to Lead(H2 L) 各MsbAi和Demon&gt；1系列的药物化学构效关系研究 化学上易于处理，可以建立可量化的先导优化标准，包括：i)EC和 Kp MSBA体外效价(IC50和lt；0.25微克/毫升)，ii)EC和Kp WT活性(16微克/毫升)，以及III)90%的HepG2细胞 50倍最低抑菌浓度对ECΔTOL C的生存能力。MSBA正在实现这些里程碑，这是一个合理的起点 未来的销售线索优化利用在AIM 2中获得的结果SAR和从AIM 3中获得的SBDD信息。 目的3.与SSGCID合作获得EC和KP MsbAi-MSBA共晶结构。的目标是 目标3活动是启动原核生物、基因技术和西雅图结构公司之间的合作 由Peter Myler博士领导的传染病基因组学中心(SSGCID)优化适用于 获得APO形式的KP MSBA以及EC和/或KP MSBA的高分辨x射线晶体结构 与MsbAis形成复合体的蛋白质。

In 2013, the CDC designated carbapenem-resistant Enterobacteriaceae (CRE) an Urgent Threat, and in 2017, the WHO designated it a Priority 1 “critical superbug”. As few therapies remain to treat CRE, the risk of “pan- resistant” CRE untreatable by any currently available antibiotic also exists. Entirely new agents with novel mechanisms of action (MOA) not cross-resistant to SOC agents languish. Development of such a ‘first-in-class’ agent also offers the potential for much-needed orally-administered CRE therapeutic, thus providing a new step-down therapy to reduce hospital stay and growing healthcare costs. Our proposal aims to develop an entirely novel therapeutic to treat life-threatening bacterial infections due to multidrug resistant (MDR) Enterobacteriaceae, including CRE. Using an innovative overexpression-based co-culture screen in Escherichia coli (Ec), we identified four structurally distinct series of small molecule inhibitors targeting MsbA, an essential and broadly conserved Gram-negative (GN) ABC transporter responsible for lipopolysaccharide (LPS) biogenesis and construction of the Gram-negative outer membrane (OM). Building upon a solid foundation of preliminary data, our Aims are: Aim (A) 1. Expanded MOA studies. Aim 1 studies will expand upon our preliminary MOA and microbiological characterization of the MsbA inhibitor (MsbAi) hits to include other GN bacteria, particularly Klebsiella pneumoniae (Kp). Specifically, we will demonstrate Kp MsbA target inhibition in (1) an in vitro biochemical assay and (2) a whole-cell context by determining MICs of our MsbAis against a Kp ΔtolC strain and subsequently selecting for MsbAisR mutants in this Kp background. Aim 2. Demonstrate chemical tractability of MsbAis. The goal of Aim 2 is to perform Hit to Lead (H2L) medicinal chemistry structure activity relationship (SAR) studies for each MsbAi and demonstrate >1 series is chemically tractable and that quantifiable pre-lead optimization criteria can be established, including i) Ec and Kp MsbA in vitro potency (IC50 < 0.25 µg/ml), ii) Ec and Kp WT activity (<16 µg/ml), and iii) > 90% HepG2 cell viability at 50X MIC against EcΔtolC. MsbAis achieving these milestones serve as a justifiable starting point for a future Lead Optimization leveraging the resulting SAR gained in Aim 2 and SBDD information from Aim 3. Aim 3. Obtain Ec and Kp MsbAi-MsbA co-crystal structures in collaboration with SSGCID. The goal of Aim 3 activities is to initiate a collaboration between Prokaryotics, Genentech, and the Seattle Structural Genomics Center for Infectious Disease (SSGCID) led by Dr. Peter Myler to optimize conditions suitable for obtaining high resolution x-ray crystal structures of Kp MsbA in the apo form as well as Ec and/or Kp MsbA proteins in complex with MsbAis.