Bioorganic chemistry of polybasic amphiphiles (PAs): Disarming bacterial defense mechanisms in Gram-negative bacteria (GNB)

低膜通透性和广泛的外排是细菌防御机制，它们协同减少化合物在革兰氏阴性菌中的积累，导致内在耐药性，阻止化合物实现(预期的)生物效应(S)。GNB具有两种具有正交膜穿透性能的细胞膜。脂多糖(LPS)包裹的外膜(OM)对大多数疏水剂是不渗透的。能够穿过OM的化合物通常是通过排列着带电氨基酸的狭窄β-Barrel蛋白(孔蛋白)来实现的。一旦进入周质，化合物就容易受到多药外排泵的影响；因此，要在GNB中累积到足以发挥活性的水平，化合物通常必须以比泵出更快的速度穿过孔道。由此产生的正交物理化学性质与外排泵的过度表达相结合，给传统的小分子带来了巨大的挑战。除了孔蛋白，另一种OM递送途径涉及多碱两亲分子(PAS)的自我促进摄取机制。PAS通过与二价阳离子与邻近的多阴离子脂多糖聚合物桥联的位置相互作用而穿过OM。这导致OM的不稳定，这被认为是导致PA和/或其他化合物自我促进吸收到GNB。*拟议研究计划的总体目标是设计、合成和研究量身定制的多功能PA的化学和生物学特性及其对化合物在GNB中积累的影响。目标1研究五种PA基支架的物理化学性质如何影响GNB中两种模型化合物1和2的膜渗透性和外流。目标2调查了(A)二价金属螯合剂的结合；(B)与铁载体的结合；(C)与膜解偶联剂的结合：(D)同功能多价态的存在和(E)PAS中异官能多价态效应如何影响1和2在GNB中的积累。目标3探讨以PA和铁载体为基础的结扎对GNB膜通透性和1和2外流的影响。长期目标是开发化学干预措施，解除GNB中的细菌防御。*拟议的研究预计将做出重大贡献，包括在与细菌系统的生物化学、生物有机和分析化学以及微生物学相关的广泛领域取得潜在突破。这些研究收集的信息将对制定化学战略产生直接和直接的强烈影响，以解除全球细菌防御机制的武装并消除全球细菌的抗药性，其多种应用包括食品和水安全、兽医保健、农业和安全。概述的计划将在细菌系统的化学生物学方面培训4名博士、2名硕士和10名UG。

Low membrane permeability and extensive efflux are bacterial defence mechanisms which synergistically reduce compound accumulation in Gram-negative bacteria (GNB) resulting in intrinsic resistance that prevents compounds from achieving their (intended) biological effect(s). GNB possess two cellular membranes with orthogonal membrane penetrating properties. The lipopolysaccharide (LPS)-coated outer membrane (OM) is impermeable for most hydrophobic agents. Compounds that are able to traverse the OM typically do so through narrow β-barrel proteins (porins) that are lined with charged amino acids. Once inside the periplasm compounds are susceptible to multidrug efflux pumps; thus, to accumulate in GNB to a level that is sufficient for activity, compounds typically must traverse porins faster than they are pumped out. The resultant orthogonal physicochemical properties combined with the overexpression of efflux pumps pose a significant challenge for traditional small molecules to overcome. In addition to porins, an alternative OM delivery pathway involves the self-promoted uptake mechanism of polybasic amphiphiles (PAs). PAs cross the OM by interacting with sites at which divalent cations crossbridge adjacent polyanionic LPS polymers. This causes a destabilization of the OM that is proposed to lead to self-promoted uptake of the PA and/or other compounds into GNB. ******The overall objective of the proposed research program is the design, synthesis and study of the chemical and biological properties of tailor made, multifunctional PAs and their effects on compound accumulation in GNB. GOAL 1 studies how the physicochemical properties of five PA-based scaffolds affect membrane permeability and efflux of two model compounds 1 and 2 in GNB. GOAL 2 investigates how (a) incorporation of bivalent metal chelators; (b) conjugation to a siderophore; (c) conjugation to membrane uncouplers: (d) the presence of homofunctional multivalency and (e) heterofunctional multivalency effects in PAs influence accumulation of 1 and 2 in GNB. GOAL 3 explores the effects of PA- and siderophore-based ligation on membrane permeability and efflux of 1 and 2 in GNB. The long-term goal is to develop chemical interventions to disarm bacterial defences in GNB. ******The proposed research is expected to make significant contributions, including potential breakthroughs in a wide variety of areas associated with biological chemistry of bacterial systems, bioorganic- and analytical chemistry and microbiology. The information gathered by these studies will have immediate, direct strong impacts on the development of chemical strategies to disarm bacterial defence mechanisms and counter antimicrobial resistance in GNB with multiple applications including food and water safety, veterinary health, agriculture and security. The outlined program will train 4 PhD, 2 MSc and 10 UG in chemical biology of bacterial systems.