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

低膜透性和广泛外排是细菌的防御机制，它们协同减少革兰氏阴性菌（GNB）中化合物的积累，从而导致内在耐药性，从而阻止化合物实现其（预期的）生物效应。GNB具有两层具有正交穿膜性能的细胞膜。脂多糖（LPS）包覆的外膜（OM）对大多数疏水剂是不可渗透的。能够穿越OM的化合物通常是通过狭窄的β桶蛋白（孔蛋白），这些蛋白与带电的氨基酸排列在一起。化合物一旦进入外质，就容易受到多药外排泵的影响；因此，要在GNB中积累到足以产生活性的水平，化合物穿过孔蛋白的速度通常必须快于它们被泵出的速度。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，对传统的小分子提出了重大挑战。除了孔蛋白外，另一种OM传递途径涉及多碱性两亲体（PAs）的自我促进摄取机制。PAs通过与二价阳离子桥接邻近的聚阴离子LPS聚合物的位点相互作用而穿过OM。这导致OM的不稳定，从而导致PA和/或其他化合物进入GNB的自我促进摄取。******拟议研究计划的总体目标是设计、合成和研究定制的多功能PAs的化学和生物学特性及其对GNB中化合物积累的影响。目标1研究五种pa基支架的理化性质如何影响GNB中两种模型化合物1和2的膜通透性和外排。目标2调查如何(a)掺入二价金属螯合剂；(b)与铁载体结合；(c)与膜解偶联剂的偶联：(d)同功能多价效应的存在和(e)异功能多价效应影响GNB中1和2的积累。目标3探讨基于PA和铁载体的结扎对GNB膜通透性和1和2外排的影响。长期目标是开发化学干预措施来解除GNB中的细菌防御。******提出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各种领域的潜在突破。这些研究收集的信息将对制定化学策略，解除GNB中的细菌防御机制和对抗抗菌素耐药性产生直接、直接的强烈影响，并具有多种应用，包括食品和水安全、兽医卫生、农业和安全。该计划将在细菌系统的化学生物学方面培养4名博士，2名硕士和10名本科。

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.