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

低膜渗透性和广泛外排是细菌防御机制，其协同减少革兰氏阴性菌（GNB）中的化合物积累，导致内在抗性，其阻止化合物实现其（预期的）生物学效应。GNB具有两种正交的细胞膜渗透性能。脂多糖（LPS）包被的外膜（OM）对大多数疏水剂是不可渗透的。能够穿过OM的化合物通常通过排列有带电氨基酸的窄β-桶蛋白（孔蛋白）这样做。一旦进入周质，化合物就容易受到多药外排泵的影响;因此，为了在GNB中积累到足以发挥活性的水平，化合物通常必须比它们被泵出更快地穿过孔蛋白。所得到的正交物理化学性质与外排泵的过表达相结合，对传统的小分子提出了重大挑战。除了孔蛋白，另一种OM递送途径涉及多元两亲物（PA）的自促进摄取机制。PA通过与二价阳离子交联相邻聚阴离子LPS聚合物的位点相互作用而穿过OM。这导致OM的不稳定，这被认为导致PA和/或其他化合物自我促进吸收到GNB中。** 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PA的化学和生物特性及其对GNB中化合物积累的影响。目标1研究了五种基于PA的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的膜渗透性和外排。目标2研究了（a）二价金属螯合剂的掺入;（B）与铁载体的缀合;（c）与膜解偶联剂的缀合;（d）PA中同功能多价和（e）异功能多价效应的存在如何影响1和2在GN B中的蓄积。GOAL 3探索了基于PA和铁载体的连接对GNB中1和2的膜渗透性和流出的影响。 长期目标是开发化学干预措施，解除巨人银行的细菌防御。 ** 拟议的研究预计将作出重大贡献，包括在与细菌系统的生物化学，生物有机化学和分析化学以及微生物学相关的各种领域取得潜在突破。 这些研究所收集的信息将对制定化学战略产生直接、直接的重大影响，以解除细菌防御机制和应对GNB的抗菌素耐药性，并具有多种应用，包括食品和水安全、兽医健康、农业和安全。概述的程序将培养4博士，2硕士和10在细菌系统的化学生物学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.