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

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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 -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.