Multidrug efflux pumps as exemplified by AcrAB-TolC from Escherichia coli are the major contributors to clinical antibiotic resistance in bacteria and to various adaptive responses during pathogenesis and chronic infections. During the previous funding period, we made major advances in two areas: 1) understanding the molecular mechanism of efflux pump assembly and the role of periplasmic membrane fusion proteins in multidrug efflux across two membranes; and 2) the development of a synergistic computational and empirical approach to discovering efflux pump inhibitors with novel mechanisms of action. We successfully applied these advances to discover inhibitors that act in a novel way, by interacting with AcrA and inhibiting the assembly of the AcrAB- TolC complex. These efflux pump inhibitors potentiate activities of multiple antibiotics in various bacteria. The major goal of the proposed research is to establish the molecular mechanisms of the new efflux pump inhibitors and to optimize these inhibitors for use in combination with specific antibiotics and against specific multidrug resistant bacteria. The underlying hypothesis is that the broad potentiation activity of the discovered inhibitors is caused by their unique mechanism that traps efflux pumps in a poorly assembled and leaky conformation. In the proposed approach, biochemical, structural and kinetic experiments will be used synergistically with advanced computations to characterize the mechanism of efflux pump inhibitors and to optimize inhibitors acting on efflux pumps of multidrug resistant Acinetobacter baumannii. To optimize inhibitors, we will apply what is to our knowledge the most comprehensive platform available. The platform utilizes a set of strains with variable efflux capacities and outer membrane permeability barriers and allows to establish structure-activity relationships separately for efflux avoidance, inhibition and permeation across the outer membrane. Successful completion of the proposed experiments will help design efflux pump inhibitors that would be effective even in the context of multiple pumps and mechanisms of antibiotic resistance.

Public Health Relevance

The emergence of multi-drug resistance in already challenging Gram-negative infections and the dry pipeline of broad-spectrum antibiotics demand alternative strategies to preserve activities of clinical antibiotics. This project is focused on development of a promising approach to inhibit multidrug efflux pumps. If successful, studies will identify novel efflux inhibitors with desired biochemical activities and primed for specific clinical applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI052293-16
Application #
9885979
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Ernst, Nancy L
Project Start
2003-03-01
Project End
2025-03-31
Budget Start
2020-04-02
Budget End
2021-03-31
Support Year
16
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Oklahoma Norman
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
848348348
City
Norman
State
OK
Country
United States
Zip Code
73019
Zgurskaya, Helen I; Rybenkov, Valentin V; Krishnamoorthy, Ganesh et al. (2018) Trans-envelope multidrug efflux pumps of Gram-negative bacteria and their synergism with the outer membrane barrier. Res Microbiol 169:351-356
Amaro, Rommie E; Baudry, Jerome; Chodera, John et al. (2018) Ensemble Docking in Drug Discovery. Biophys J 114:2271-2278
Hwang, Hyea; Paracini, Nicolò; Parks, Jerry M et al. (2018) Distribution of mechanical stress in the Escherichia coli cell envelope. Biochim Biophys Acta Biomembr 1860:2566-2575
Picard, Martin; Tikhonova, Elena B; Broutin, Isabelle et al. (2018) Biochemical Reconstitution and Characterization of Multicomponent Drug Efflux Transporters. Methods Mol Biol 1700:113-145
López, Cesar A; Travers, Timothy; Pos, Klaas M et al. (2017) Dynamics of Intact MexAB-OprM Efflux Pump: Focusing on the MexA-OprM Interface. Sci Rep 7:16521
Yue, Zhi; Chen, Wei; Zgurskaya, Helen I et al. (2017) Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump. J Chem Theory Comput 13:6405-6414
Haynes, Keith M; Abdali, Narges; Jhawar, Varsha et al. (2017) Identification and Structure-Activity Relationships of Novel Compounds that Potentiate the Activities of Antibiotics in Escherichia coli. J Med Chem 60:6205-6219
Abdali, Narges; Parks, Jerry M; Haynes, Keith M et al. (2017) Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump. ACS Infect Dis 3:89-98
Ntreh, Abigail T; Weeks, Jon W; Nickels, Logan M et al. (2016) Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC. J Bacteriol 198:3176-3185
Weeks, Jon W; Nickels, Logan M; Ntreh, Abigail T et al. (2015) Non-equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa. Mol Microbiol 98:343-56

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