# Transport Mechanisms and Inhibition of Efflux Pumps in Pathogenic Organisms

> **NIH NIH R01** · NEW YORK UNIVERSITY · 2024 · $73,396

## Abstract

Project Summary
 The long-term goal of our research is to develop first-in-class, protein-based inhibitors against
human bacterial pathogens by directly blocking efflux pumps. Drug resistant bacteria pose an
urgent global health challenge by reducing the effectiveness of antibiotics used to treat infections
in humans and animals. The broadest resistance mechanism against antibiotics are efflux pumps,
which transport drugs out of the cytoplasm and reduce toxicity to the organism. While it is known
that efflux pumps display broad specificity to structurally distinct compounds, the mechanisms of
polyspecific drug binding and ion-coupled transport remain unanswered questions in the field.
Given the promiscuity of efflux pump binding to structurally distinct drugs, it is also unclear
whether potent and selective efflux pump inhibitors can be designed to target specific classes of
efflux pumps. The specific goals of this project are to discover novel mechanisms of active
transport in drug resistant Staphylococcus aureus and to harness this knowledge to design
selective inhibitors toward efflux pumps. Our proposal is strongly motivated by our recent
discovery of antibody fragments (Fabs) that bind the Staphylococcus aureus efflux pump NorA
and successful determination of high-resolution cryoEM structures using the Fabs as fiduciaries.
The structures revealed that the Fabs insert a loop into the substrate binding pocket from the
extracellular side, which suggests a design path toward protein- and peptide-based inhibitors.
This interaction is facilitated by an electrostatic interaction between a positively charged arginine
on the Fab and two essential anionic residues within NorA. Building on these preliminary data,
we propose to carry out four Specific Aims. Aim 1 will develop a hybrid approach of cryo-electron
microscopy and NMR spectroscopy to comprehensively study the transport cycle of NorA. Aim 2
will seek to determine the molecular basis for polyspecific drug binding. Aim 3 will design and
characterize protein-based inhibitors that target the accessible, outward-open conformation of
NorA. Aim 4 will develop peptides that miniaturize the antibody loops observed in the binding
pocket of NorA. We have assembled an interdisciplinary team with expertise in structural biology,
protein engineering, microbiology, chemical synthesis, and computational chemistry to rapidly
answer fundamental questions about multidrug transport and inhibition of efflux pumps. All of the
approaches applied to NorA will be translatable to other transporter systems.

## Key facts

- **NIH application ID:** 10936764
- **Project number:** 3R01AI165782-03S1
- **Recipient organization:** NEW YORK UNIVERSITY
- **Principal Investigator:** SHOHEI KOIDE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $73,396
- **Award type:** 3
- **Project period:** 2021-12-01 → 2026-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10936764

## Citation

> US National Institutes of Health, RePORTER application 10936764, Transport Mechanisms and Inhibition of Efflux Pumps in Pathogenic Organisms (3R01AI165782-03S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10936764. Licensed CC0.

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