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.