Structure of a bacterial 'drug pump' reveals new way to counter
hospital-borne infection

Date:
March 31, 2022
Source:
NYU Langone Health / NYU Grossman School of Medicine
Summary:
By revealing the structure of a protein used by bacteria to
pump out antibiotics, a research team designed an early-stage
therapeutic that sabotages the pump and restores the effectiveness
of antibiotics.



FULL STORY ==========================================================================
By revealing the structure of a protein used by bacteria to pump out antibiotics, a research team designed an early-stage therapeutic that
sabotages the pump and restores the effectiveness of antibiotics.


==========================================================================
Led by researchers from New York University, NYU Grossman School of
Medicine, and NYU Langone's Laura and Isaac Perlmutter Cancer Center,
the new study used advanced microscopy to "see" for the first time the structure of NorA, a protein that the bacterial species Staphylococcus
aureus uses to pump out widely used antibiotics before they can kill them.

Efflux pumps represent one mechanism by which S. aureus has evolved
resistance to fluoroquinolones, a group of more than 60 approved
antibiotics that includes norfloxacin (Noroxin), levofloxacin (Levaquin),
and ciprofloxacin (Cipro).

Fluoroquinolones are now ineffective against some drug-resistant bacterial strains, including methicillin-resistance S. aureus (MRSA), a major cause
of death among hospitalized patients when infections become severe, the researchers say. For this reason, the field has sought to design efflux
pump inhibitors, but early attempts have been hindered by side effects.

"Instead of trying to find a new antibiotic, we hope to make the most
widely used antibiotics over the last few decades, rendered ineffective
by bacterial resistance, highly effective again," says first study author
Doug Brawley, PhD.

He completed his doctoral thesis in the laboratories of senior authors
Nate Traaseth, PhD, a professor in the Department of Chemistry at New
York University, and Da-Neng Wang, PhD, a professor in the Department
of Cell Biology at NYU Grossman School of Medicine.

Antibodies to the Rescue Published online March 31 in the journal Nature Chemical Biology, the study builds on advances in antibody technology development in recent years. Invading bacteria trigger the body's immune
system to make many slightly different antibodies, proteins shaped to
attach to and neutralize specific invaders.



==========================================================================
For the current study, the research team used antibodies to overcome a challenge that had kept the structure of NorA from being analyzed. Brawley worked for years to fine-tune the expression and purification conditions
needed for this analysis, but the NorA molecule is compact and barely detectable even with advanced cryo-electron microscopy (cryo-EM).

As a solution, the researchers screened a large collection of synthetic antibodies -- assembled by the lab of senior study author Shohei
Koide, PhD, professor in the Department of Biochemistry and Molecular Pharmacology at NYU Grossman School of Medicine -- to find the ones that attached most tightly to NorA. By attaching the antibodies to NorA, the
team effectively doubled the size of the molecule, which enhanced the
cryo-EM images and revealed the NorA pump's structure for the first time.

The work also revealed the site where the team's lead antibody docked
into NorA, like a key into a lock. The team was surprised to find that
the place where this antibody fit into NorA was the same place that NorA latches onto and removes antibiotics. These observations suggested that
the antibody could block the pump, enable antibiotics to remain inside bacterial cells, and interfere with bacterial growth.

From the cryo-EM structure, the team also realized that the part of the antibody most deeply embedded in NorA's binding cavity was a short,
looping peptide, a segment of protein building blocks. "We became
excited that an isolated peptide corresponding to the loop by itself
might inhibit NorA," says Traaseth. The team found that this peptide
(termed NPI-1) functioned as an efflux pump inhibitor (EPI) and reduced antibiotic-resistant S. aureusgrowth in dishes with nutrients (cultures)
by more than 95 percent at high concentrations when combined with the antibiotic norfloxacin.

The structural analysis also showed that the EPI had many interactions
with protein building blocks in the structural pocket where NorA attaches
to antibiotic molecules. "This makes it highly unlikely that bacteria
could develop resistance to such a treatment, because they would have
to randomly evolve to somehow defeat the EPI without taking away the
ability of the efflux pump site to grab antibiotics," says Wang.



========================================================================== Moving forward, the team is working to improve the design of their
EPI. Each residue of NPI-1 can be optimized for greater potency and to
reduce any potential side effect, say the authors. Their strategy for developing synthetic antibodies to NorA-like efflux pumps may help to
discover EPIs against other pathogens known to depend on pumps, which includeStreptococcus pneumonia and Mycobacterium tuberculosis.

"The discovery of this new way to inhibit MRSA demonstrates that five
labs from four departments -- with complementary expertise in structural biology, protein engineering, peptide chemistry, and microbiology --
can collaborate to accomplish what none could alone," adds Koide.

Along with Brawley, Traaseth, Wang, and Koide, authors of the study
were David Sauer and Jinmei Song of the Department of Cell Biology;
Jianping Li, Ganesh Jedhe, Tiffany Suwatthee, and Paramjit Arora of the Department of Chemistry; Xuhui Zheng and Victor Torres in the Department
of Microbiology, Akiko Koide in the Department of Medicine, and Zheng
Liu in the Cryo-Electron Microscopy Facility.

This work was supported by National Institutes of Health grants
R01AI165782, R01AI108889, R01NS108151, R01GM121994, R01DK099023,
R01AI099394, R01AI105129, R01AI137336, R01AI140754, R21AI149350,
R35GM130333, and T32-GM088118; National Science Foundation award
MCB 1506420; American Cancer Society Postdoctoral Fellowship 129844-PF-17-135-01-TBE, and Department of Defense Horizon Award
W81XWH-16-1- 0153. The research team also acknowledges the investment
by NYU Grossman School of Medicine in the cryo-EM Core facility, which
was essential to the study's finding.

New York University has filed a patent application on the antibodies
described in this work. Koide holds equity in and receives consulting
fees from Black Diamond Therapeutics and receives research funding from Puretech Health and Argenx BVBA. Torres is an inventor on patents and
patent applications filed by NYU, which are currently under commercial
license to Janssen Biotech Inc. These relationships are being managed
in accordance with the policies of NYU Langone Health.


========================================================================== Story Source: Materials provided by NYU_Langone_Health_/_NYU_Grossman_School_of_Medicine.

Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Douglas N. Brawley, David B. Sauer, Jianping Li, Xuhui Zheng, Akiko
Koide, Ganesh S. Jedhe, Tiffany Suwatthee, Jinmei Song, Zheng Liu,
Paramjit S. Arora, Shohei Koide, Victor J. Torres, Da-Neng Wang,
Nathaniel J. Traaseth. Structural basis for inhibition of the
drug efflux pump NorA from Staphylococcus aureus. Nature Chemical
Biology, 2022; DOI: 10.1038/s41589-022-00994-9 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/03/220331121241.htm

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