# Subproject 2: Identification of Pathways that Can be Targeted for the Development of Novel Therapies for MRSA

> **NIH NIH P01** · MASSACHUSETTS EYE AND EAR INFIRMARY · 2024 · $401,498

## Abstract

SUMMARY
Methicillin-resistant S. aureus (MRSA) are leading causes of serious community and hospital-associated
infection. In addition to acquiring antibiotic resistance, S. aureus converts to a persistent antibiotic-tolerant form
in which traditional treatments are powerless. Antibiotic-tolerant persister cells are responsible for recalcitrant
chronic infections. To address the urgent need for new anti-MRSA therapeutics, we developed a C. elegans-
MRSA screening platform to identify compounds with activity against MRSA. The assay identifies compounds
with both in vivo efficacy and low host toxicity. We combined a whole animal in vivo screen of ~82,000 synthetic
compounds for ones that exhibit potential anti-MRSA activity with in vitro screens for compounds with the ability
to kill the stubborn persister subpopulation. We have focused on four compounds, CD437, nTZDpa, bithionol,
and PQ401, all four of which eradicate both actively growing MRSA and stationary phase MRSA persister cells
by disrupting their membrane lipid bilayers. These compounds exhibit fast killing rates, synergism with other
antibiotics, extremely low probabilities of resistance selection, and high selectivity for bacterial over mammalian
membranes. Using bithionol and nTZDpa and analogs of these compounds, we found that anti-persister activity
of these compounds positively correlates with their ability to increase membrane fluidity. All four compounds
have been previously studied as potential therapeutics and bithionol is a former FDA-approved anthelmintic.
Despite the potential of membrane-disrupting compounds to kill persisters, it is generally thought that their
therapeutic applications are limited due to a lack of selectivity for bacterial over mammalian membranes.
However, our results show that the presence of cholesterol in mammalian but not in bacterial membranes
provides an important opportunity to identify/design non-toxic anti-persister membrane-disrupting compounds.
We therefore propose the following two aims to identify features of membrane-disrupting compounds that
correlate with their ability to kill persisters but not disrupt mammalian membranes. In Aim 1, we will utilize
biochemical, physiological, biophysical, and cryo-EM techniques to 1) further investigate the correlation between
the ability of compounds to kill persisters and their ability to increase membrane fluidity, and 2) to identify the
mechanisms of action and targets of membrane-disrupting compounds. We also propose to test the efficacy of
membrane-disrupting compounds in a murine S. aureus subcutaneous abscess infection model in collaboration
with the Hooper lab and determine whether the compounds also target E. faecalis cell membranes. In Aim 2, we
will utilize high throughput microfluidics technology developed in the Paulsson laboratory (Core B) and referred
to as “mother machines” to: 1) isolate rare MRSA persister cells in growing cultures, and 2) in collaboration with
the Walker lab, use Tn-seq ...

## Key facts

- **NIH application ID:** 10769878
- **Project number:** 5P01AI083214-16
- **Recipient organization:** MASSACHUSETTS EYE AND EAR INFIRMARY
- **Principal Investigator:** ELEFTHERIOS MYLONAKIS
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $401,498
- **Award type:** 5
- **Project period:** 2009-09-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10769878

## Citation

> US National Institutes of Health, RePORTER application 10769878, Subproject 2: Identification of Pathways that Can be Targeted for the Development of Novel Therapies for MRSA (5P01AI083214-16). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10769878. Licensed CC0.

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