# Exploiting Riboswitch Sensors to Reveal Antibiotics Uptake and Retention in Gram Negative Bacteria

> **NIH NIH R01** · YALE UNIVERSITY · 2020 · $1,114,844

## Abstract

ABSTRACT
Infections caused by MDR Pseudomonas aeruginosa and other Gram-negative pathogens challenge clinicians
to find safe and effective antibiotic regimens that can eradicate these opportunistic pathogens from the frail,
often immunocompromised hosts that they target. Two features of the P. aeruginosa cell envelope - its limited
permeability to small molecules and the large number of both constitutive and inducible efflux systems it
contains - render this pathogen intrinsically resistant to many available antimicrobials and contribute to
acquired resistance toward the small set of existing anti-Pseudomonal antibiotics. This seriously limits the
ability to identify “hits” with antibiotic activity using whole-cell assays - as compounds that penetrate and can
inhibit key metabolic pathways are often effluxed out before they measurably inhibit bacterial growth or
viability. Approaches that identify novel small molecule inhibitors of key bacterial enzymes often fail when
these small molecules cannot achieve effective intrabacterial concentrations - and our understanding of the
chemistries that would allow for penetration and retention is woefully incomplete.
In this application we use a diverse array of riboswitches, sensitive and specific RNA-based small molecule
sensors, as rapid and quantitative indicators that bacterial physiology has been perturbed. By multiplexing
several riboswitches that report on accumulation of the alarmones ZTP and ppGpp, as well as the toxic product
of increased SAM utilization, SAH, we can effectively screen for “signatures” of a bacterial response to sub-
MIC levels of small molecules. Our approach places these riboswitch reporters in isogenic MDR and efflux-
deficient P. aeruginosa strains, simultaneously yielding information about both physical and structural chemical
features that allow penetration and efflux-avoidance and identifying “hit” molecules that can be developed as
leads for new antibacterial agents. Our medicinal chemistry approach will build on both types of knowledge,
allowing novel anti-Pseudomonal compounds to be identified and optimized.

## Key facts

- **NIH application ID:** 9856975
- **Project number:** 5R01AI136794-03
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** RONALD R BREAKER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $1,114,844
- **Award type:** 5
- **Project period:** 2018-02-19 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9856975

## Citation

> US National Institutes of Health, RePORTER application 9856975, Exploiting Riboswitch Sensors to Reveal Antibiotics Uptake and Retention in Gram Negative Bacteria (5R01AI136794-03). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/9856975. Licensed CC0.

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