# Structural Determinants of Permeation Barriers in Escherichia coli

> **NIH NIH R01** · UNIVERSITY OF VIRGINIA · 2024 · $607,833

## Abstract

PROJECT SUMMARY
Antibiotics represent the most successful class of drugs developed by modern science. They have spurred
numerous medical advances by facilitating invasive surgeries with minimal risk of infection. Today, a major
healthcare crisis looms as we are fast approaching a post-antibiotic era. Historically, it has proven to be much
more difficult to find agents that are active against Gram-negative pathogens compared to Gram-positive
pathogens. The primary reason is that Gram-negative bacteria have a unique asymmetric outer membrane (OM)
in addition to an inner membrane. The targets for most antibiotics reside beyond the OM, and thus these
molecules need to penetrate the OM to be active. Yet, the OM is uniquely effective in blocking the translocation
of small molecules, thus creating a major challenge for the field.
The Golden Era of antibiotics leveraged naturally abundant small molecules that were readily identified using
traditional methods; however, this methodology has proven to be much more difficult to be further mined for new
antibiotics during the past several decades. The next phase of antibiotic drug discovery has the potential to be
supported by our increasing collection of proteomics, genomics, and metabolomics data that will reveal promising
drug targets. Academia and industry could potentially exploit these data sets to design small molecule agents
that are potent and of high specificity. To accomplish this, the field fundamentally requires guiding principles
describing the molecular determinants of permeation into bacterial cells akin to the Lipinski’s rules of 5 (Ro5).
We propose to develop a novel fluorescence assay that measures the accumulation of small molecules in Gram-
negative pathogens based on a combination of HaloTag expression and anchoring of a biorthogonal epitope
within HaloTag. Our team will systematically (testing established antibiotics with known permeation profiles) and
broadly (screening a unique library of small molecules modified with an azide tag) apply this approach to
measurably grow our fundamental understanding of the molecular determinants of permeation. Additionally, we
will utilize the same platform to provide unprecedented spatial resolution of the distribution of small molecules
(including known antibiotics) in subcellular compartments.

## Key facts

- **NIH application ID:** 10851825
- **Project number:** 5R01AI178975-02
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** Marcos M. Pires
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $607,833
- **Award type:** 5
- **Project period:** 2023-05-31 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10851825

## Citation

> US National Institutes of Health, RePORTER application 10851825, Structural Determinants of Permeation Barriers in Escherichia coli (5R01AI178975-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10851825. Licensed CC0.

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