# Discovery of Gram-negative permeable chemical probes for tRNA methylation

> **NIH NIH R01** · THOMAS JEFFERSON UNIVERSITY · 2020 · $576,878

## Abstract

Project Summary. Gram-negative (Gram (-)) bacteria are intrinsically resistant to drugs, due to a double
membrane structure that acts as a permeability barrier to drugs and as an anchor for efflux pumps. Many Gram
(-) bacteria have developed multi-drug resistance, which poses one of the most pressing issues in modern
medicine. Antibiotics are barred and extruded from cells and cannot reach high enough intracellular
concentrations to exert a therapeutic effect. While efforts have focused on targeting one membrane protein at a
time, resistance mutations can quickly develop. We propose to target the m1G37-tRNA methylation catalyzed
by TrmD to inhibit biosynthesis of multiple membrane proteins simultaneously, thus reducing drug barrier and
efflux and accelerating bactericidal action. TrmD is a bacteria-specific S-adenosyl-methionine (AdoMet)-
dependent methyl transferase that controls accuracy of the protein-synthesis reading frame. Loss of TrmD
increases +1 frameshifts and terminates protein synthesis prematurely. We have discovered that genes for
multiple membrane proteins and efflux pumps in E. coli and other Gram (-) bacteria contain TrmD-dependent
codons near the start of the reading frame. We hypothesize that targeting TrmD will reduce protein synthesis of
all of these genes. By reducing multiple membrane- and efflux-proteins at once, we propose that targeting
TrmD offers a novel solution to an unmet need. While AstraZeneca (AZ) has attempted to target TrmD, the
isolated hits lacked the cell-permeability needed to exhibit an antibacterial effect. We hypothesize that
successful targeting must identify compounds that are cell-permeable and selective for TrmD over the human
counterpart Trm5. To test this hypothesis, we have developed and optimized a cell-based fluorescence assay
for E. coli TrmD (EcTrmD), in which we will mix a 1:1 ratio of an E. coli mCherry (mCh)-expressing strain
dependent on TrmD for survival and a separate YFP-expressing strain dependent on Trm5 for survival to
discover cell-permeable compounds that selectively inhibit the TrmD-dependent but not the Trm5-dependent
strain. In Aim 1, we will use this cell-based assay, which is high-throughput screening (HTS)-ready, in a large-
scale campaign to discover cell-permeable and selective inhibitors of EcTrmD. We will screen a diverse
collection of ~180,000 compounds and a collection of 10,000 natural products to identify inhibitors and remove
false positives. In Aim 2, we will assess hits in secondary assays to determine their potency and mechanism of
action. We will fractionate natural products to active compounds. We will also test hits on Gram (-) bacteria
Salmonella and Pseudomonas aeruginosa. In Aim 3, we will use whole-cell assays to identify hits that inhibit
cell growth and display TrmD-deficient phenotypes. We will assess initial structure-activity relationship (SAR)
of each cluster of hits by analysis of ~20 analogs selected from in silico modeling in our TrmD crystal struc...

## Key facts

- **NIH application ID:** 9857551
- **Project number:** 5R01AI139202-02
- **Recipient organization:** THOMAS JEFFERSON UNIVERSITY
- **Principal Investigator:** Ya-Ming Hou
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $576,878
- **Award type:** 5
- **Project period:** 2019-02-01 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9857551

## Citation

> US National Institutes of Health, RePORTER application 9857551, Discovery of Gram-negative permeable chemical probes for tRNA methylation (5R01AI139202-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9857551. Licensed CC0.

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