# Administrative Supplement for GM126210 - Probe discovery for tRNA methylation

> **NIH NIH R01** · THOMAS JEFFERSON UNIVERSITY · 2020 · $11,310

## Abstract

Multi-drug resistance is one of the most pressing issues in treating bacterial infections. Antibiotics are
extruded from cells and cannot reach high enough intracellular concentrations to exert a therapeutic effect.
This problem is most formidable with Gram-negative (Gram (-)) bacteria, due to their double-membrane
structure. While efforts have focused on targeting one efflux pump at a time, resistance mutations can quickly
develop. We propose to target the m1G37-tRNA methylation catalyzed by TrmD to inhibit protein synthesis of
multiple pumps simultaneously, thus reducing drug efflux and accelerating bactericidal action. TrmD is a
bacteria-specific S-adenosyl-methionine (AdoMet)-dependent methyl transferase that controls the accuracy of
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 medical need.
While AstraZeneca (AZ) has attempted to target TrmD, progress has stalled, because isolated inhibitors lacked
both the selectivity against the human counterpart (Trm5) and the activity against bacterial growth. We
hypothesize that successful targeting must explore novel chemical space and diversity to capture the unique
conformation of AdoMet when bound to TrmD. To test this hypothesis, our multi-PI team will use E. coli TrmD
(EcTrmD) as a model and apply a series of high-throughput screening (HTS) assays, each unique to our
team, to isolate potent and selective inhibitors. In Aim 1, we will use an enzyme-based fluorescence assay to
isolate active inhibitors of EcTrmD. This fluorescence assay is HTS-ready, has all of the required reagents in
hand, and exhibits advantages over the radioactivity-based (3H-AdoMet) assay. We will screen the collection of
~370,000 compounds in the NCATS SMR (small molecular repository) at Sanford Burnham Prebys (SBP) and
will apply human Trm5 in a counter screen to remove non-selective compounds. In Aim 2, we will use
cheminformatics to prioritize hits. We will assess hits in a multitude of secondary assays to determine their
inhibition potency and modality. In Aim 3, we will screen hits with our whole-cell assays to isolate compounds
that inhibit cell growth and display phenotypes specific to TrmD deficiency, including reduced drug efflux. We
will assess the structure-activity relationship of each hit by analysis of ~20 analogs from commercial vendors
and determine the binding modality using a computer-aided approach based on our ternary TrmD crystal
structure in complex with a bound tRNA and sinefungin (a non-reactive analog of AdoMet). We will determine
hits for ...

## Key facts

- **NIH application ID:** 10146603
- **Project number:** 3R01GM126210-03S1
- **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:** $11,310
- **Award type:** 3
- **Project period:** 2018-06-01 → 2021-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10146603

## Citation

> US National Institutes of Health, RePORTER application 10146603, Administrative Supplement for GM126210 - Probe discovery for tRNA methylation (3R01GM126210-03S1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10146603. Licensed CC0.

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