While bacterial infections present major threats to human health, antibacterial discovery has been difficult, particularly with the single-enzyme-based strategy. Two main reasons stand out: the occurrence of resistance mutations in the single enzyme target, and the insufficient chemical diversity of compound libraries used for screening inhibitors of the single enzyme target. A recent genome-wide analysis has ranked the enzyme TrmD as a leading antibacterial target, because it is essential for bacterial growth, broadly conserved across bacterial species, distinct from its human counterpart, and has a """"""""druggable"""""""" site that drug-like molecules mimicking S- adenoscyl methionine (AdoMet) can bind to. TrmD is unlike targets of clinical antibiotics (the ribosome, DNA gyrase and topoisomerases, and cell-wall biosynthesis enzymes). Instead, TrmD is a tRNA enzyme that modifies G37 to m1G37 using AdoMet as the methyl donor. We hypothesize that TrmD is attractive for singe- enzyme-based drug discovery;because targeting TrmD would reduce bacterial efflux, allowing intracellular accumulation of multiple drugs for rapid cell killing before the occurrence of resistance. We also suggest that drugs targeting TrmD must explore novel chemical space and diversity. While pharmaceutical companies AstraZeneca (AZ) and GlaxoSmithKline (GSK) have made intense efforts to target TrmD as a member of growth-essential enzymes in bacteria, their anti-TrmD program has stalled, in part due to the use of radioactive 3H-AdoMet in a high-throughput screening (HTS) assay. We propose instead to develop and optimize a novel fluorescence assay that is more robust and cost-effective and is based on a principle different from that of the 3H assay. The development of this fluorescence assay will enable discovery of novel classes of inhibitors to probe how targeting TrmD can cause collateral damage on bacterial efflux in an innovative growth arrest mechanism distinct from the mechanisms of antibiotics in clinical use. Using E. coli TrmD (EcTrmD) as a model, preliminary work has validated the robustness and amenability of the fluorescence assay to the HTS format.
Aim 1 will further improve parameters of the assay.
Aim 2 will validate the assay for HTS-ready by collaboration with the NSRB/ICCB-Longwood (NSRB/ICCB-L) screening facility at Harvard. Following the validation, we will launch a large-scale screening campaign at Harvard to screen ~500,000 compounds from diverse chemical libraries. False positives will be removed in counter screens and the hit pool will be screened using secondary, tertiary, and phenotypic assays.
Aim 3 will validate hits for the ability of targeting EcTrmD in the whole cell and will improve qualities of hits by chemical optimization based on our recently developed tRNA-bound crystal structure of TrmD in complex with sinefungin (a non-reactive analog of AdoMet). Hits with desired criteria will be tested for growth arrest and in vivo efficacy. The identified hits will serve as chemical probes to understand the growth-arrest mechanism of targeting TrmD and as leads for antibiotic discovery to address the global burden of bacterial infectious disease.

Public Health Relevance

Despite major threats of bacterial infections to public health, single-enzyme-based antibacterial discovery has been challenging, due in part to the occurrence of resistance mutations in the single enzyme target and in part to the limited diversity of compound libraries used for screening inhibitors of the single enzyme. We hypothesize that the tRNA-modification enzyme TrmD is attractive for addressing these problems, because its targeting would require exploration of new chemical diversity of drugs and because these drugs would decrease the expression of efflux pumps and hence sensitize to the action of other drugs, possibly before the occurrence of resistance to the TrmD drug. We propose to develop a novel HTS assay for EcTrmD in a large screening campaign to identify inhibitors that can be used to test our hypothesis and to serve as leads for developing antibiotics with mechanism of action distinct from those in clinical use.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Fabian, Miles
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Thomas Jefferson University
Schools of Medicine
United States
Zip Code
Masuda, Isao; Takase, Ryuichi; Matsubara, Ryuma et al. (2018) Selective terminal methylation of a tRNA wobble base. Nucleic Acids Res 46:e37
Oprescu, Stephanie N; Chepa-Lotrea, Xenia; Takase, Ryuichi et al. (2017) Compound heterozygosity for loss-of-function GARS variants results in a multisystem developmental syndrome that includes severe growth retardation. Hum Mutat 38:1412-1420
Masuda, Isao; Igarashi, Takao; Sakaguchi, Reiko et al. (2017) A genetically encoded fluorescent tRNA is active in live-cell protein synthesis. Nucleic Acids Res 45:4081-4093
Hou, Ya-Ming; Matsubara, Ryuma; Takase, Ryuichi et al. (2017) TrmD: A Methyl Transferase for tRNA Methylation With m1G37. Enzymes 41:89-115
Hou, Ya-Ming (2016) Single-Turnover Kinetics of Methyl Transfer to tRNA by Methyltransferases. Methods Mol Biol 1421:79-96
Ardell, David H; Hou, Ya-Ming (2016) Initiator tRNA genes template the 3' CCA end at high frequencies in bacteria. BMC Genomics 17:1003
Gall, Aaron R; Datsenko, Kirill A; Figueroa-Bossi, Nara et al. (2016) Mg2+ regulates transcription of mgtA in Salmonella Typhimurium via translation of proline codons during synthesis of the MgtL peptide. Proc Natl Acad Sci U S A 113:15096-15101
Falk, Marni J; Gai, Xiaowu; Shigematsu, Megumi et al. (2016) A novel HSD17B10 mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression. RNA Biol 13:477-85
Christian, Thomas; Sakaguchi, Reiko; Perlinska, Agata P et al. (2016) Methyl transfer by substrate signaling from a knotted protein fold. Nat Struct Mol Biol 23:941-948
Gamper, Howard B; Masuda, Isao; Frenkel-Morgenstern, Milana et al. (2015) Maintenance of protein synthesis reading frame by EF-P and m(1)G37-tRNA. Nat Commun 6:7226

Showing the most recent 10 out of 23 publications