Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N1G37) Methyltransferase (TrmD) Inhibitors.

Bacterial tRNA modification synthesis pathways are critical to cell survival under stress and thus represent ideal mechanism-based targets for antibiotic development. One such target is the tRNA-(N1G37) methyltransferase (TrmD), which is conserved and essential in many bacterial pathogens. Here we developed and applied a widely applicable, radioactivity-free, bioluminescence-based high-throughput screen (HTS) against 116350 compounds from structurally diverse small-molecule libraries to identify inhibitors of Pseudomonas aeruginosa TrmD ( PaTrmD). Of 285 compounds passing primary and secondary screens, a total of 61 TrmD inhibitors comprised of more than 12 different chemical scaffolds were identified, all showing submicromolar to low micromolar enzyme inhibitor constants, with binding affinity confirmed by thermal stability and surface plasmon resonance. S-Adenosyl-l-methionine (SAM) competition assays suggested that compounds in the pyridine-pyrazole-piperidine scaffold were substrate SAM-competitive inhibitors. This was confirmed in structural studies, with nuclear magnetic resonance analysis and crystal structures of PaTrmD showing pyridine-pyrazole-piperidine compounds bound in the SAM-binding pocket. Five hits showed cellular activities against Gram-positive bacteria, including mycobacteria, while one compound, a SAM-noncompetitive inhibitor, exhibited broad-spectrum antibacterial activity. The results of this HTS expand the repertoire of TrmD-inhibiting molecular scaffolds that show promise for antibiotic development.

[1]  Matthias Rarey,et al.  Automated Drawing of Structural Molecular Formulas under Constraints , 2004, J. Chem. Inf. Model..

[2]  Utkarsh Kapoor,et al.  Understanding RNA modifications: the promises and technological bottlenecks of the ‘epitranscriptome’ , 2017, Open Biology.

[3]  G. Vogel.  Meet WHO’s dirty dozen: The 12 bacteria for which new drugs are most urgently needed , 2017 .

[4]  J. Lescar,et al.  Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA (Guanine37-N1)-Methyltransferase (TrmD) by Restructuring the Active Site with a Tyrosine-Flipping Mechanism , 2019, Journal of medicinal chemistry.

[5]  Timothy R. Walsh,et al.  Tackling antibiotic resistance , 2011, Nature Reviews Microbiology.

[6]  J. I. Sulkowska,et al.  Restriction of S-adenosylmethionine conformational freedom by knotted protein binding sites , 2020, PLoS computational biology.

[7]  A. Byström,et al.  A primordial tRNA modification required for the evolution of life? , 2001, The EMBO journal.

[8]  Woody Sherman,et al.  Large-Scale Systematic Analysis of 2D Fingerprint Methods and Parameters to Improve Virtual Screening Enrichments , 2010, J. Chem. Inf. Model..

[9]  Regina Z. Cer,et al.  IC50-to-Ki: a web-based tool for converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding , 2009, Nucleic Acids Res..

[10]  Thomas J. Begley,et al.  tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence , 2016, Nature Communications.

[11]  Katrin Stierand,et al.  Molecular complexes at a glance: automated generation of two-dimensional complex diagrams , 2006, Bioinform..

[12]  S. Ehrlich,et al.  Essential Bacillus subtilis genes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Michelle F. Richter,et al.  Predictive compound accumulation rules yield a broad-spectrum antibiotic , 2017, Nature.

[14]  Jack Snoeyink,et al.  Nucleic Acids Research Advance Access published April 22, 2007 MolProbity: all-atom contacts and structure validation for proteins and nucleic acids , 2007 .

[15]  S. Yokoyama,et al.  Structural basis for methyl-donor–dependent and sequence-specific binding to tRNA substrates by knotted methyltransferase TrmD , 2015, Proceedings of the National Academy of Sciences.

[16]  J. Lescar,et al.  Backbone resonance assignment for the N-terminal region of bacterial tRNA-(N1G37) methyltransferase , 2018, Biomolecular NMR assignments.

[17]  Woody Sherman,et al.  Analysis and comparison of 2D fingerprints: insights into database screening performance using eight fingerprint methods , 2010, J. Cheminformatics.

[18]  Ning Gao,et al.  Selective inhibitors of bacterial t-RNA-(N(1)G37) methyltransferase (TrmD) that demonstrate novel ordering of the lid domain. , 2013, Journal of medicinal chemistry.

[19]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[20]  Thomas J. Begley,et al.  tRNA modifications regulate translation during cellular stress , 2014, FEBS letters.

[21]  Shigeyuki Yokoyama,et al.  Tertiary structure checkpoint at anticodon loop modification in tRNA functional maturation , 2009, Nature Structural &Molecular Biology.