Novel approaches to discovering inhibitors of essential bacterial enzymes

Abstract

Antimicrobial resistance (AMR) is a major threat to human health and continues to increase, with common clinical infections developing resistance to ‘last resort’ antibiotics. In 2050, it is predicted 10 million people a year will die as a result of AMR; thus, it is hard to overstate the immediate risks and the unmet need for novel antibiotics. A common approach for the discovery of novel antimicrobials is through the identification of compounds that inhibit essential bacterial enzymes. Such enzymes are often found ubiquitously throughout the bacterial domain and are involved in essential cellular processes. One such targetable enzyme is tRNA-(N1G37) methyltransferase (TrmD), which contributes in the posttranscriptional modification of tRNA.

TrmD methylates guanine positioned at nucleotide 37 (located in the anticodon arm) of specific tRNAs at the N1 position. This modification is vital for reading frame maintenance to ensure the fidelity of a newly synthesized protein sequence. In addition to its essentiality, TrmD displays distinct structural and biochemical differences to the human Trm5 equivalent thus making it an ideal antibacterial candidate. Multiple industrial and academic groups have identified potent in vitro inhibitors through classical radiochemical assays. Unfortunately, these compounds normally show limited antimicrobial activity within whole cells, thought to be a result of insufficient cellular penetration and excessive bacterial efflux. It is hypothesized an intracellular high-throughput assay would overcome this current bottleneck and result in the discovery of highly potent TrmD inhibitors with sufficient cell uptake properties; however, current radiochemical assays are unable to measure intracellular TrmD inhibition specifically. With the use of molecular beacons (MBs), a novel fluorescence based activity assay to monitor TrmD activity in vitro has been developed and validated through a variety of orthoganol techniques. In addition, the analysis of known TrmD inhibitors utilizing this fluorescence assay correlated well with previously published data that utilized radiochemical methodology. Furthermore, with the use of trmD knockdown mutants, the application of this assay to measure intracellular TrmD activity has been explored, through the measurement of m¹G37 levels in purified samples of E. coli tRNA^Leu_UAG and within fixed E. coli cells with the use of fluorescence in situ hybridization (FISH).

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Identifiers

ORCID for Peter L. Roach: orcid.org/0000-0001-9880-2877

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