A Continuous Assay For Monitoring The Synthetic And Proofreading Activities Of Multiple Aminoacyl-Trna Synthetases For High-Throughput Drug Discovery

Keywords

aaRSs; Aminoacyl-tRNA synthetase; aminoacylation; drugs; editing; High-throughput screening; mechanism of translation; methods; proofreading; protein-RNA interactions; Therapeutics; translation; tRNA; TRNA

Abstract

Aminoacyl-tRNA synthetases (aaRSs) catalyze the aminoacylation of tRNAs to produce the aminoacyl-tRNAs (aa-tRNAs) required by ribosomes for translation of the genetic message into proteins. To ensure the accuracy of tRNA aminoacylation, and consequently the fidelity of protein synthesis, some aaRSs exhibit a proofreading (editing) site, distinct from the aa-tRNA synthetic site. The aaRS editing site hydrolyzes misacylated products formed when a non-cognate amino acid is used during tRNA charging. Because aaRSs play a central role in protein biosynthesis and cellular life, these proteins represent longstanding targets for therapeutic drug development to combat infectious diseases. Most existing aaRS inhibitors target the synthetic site, and it is only recently that drugs targeting the proofreading site have been considered. In the present study, we developed a robust assay for the high-throughput screening of libraries of inhibitors targeting both the synthetic and the proofreading sites of up to four aaRSs simultaneously. Thus, this assay allows for screening of eight distinct enzyme active sites in a single experiment. aaRSs from several prominent human pathogens (i.e., Mycobacterium tuberculosis, Plasmodium falciparum, and Escherichia coli) were used for development of this assay.

Publication Date

5-4-2018

Publication Title

RNA Biology

Volume

15

Issue

4-5

Number of Pages

659-666

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1080/15476286.2017.1397262

Socpus ID

85045462801 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/85045462801

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