Techniques for Screening Translation Inhibitors

Abstract

The machinery of translation is one of the most common targets of antibiotics. The development and screening of new antibiotics usually proceeds by testing antimicrobial activity followed by laborious studies of the mechanism of action. High-throughput methods for new antibiotic screening based on antimicrobial activity have become routine; however, identification of molecular targets is usually a challenge. Therefore, it is highly beneficial to combine primary screening with the identification of the mechanism of action. In this review, we describe a collection of methods for screening translation inhibitors, with a special emphasis on methods which can be performed in a high-throughput manner.

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

“…However, their clinical use is still limited due to the cytotoxicity related side effects. Rapid development of high throughput screening tech niques [303], as well as machine learning [304] and com puter modeling, in combination with modern methods of structural analysis and chemical synthesis [84, 237 239] has given hope for rapid progress in the development of new drug derivatives with improved therapeutic proper ties. The impact of systems biology approaches on the search for and characterization of new inhibitors will also undoubtedly increase.…”

Safe and easy evaluation of tmRNA-SmpB-mediatedtrans-translation in ESKAPE pathogenic bacteria

“…Because of its biological properties, transposition of trans-translation into a nonhazardous system that could allow for rapid and easy evaluation of its activity would greatly help in the search for new antibiotics which target this system. While there are routine methods for screening the antimicrobial activity of compounds from chemical libraries, a combination of this primary screening with the specification of a molecular target is much harder to implement (Osterman et al, 2016). An ideal method would allow not just the identification of the targeted cellular process, but also its level of specificity toward a bacterial genus or species.…”

“…Furthermore, an easy quantitative and rapid analysis of the process should be possible even in small volumes. Reporter assays are the best candidates for efficient initial high-throughput screening (HTS) methods, as they can be quick and automated, as well as quite useful for screening unpurified mixtures of natural extracts (Osterman et al, 2016). Accordingly, we recently used a commercial reconstituted in vitro translation system (PURExpress) to create a reliable in vitro reporter system that detects the E. coli trans-translation activity (Guyomar et al, 2020).…”

Overview of 17,856 Compound Screening for Translation Inhibition and DNA Damage in Bacteria

“…An alternative to these methods is application of the reporter strains that respond to particular functional type of the inhibitor by upregulation of the gene, whose product is easy to detect [14,22]. Among the set of possible reporter genes, those coding for fluorescent proteins are preferable due to the lack of any reagent requirements, making application of other reporters, such as luciferases [18], much more expensive.…”

“…For example, there are reporters aimed in detection of beta lactams [24], tetracyclines [7], and macrolides [2,11]. Detection of specific classes of known antibacterials is of use, but it is not applicable for the discovery of new antibacterials [14,22].…”