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High-throughput Screening Assays for Antibacterial Drug Discovery Employing RNA Targets

Bacterial riboswitches are natural aptamers in UTR region of a mRNA molecule that can directly bind a small target molecule and effect the gene expression. For time being 12 bacterial riboswitch classes are discovered that regulate gene expression through translation prevention, transcription termination, and self-cleavage. The encounter of one or more riboswitch classes in 15 Human bacterial pathogens raises the hopeful of using them as novel targets for antibacterial drug discovery. The demand for novel antibiotics is constantly increasing since more Human bacterial pathogen stems are becoming resistant to long time used antibiotics. In this regard novel antibacterial drug targets may be valuable to pharmaceutical industry and may be a commercial success.

Figure 1. The secondary structures of wild-type and FRET glmS ribozymes. (A) An unimolecular glmS ribozyme from B subtilis. Arrow identifies the site of ribozyme-mediate cleavage stimulated by GlcN6P (1). (B) A bimolecular glmS ribozyme construct derived from S. aureus. This construct differs from the wild-type glmS ribozyme due to truncation of the P1 stem and the use of a 15-nucleotide substrate RNA (shown in grey). The substrate is labeled with a Cy3 acceptor at its 5`-end and a 5/6-FAM donor at its 3`-end.The FRET ribozyme construct was used for HTS arrays.

I have been involved in the development and application of an assay for glmS riboswitch. The glmS riboswitch is a ribozyme that cleaves itself in the presence of glucose amine 6-phosphate. It is present in 4 Human bacterial pathogens. It was straight forward to develop a cleave-based high-throughput screening assay for the glmS riboswitch using FRET (Fig. 1). The rest riboswitch classes only bind a small target molecule without self-cleavage. Thus, the development of a cheap and effective biochemical high-throughput screening assay for 11 riboswitch classes is not a straight forward task. The application of computational high-throughput screen assays for those riboswitch classes may be a valuable option for the riboswitch-based antibacterial drug discovery. The automated pipeting system used for HTS arrays is shown in Fig.2.

Figure 2. A pipeting robot system for fully automated high-throughput screening arrays.

1. Kennet Blount, Izabela Puskarz, Robert Penchovsky, Ronald Breaker - Development and application of a high-throughput assay for glmS Riboswitch Activators – 2006, RNA Biolоgy, 15558584, Q1 (Biochemistry, Genetics and Molecular Biology), IF – 5,546

2. Robert Penchovsky, Stoilova C.C. - Riboswitch-based antibacterial drug discovery using high-throughput screening methods – 2013, Expert Opinion on Drug Discovery, 1746-0441, Q1 (Pharmacology, Toxicology and Pharmaceutics) , IF – 4,676

3. Robert Penchovsky & Martina Traykovska - Designing drugs that overcome antibacterial resistance: where do we stand and what should we do? – 2015, Expert opinion on drug discovery, Q1 (Pharmacology, Toxicology and Pharmaceutics), IF – 4,66

4. Aikaterini Valsamatzi-PanagiotouKatya B. Popova & Robert Penchovsky - Chapter 9: Drug Discovery for Targeting Drug Resistant Bacteria – 2020, Sustainable Agriculture Reviews 46, Mitigation of Antimicrobial Resistance Vol. 1, Tools and Targets:.

5. Aikaterini Valsamatzi-PanagiotouKatya B. Popova & Robert Penchovsky - Chapter 1: Strategies for prevention and containment of antimicrobial resistance – 2020, Sustainable Agriculture Reviews 49 Mitigation of Antimicrobial Resistance Vol. 2, Natural and Synthetic Approaches.