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Thesis_Myers_Mason_Final_220421.pdf (3.12 MB)

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Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA

Myers, Mason Thomas
http://orcid.org/0000-0002-8333-1424
http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1619173211823351

Abstract Details

2021, Bachelor of Science (BS), Ohio University, Chemistry.
As the threat of antibiotic resistant infections and outbreaks looms, there has been a reinvigorated interest in identifying new therapeutics to target alternative targets in species primed for developing resistance. One such target is the antiterminator sequence of the T-box riboswitch, an important regulatory motif that acts as an `on’ switch for important protein synthesis genes in Gram-positive bacteria. The antiterminator is kinetically favored in transcription of T-box regulated genes, but is thermodynamically unfavored to its counterpart terminator sequence, which contains many of the same nucleotides and prevents gene expression through transcription termination or enveloping the Shine-Dalgarno sequence, preventing translation. The antiterminator is stabilized through interaction with the acceptor end of uncharged tRNA, and as such is responsive to the cellular concentration ratio of charged and uncharged tRNA. As a thermodynamically unstable and highly conserved regulatory element, the T-box antiterminator has been the focus of drug-design efforts to create ligands that would preclude or destabilize tRNA binding to the antiterminator, disrupting protein biogenesis ultimately leading to cell death. In an effort to devise a new primary, high-moderate throughput compound screening to find small molecules which bind to the antiterminator mechanism of the T-box riboswitch with high specificity, this thesis investigates a hybrid assay combining computational and experimental techniques. Computational docking of libraries of compounds using a receptor grid developed from the antiterminator NMR solution structure (PDB: 1N53) is used to identify a selection of compounds with favorable chemical features which bind to the antiterminator with high selectivity and strong bonding values. These compounds can then be tested in a single temperature fluorescence assay against three similar, but structurally disparate models based on the T-box antiterminator to identify ligand affinity and binding specificity, important aspects of drug discovery research. Agreement between computational and experimental techniques will lead to the identification of common structures or trends in the molecules tested which effectively bind and modulate the antiterminator structure, enhancing the foundational knowledge required for pharmacophore development of a T-box antiterminator inhibitor. Two compound libraries, the MedChemExpress FDA-approved plus library and the ZINC database natural metabolite subset, were tested using this combined assay approach. In addition, a selection of laboratory compounds known to bind RNA were also tested in an inverse sequence of the assay, completing fluorescence screenings followed by computational assays. Of the candidate molecules identified in computational screenings , four compounds were tested in the experimental assays, two from each library. One of these compounds, amodiaquine, was found bind to the T-box antiterminator with structure -dependent specificity, and the compounds screened from the laboratory `training’ set all had structure-dependent specificity. The results of this project indicate that while specificity is not well determined by computational screening of a compound library, annotation of compound interaction by receptor regions improves prediction of relative ligand binding strengths. In addition, the quinoline ring system appears to be an intriguing moiety for RNA drug design, appearing in multiple compounds that bind the antiterminator to affect its structure in a model-dependent manner. In sum, the results of this assay support the combination of computational and experimental assays to better predict RNA-small molecule binding in drug-discovery efforts targeting the T-box riboswitch.
Jennifer V. Hines, Dr. (Advisor)
96 p.
Biochemistry; Chemistry
RNA; Drug Discovery; T-box Riboswitch; Computational Screening; Fluorescence; Ligand Binding

Recommended Citations

Citations

  • Myers, M. T. (2021). Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA [Undergraduate thesis, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1619173211823351

    APA Style (7th edition)

  • Myers, Mason. Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA . 2021. Ohio University, Undergraduate thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1619173211823351.

    MLA Style (8th edition)

  • Myers, Mason. "Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA ." Undergraduate thesis, Ohio University, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1619173211823351

    Chicago Manual of Style (17th edition)

ouhonors1619173211823351
118
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This open access ETD is published by Ohio University Honors Tutorial College and OhioLINK.