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

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

Bibliographic Details
Main Author: Myers, Mason Thomas
Language:English
Published: Ohio University Honors Tutorial College / OhioLINK 2021
Subjects:
Biochemistry
Chemistry
RNA
Drug Discovery
T-box Riboswitch
Computational Screening
Fluorescence
Ligand Binding
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1619173211823351
id ndltd-OhioLink-oai-etd.ohiolink.edu-ouhonors1619173211823351
record_format oai_dc
collection NDLTD
language English
sources NDLTD
topic Biochemistry
Chemistry
RNA
Drug Discovery
T-box Riboswitch
Computational Screening
Fluorescence
Ligand Binding
spellingShingle Biochemistry
Chemistry
RNA
Drug Discovery
T-box Riboswitch
Computational Screening
Fluorescence
Ligand Binding
Myers, Mason Thomas
Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA
author Myers, Mason Thomas
author_facet Myers, Mason Thomas
author_sort Myers, Mason Thomas
title Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA
title_short Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA
title_full Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA
title_fullStr Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA
title_full_unstemmed Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA
title_sort combining primary specificity screenings for drug discovery targeting t-box antiterminator rna
publisher Ohio University Honors Tutorial College / OhioLINK
publishDate 2021
url http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1619173211823351
work_keys_str_mv AT myersmasonthomas combiningprimaryspecificityscreeningsfordrugdiscoverytargetingtboxantiterminatorrna
_version_ 1719458397365993472
spelling ndltd-OhioLink-oai-etd.ohiolink.edu-ouhonors16191732118233512021-08-03T07:17:10Z Combining Primary Specificity Screenings for Drug Discovery Targeting T-box Antiterminator RNA Myers, Mason Thomas Biochemistry Chemistry RNA Drug Discovery T-box Riboswitch Computational Screening Fluorescence Ligand Binding 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. 2021-05-18 English text Ohio University Honors Tutorial College / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1619173211823351 http://rave.ohiolink.edu/etdc/view?acc_num=ouhonors1619173211823351 restricted--full text unavailable until 2023-05-01 This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

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