Description
TitleDual-targeted inhibitors of bacterial RNA polymerase
Date Created2020
Other Date2020-10 (degree)
Extent1 online resource (xiv, 105 pages) : illustrations
DescriptionResistance to antibacterial agents has become a grave threat to public safety and human health. In this work, in hopes of helping to combat this ever-increasing threat, we have designed, synthesized, and characterized two novel classes of compounds that inhibit the bacterial RNA polymerase (RNAP) enzyme and, as a result, inhibit bacterial growth. These compounds are dual-targeted inhibitors of bacterial RNAP. Through the covalent conjugation of two non-identical bacterial RNAP inhibitors, each having a distinct binding site on the bacterial RNAP enzyme, we have created compounds that can bind to two different sites on the enzyme, either in parallel or simultaneously. As a result, these dual-targeted inhibitors are able to overcome a resistance substitution that would prevent a single-target inhibitor from binding to the enzyme – making it much more difficult for a bacterium to develop resistance to these novel compounds.
The first dual-targeted inhibitors examined in this work, Class I inhibitors, are composed of a first moiety, which binds to the rifamycin/sorangicin (Rif/Sor) binding site on bacterial RNAP, that is covalently-linked to a second moiety, which binds to the immediately adjacent GE23077 (GE) binding site on bacterial RNAP. The two moieties in one molecule of a Class I dual-targeted inhibitor are able to interact simultaneously with one molecule of bacterial RNAP through the two neighboring binding sites. Several Class I dual-targeted inhibitors were synthesized through the conjugation of Rif-derivatives or Sor-derivatives to derivatives of GE, to yield RifaGEs or SoraGEs, respectively. Biochemical experiments demonstrate that Class I dual-targeted inhibitors potently inhibit Escherichia coli (E. coli) RNAP, and are able to overcome resistance arising from a substitution in either the Rif/Sor binding site or the GE binding site. The crystal structure of Thermus thermophilus (Tth) RNAP in complex with a SoraGE demonstrates that both moieties on Class I dual-targeted inhibitor interact simultaneously with each of their binding sites on RNAP.
Class II dual-targeted inhibitors, the other primary focus of this study, are composed of a first moiety, which binds to the Rif binding site, that is covalently-linked to a second moiety, which binds to the non-adjacent binding site of the N-aroyl-N-aryl-phenylalaninamides (AAPs) on bacterial RNAP. Since the binding sites are non-adjacent, two molecules of a Class II dual-targeted inhibitor are able to bind simultaneously to every one molecule of bacterial RNAP. Several Class II dual-targeted inhibitors were been synthesized through the conjugation of Rif-derivatives to AAP-derivatives, to yield RifaAAPs. Biochemical experiments demonstrate that RifaAAPs potently inhibit Mycobacterium tuberculosis (Mtb) RNAP, exhibit potent antibacterial activity against Mtb in culture, and overcome resistance arising from a substitution in either the Rif binding site or the AAP binding site. The crystal structure of Mtb RNAP in complex with a RifaAAP indicates that every one molecule of bacterial RNAP has two molecules of RifaAAP bound to it, one located at the Rif binding site and one located at the AAP-binding site.
This work provides a basis for developing new inhibitors of bacterial RNAP that can overcome the most common mechanism of rifampin-resistance in clinical isolates. Dual-targeted inhibitors may play an important role in development of new antibiotics that could overcome the serious threat of clinical antibiotic resistance.
NotePh.D.
NoteIncludes bibliographical references
Genretheses, ETD doctoral
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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