Dual-targeted inhibitors of bacterial RNA polymerase
Creators: name: Lin, Chih-Tsung
Publisher: name: No Publisher Supplied
Year: 2020

description: Resistance 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.