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Title: Novel inhibitors of RNA polymerase and mechanisms of their action
Author: Ceccaroni, Lucia
ISNI:       0000 0004 7961 0564
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2018
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Drug resistance is an increasing global problem, with very few novel compounds in development, and transcription is a potent target for novel antibiotics. In this work, we set out to search for novel compounds inhibiting RNA polymerase (RNAP) and to investigate their mode of action, in collaboration with a university-based bio-tech company, DemurisTM. DemurisTM lab screened a vast collection of Actinomycete strains, and identified strains that produced compounds potentially targeting transcription. The purified compounds were then analysed by us in vitro. Hundreds of compound were tested throughout this project. Most of the compounds were identified as known relatives of Rifampicin, a widely used antibiotic that targets bacterial RNAP. Some compounds, however, were identified as distinct molecules with potentially interesting modes of actions, and were chosen for further characterization. We found that compound DEM31376, later identified as antibiotic Madurastatin C1, inhibited transcription initiation. However, a secondary metabolite, GE23077, was found to be co-produced and co-purified with it. GE23077 is known to inhibit transcription by unknown mechanism. We showed that the mixture (likely GE23077) inhibits binding of the initiating nucleotide in the "i" site of the RNAP active centre. DEM10430 was identified as the sequence dependent DNA intercalator Echinomycin. It prevents translocation of RNAP.We found that DEM10430 does not inhibit transcription initiation but blocks translocation of RNAP during elongation of transcription 1 bp upstream of its binding site. DEM30355B1 is a novel derivative of antibiotic Kanglemycin A that belongs to the Ansamicyn family, and which spontaneously degrades into a secondary form, DEM30355/B2. We showed that both forms inhibit transcription initiation by blocking synthesis of RNA longer than 4 nucleotides, similar to Rifampicin. Interestingly, however, DEM30355B1 displayed activity against Rifampicin-resistant RNAPs, suggesting its potential clinical usefulness. Our study suggests that looking for new antibiotics targeting transcription is still promising.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available