Chemical biology evaluation of bacterial transcription Inhibitors

Abstract

Antibiotic resistance poses global health threat and it is urgent to identify new targets for novel antibacterial agent development. Bacterial transcription as a novel target has been identified and two new classes of compounds with antibacterial activities that target on different essential factors in bacterial transcription were synthesized. Herein, this thesis focuses on the biological evaluation on the two classes of bacterial transcription targeting inhibitors, i.e. the C3 derivatives and the MC4 derivatives. C3 derivatives are a novel class of transcription targeting inhibitors that can inhibit the bacterial transcription initiation by inhibition of the interaction between the clamp helix (CH) region of β' subunit of bacterial RNA polymerase (RNAP) and region 2.2 of σ factor through targeting the RNAPβ'- CH. Compounds that are designed to target the necessary β'- CH to inhibit the formation of holoenzyme are potential to be developed into new antibacterial drugs. Biological evaluation on these compounds was performed. The fluorescence images of live bacterial Bacillus subtilis cells were captured to analyze the mechanism of action of the compounds. Compounds were found to have mechanism of action consistent to the inhibition of bacterial transcription initiation. MC4 is a lead compound with antibacterial activity. The derivatives were designed to target the essential antitermination factor NusB. The interaction between NusB and NusE are critical for the formation of antitermination complex with bacterial RNAP. Compounds are designed to target NusB for the inhibition of NusB-NusE interaction and hence the rRNA synthesis in bacteria. Several biological evaluations of MC4 derivatives were performed to analyze their interaction with NusB. For instance, circular dichroism was measured to study the effect of compounds on the secondary structure of NusB. ITC assay was attempted to determine the dissociation constants of compounds and native mass spectrometry as a complementary method for determination. Fluorescence microscopic images of treated live B. subtilis cells were captured to analyze the mechanism of action of the compounds.