| Author: | Zheng, Yingbo |
| Title: | Development of bacterial transcription inhibitors targeting the RNA polymerase clamp-helices region |
| Advisors: | Ma, Cong (ABCT) |
| Degree: | Ph.D. |
| Year: | 2023 |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | 309 pages : color illustrations |
| Language: | English |
| Abstract: | Bacterial infection has been identified as the primary cause of several common human diseases. Classic antibiotics, which experienced “the golden age” in the mid-twentieth century, have become inefficient due to the rapid development of antibiotic-resistant bacteria. N protein utilization substance G (NusG) is an essential bacterial factor that regulates transcription, including elongation and termination processes. As a conserved transcription factor, its interactions with RNA polymerase (RNAP) have been observed to maintain a consistent binding model across different bacteria. Considering these features, their interactions have the potential to serve as a novel and promising target for the discovery of small-molecule antibiotics. The discovery and rational design of antibiotics targeting the protein-protein interactions (PPIs) between NusG and RNAP are based on the X-ray crystal structure of E. coli NusG in complex with RNAP. We generated four pharmacophores from predicted key residues on clamp helices of the β′ subunit of RNAP (β′-CH) which contribute to the formation of PPIs with NusG. Using this pharmacophore model, we conducted the virtual screening to discover a promising lead compound, AW00783, from the drug-like MiniMaybridge database. Testing its antimicrobial activities revealed a minimum inhibitory concentration (MIC) value of 256 µg/mL against Gram-positive strains. Modifications to this lead compound have been classified into two categories: aminoethyl and aromatic derivatives. Seventy-two compounds have been synthesized and tested for their antimicrobial activities against selected Gram-positive and Gram-negative strains. Among these derivatives, most exhibited antimicrobial activities against Gram-positive strains, while only specific compounds from the aminoethyl derivatives showed activities against Gram-negative strains. Compound G1-36 from aminoethyl derivatives exhibited the best antimicrobial activity against Gram-negative Escherichia Coli with the MIC value of 8 µg/mL. Meanwhile, compound GII-16 from aromatic derivatives showed the best activity with the MIC value of 1 µg/mL against Gram-positive Staphylococcus aureus. SARs analyses were conducted on these derivatives, predicting the trifluoromethyl and hydroxy groups that contribute to interactions with RNAP. In silico docking simulation displayed the possible docking models of candidate compounds, including GI-19, GI-36, GI-39 and GII-16. Although their docking models showed some differences, it provided structural evidence supporting the hypothesis of necessary functional groups targeting the clamp helices. Further cytotoxicity assay and fluorescence microscopy experiment were conducted on candidate compounds from the aminoethyl derivatives. In vitro cytotoxicity assay demonstrated their mild cytotoxicity, with a 50% cytotoxic concentration (CC50) value of 10-40 μM. Additionally, fluorescence microscopy demonstrated that these candidate compounds were capable of disrupting the normal localization of bacterial transcription complexes at the cellular level. They were also tested and found to exhibit good antimicrobial activities against selected Bacillus subtilis strains. |
| Rights: | All rights reserved |
| Access: | open access |
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