Bacteriophage-derived Inhibitors of the Bacterial RNA Polymerase

Many activities that allow organisms to grow, remain alive and viable rely on molecules resembling small machines. In bacteria, the machine which is responsible for accessing information contained in the genes is the RNA polymerase (RNAp), and the process by which the RNAp accesses the information contained in the genes is collectively called gene expression. Therefore, since the RNAp carries out an essential function necessary for the survival of the bacterial cell, the RNAp is also an attractive target for drugs to treat bacterial infections. For instance, the clinically widely used antibiotic rifampicin inhibits the RNAp. However, the frequent emergence of rifampicin-resistant harmful bacteria necessitates research into new compounds to combat bacterial infections by inhibiting the RNAp. Our research focuses on naturally-occurring small proteins which are potent inhibitors of the RNAp and display several of the desirable features for potential development into novel antibiotic-like compounds for inhibiting the RNAp. Here, we will study precisely how three such naturally-occurring RNAp inhibitors, called Gp2, P7 and Gp5.7 inhibit the RNAp from the bacterium Escherichia coli (for Gp2 and Gp5.7) and Xanthomonas orzyae (a bacterial pathogen that causes rice blight). We will employ structural biology to unravel how Gp2, Gp5.7 and P7 look like and interact with the RNAp. We will employ state-of-the art biochemistry and molecular biology to study how Gp2, Gp5.7 and P7 inhibit the RNAp. We will employ a technique called "next-generation sequencing" to study the impact of Gp2 on RNAp activity, i.e. gene expression, at a whole-cell level. In summary, our results will (1) provide novel insights into how the activity of the bacterial RNAp can be controlled, in this case inhibited, by non-bacterial and naturally-occurring factors; (2) establish a solid framework and industrial links for development of novel antibiotic-like lead compounds targeting the bacterial RNAp and (3) provide useful resources for synthetic biology applications.

All RNA synthesis in bacteria is accomplished by the large multisubunit enzyme RNA polymerase (RNAp) by a process called transcription. Since transcription is a major regulatory checkpoint during bacterial gene expression the RNAp is often targeted by many transcription factors (TFs) which modulate its activity to activate or repress the transcription of genes. The bacterial RNAp is also a proven antimicrobial target and is inhibited by the antibiotic rifampicin. Bacteriophages (phages) are viruses that infect bacteria. Not surprisingly, the bacterial RNAp is commonly targeted by phage encoded TFs which modulate bacterial RNAp activity to serve viral needs. Some phage TFs are potent inhibitors of the bacterial RNAp (hereafter called pTF-I). Understanding how pTF-I interact with the bacterial RNAp, the mechanisms by which they inhibit the RNAp and the impact they have on bacterial gene expression at a global level are important and as yet unaddressed questions in the field of bacterial gene regulation. Results from such studies could provide novel insights into strategies that control bacterial gene expression as well as tools and inspiration for synthetic biology and antibacterial drug discovery. Here, we propose to study the mechanism of action of three potent pTF-Is from structural, biochemical and global perspectives. For one pTF-I, called Gp2, we will explore the feasibility of identifying and evaluating synthetic drug-like compounds that mimic Gp2 in collaboration with our lead drug discovery services partner Domainex. Furthermore, we will explore the effect of Gp2 on RNAp activity in E. coli at a genome-wide scale. In summary, our results will provide novel mechanistic insights into how the bacterial RNAp is subjected to regulation by non-bacterial TFs, establish a solid framework for the development of structurally novel antibacterial lead compounds and potentially identify novel antibacterial targets and provide useful resources for synthetic biology.

The research proposed focuses on how naturally-occurring non-bacterial factors (derived from bacteriophage (phage) i.e. viruses that infect bacteria) inhibit an important conserved bacterial enzyme, called RNA polymerase (RNAp), which is responsible for all RNA synthesis in bacteria. In addition, we will collaborate with our lead drug discovery partners Domainex to use one such non-bacterial factor as an inspiration for screening and evaluating novel synthetic lead drug-like compounds that have the potential to be developed into a novel type(s) of antibiotic. Therefore, in addition, to contributing to advancing our understanding of bacterial gene regulatory processes and systems in general, we expect that results will also be beneficial to those seeking to exploit such knowledge for the identification and/or development of novel targets for antibacterial drugs to combat or prevent diseases caused by pathogenic bacteria. Thus, the proposed research will impact microbiology research in the academic and industrial settings. To this end, we will ensure that results are rapidly published in peer-reviewed journals and by presenting our work at national and international research meetings. Where possible we will make our findings available to the wider public (by publication in popular science magazine etc.). Any possibilities for commercial exploitation (i.e. evaluation of targets for drug discovery etc.) will be realised via locally existing structures (e.g. Imperial Drug Discovery Facility, IC Innovations etc.) and through existing links with Domainex (our lead drug discovery services partner).

In the proposed research we will employ state-of-the-art biochemical, structural biology and post-genomic methods to study how the phage encoded RNAp inhibitors operate at structural, mechanistic and system-wide level. This provides an excellent platform to enhance training of the associated PDRAs as well as undergraduate/postgraduate students, who will contribute to the proposed research (e.g. by conducting mini-projects etc.). Such trained PDRAs (and associated PhD, masters and undergraduate students) will represent the "next generation" of experimental bioscientists and are likely to benefit the biotechnology and pharmaceutical industries, as well as the academic base in the UK and its representation abroad. We therefore anticipate medium term economic benefits arising from a well-trained UK research base, reflected in maintaining internationally competitive and research intensive universities and associated industries.

Since one aspect of our research involves liaising with UK bioindustry to screen and evaluate synthetic antibiotic-like novel lead compounds against bacterial RNAp, the proposed research could potentially inform new antibiotic development by the pharmaceutical industry, and therefore has the potential to impact the government and healthcare policies in the long term.