Rational design and molecular characterisation of novel bacterial RNA polymerase inhibitors

Bacterial infections are a major cause of human disease. Until recently most infections were easily treated with a range of antibiotics discovered and developed during the golden age of antibiotic discovery between 1940 and 1970. However, there has now been a dramatic rise in the incidence of bacterial resistance to existing antibiotics, leading to the possibility of returning to a pre-antibiotic era when only palliative care could be administered. A disturbing feature has been the emergence of bacteria multiply resistant to antibiotics, the so-called superbugs. Two very important examples of such bacteria are methicillin-resistant S.aureus (MRSA) and multiple drug resistant tuberculosis (MDR TB). New antibiotics are urgently required to combat these bacteria. The identification and characterisation of new inhibitors of RNAP, an essential bacterial enzyme, as proposed in this grant application, will provide new approaches to relieve the burden of disease caused by infectious drug resistant bacteria, . The newly identified inhibitors, or molecules subsequently derived from first-generation inhibitors, could become candidates for development as antibiotics. Ultimately it is hoped that new antibiotics, identified by the proposed research, will be used to treat life-threatening bacterial infections in man such as MRSA and TB. We anticipate that this will include patients suffering from these infections in the United Kingdom.

Prokaryotic RNA polymerase (RNAP) is an important target for antibacterial chemotherapy since this enzyme is essential for bacterial growth and survival and possesses features that distinguish it from mammalian counterparts. However, bacterial RNAP remains underexploited as an antibacterial drug target. During preliminary work to dentify new RNAP inhibitors we have employed commercial preparations of purified E. coli RNAP. In view of the clinical problems associated with multiple antibiotic resistant strains of S. aureus (e.g MRSA) and M. tuberculosis we now intend to develop RNAP molecular screening systems focused on these specific pathogens. We will purify recombinant RNAP from these organisms by affinity chromatography for in vitro screening of inhibitors. We will also develop recombinant systems for controlled down-regulation of RNAP in S. aureus to generate a cellular screening system specifically sensitised to RNAP inhibitors. Novel RNAP inhibitors will be generated in the School of Chemistry, University of Leeds, by virtual high throughput screening and de novo ligand design using the published high resolution crystal structure of Thermus thermophilus RNAP and appropriate homology models of the S. aureus and M.tuberculosis RNAP enzymes. Promising inhibitors, selected on the basis of their activity in the RNAP screens described above, will be evaluated for anti-microbial activity against clinical isolates of S.aureus and M. tuberculosis. The staphylococcal studies will be performed at Leeds and the MTB studies with our collaborators at the Novartis Institute for Tropical Diseases (NITD), Singapore. RNAP from T. thermophilus will be purified to reproduce the published crystallisation, as well as to begin co-crystallisation experiments with ligands selected as inhibitor leads. The Novartis Institutes for Biomedical Research (NIBR) in Cambridge, Mass, will be an additional collaborator. NIBR (and NITD) will advise on the possible further development of promising inhibitors.