An inhibited state of bacterial RNA polymerase as a framework for antibiotic design.

Successfully managing bacterial infections in humans and in animals has been possible through the use of antibiotics, agents that kill the bacteria and or stop them from growing. Resistance to these compounds is now wide spread, in part because bacteria mutate to evade the actions of these agents and in part because for all known natural antibiotics, a resistance mechanism is genetically encoded in the organism which makes the anti bacterial compound. These intrinsic resistance mechanisms are portable and can transfer between bacteria to help spread drug resistance.

Recently we have unraveled, in great detail, how one natural protein inhibitor works to prevent an essential bacterial machinery from functioning. This machine accesses and converts the genetic information stored in DNA in a process called transcription which eventually allows proteins to be made. Proteins carry out the majority of cellular activities. Therefore, without the proper function of this essential machine, the bacteria can't survive. Interestingly this machine is relatively conserved among bacteria but very different from humans and animals, so it is a good target for broad spectrum antibiotics to work. We plan to use our newly acquired in-depth knowledge to design new non-native compounds that will inhibit and/or kill bacteria. To do so we will use methods that allow us to look at how these molecules are organized in 3-dimension to understand the exact detailed interactions at the molecular and atomic levels, and use this information to design small inhibitors of this essential machinery.

We have a wide range of methods to study in mechanistic details this essential machinery, called the bacterial multi-subunit RNA polymerase, and so can work out how best to inhibit it based on our knowledge of a novel naturally occurring factor which normally inhibits the machine. This approach has not been previously taken, rather naturally occuring small molecules have been sought which inhibit the RNA polymerase machine.

RNA polymerases are essential enzymes required to access information stored in DNA via the process of transcription. The bacterial RNA polymerase is a validated anti microbial target, although only a handful of antibiotics are available to inhibit it. Our X-ray crystallographic determination of a 3.8 Å structure of a previously undescribed inhibited state of the E. coli RNA polymerase (RNAP), comprising of RNAP with the full length inhibitory factor sigma54, now places us in a unique position to derive novel low molecular weight inhibitors of this essential enzyme. Importantly, we identify at least five hotspots within RNAP that can be inhibited and in our structures, these hotspots, some of them are widely separated on RNAP, are inhibited by distinct structural modules/domains of sigma54. This new discovery therefore opens up avenues to design small peptides/molecules to target previously unexplored areas in RNAP. We plan to extend our structural studies to obtain further inhibited states of the RNAP and gain insights into the organization of surfaces between sigma54 and the RNA polymerase core enzyme. We will determine new crystal structures using fragments of the sigma54 protein based on our structures of the full length protein in order to define precisely molecular details on specific inhibitory states. Using biochemical approaches we will define surfaces of RNAP that can accommodate inhibiting in trans acting amino acid sequences as leads for new small molecule inhibitors of bacterial transcription.

Many important scientific advances are only found to be useful many years after the original discovery. However, our recent work on the structures and molecular mechanisms of transcription inhibition by sigma54 provides a unique opportunity to explore our new knowledge for designing novel antibiotics that target the central transcription machinery, the RNA polymerase itself. In addition to develop potential cross spectrum antibiotics due to conserved and essential nature of RNAP, sigma54 and its activators are also involved in bioremediation by a number of bacteria. A detailed understanding of these gene regulation systems could be exploited to enhance bioremediation activities.

The interdisciplinary approach of the collaborating groups, ranging from x-ray crystallography, peptide and fragments design, in vitro biochemical and biophysical characterisation, in vitro and in vivo transcription studies, will greatly enhance training of the associated RAs, especially with respect to their ability to work within interdisciplinary teams. Such trained RAs (and associated PhD, masters and undergraduate students) are likely to benefit the biotechnology and pharmaceutical industries, as well as the academic base in the UK and abroad. We therefore anticipate medium term economic benefits arising from a well-trained UK and international research base, reflected in maintaining internationally competitive research intensive universities and associated industries.

We will interact using journal clubs, seminars and workshops with the Industry Clubs at ICL, as well as the MRC CMBI at ICL to show how our earlier studies on gene control mechanism now places us in a world leading position to work out new ways to inhibit the multi subunit bacterial RNA polymerase to mange bacterial pathogens of animals and humans, and the threat of increasing AMR.