MICA: Novel mode of RNA polymerase inhibition by a new natural rifamycin, which is active against rifampicin-resistant RNA polymerases and bacteria

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Rifamycins are a family of mainly semisynthetic antibiotics (including rifampicin, RIF) that inhibit bacterial RNA polymerase (RNAP), the essential enzyme accomplishing the first step of gene expression - transcription. RIF is a key drug in the treatment of tuberculosis - the leading cause of death by infectious disease, with 10.4 million new cases and 1.7 million deaths annually. Development of RIF-resistance by the causative agent of tuberculosis bacterium Mycobacterium tuberculosis is the major problem in treatment of tuberculosis, with 600,000 new RIF-resistant TB cases reported each year. Furthermore, RIF-resistant M. tuberculosis can also develop further resistance to other first, second and third line antibiotics (multi-drug-resistance (MDR-Mtb) and extensive-drug-resistance (XDR-Mtb)), resulting in 240,000 additional deaths per year. Therefore, overcoming RIF-resistance would be an important step in treatment of drug-resistant tuberculosis. However the development of new rifamycins has stalled, due to both RIF-resistance problems and the fact that the areas of the molecule known to allow semi-synthetic modification, without loss of activity, have been limited to those originally discovered in the 1950s. Previous attempts to introduce modifications on other positions resulted in inactivation of the antibiotic. Recently, MICA co-applicants and the Industrial Partner Demuris Ltd. discovered a new natural rifamycin, referred to as B1, which carries unique substituents in previously underexplored regions of the molecule. Remarkably, B1 is up to three orders of magnitude more effective against RIF-resistant RNA polymerases, with the most clinically frequent mutations having no significant effect on its efficacy, and also showed antibiotic activity against RIF-resistant MDR-Mtb. Furthermore, unlike any other rifamycins, that sterically block propagation of the growing nascent RNA through RNAP, B1 strongly inhibits synthesis of the initial dinucleotide, indicating that it has a different mode of action to previous rifamycins. However the mechanisms of inhibition of RNAP and of overcoming RIF-resistance by B1 are not known. Finally we found that B1 is also tolerant to ADP-ribosylation, a modification of the rifamycins that inactivates them leading to RIF-resistance - a poorly understood resistance mechanism adopted by some Mycobacteria. It is, however, not clear if B1 cannot be ADP-ribosylated or if its binding to RNAP is not affected by the modification. Understanding the mode of action of RNAP inhibitors at the molecular level has proven to be a powerful research tool in understanding functions of RNAP itself. We therefore propose a fundamental study into understanding the mechanisms of B1 action, and to use B1 (and its derivatives) as molecular probes to shed new light on the early steps of the complex process of initiation of RNA synthesis, plasticity of RNAP active centre, and basic principles of RIF-resistance. Employing a combination of organic chemistry, molecular biology and crystallography, we will modify unique groups of B1 and test the resulting derivatives in a very wide range of in vitro transcription experiments. Derivatives will be crystallised with transcription promoter open complex and RNAP holoenzymes carrying the clinically most frequent RIF-resistant mutations (which has already been successful with B1). Furthermore we will analyse in vitro ADP-ribosylation and its consequences on transcription. This information will help the understanding of the basic processes at the very first steps of RNA synthesis and the plasticity of RNAP active centre, providing essential knowledge towards overcoming RIF-resistance in the future. These studies may identify new targets for antimicrobials, and will be also important for future development of B1 or its derivatives as antibiotics against IRF-resistant Mtb by the Industrial Partner.