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

Lead Research Organisation: Newcastle University
Department Name: Biosciences Institute

Abstract

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.

Technical Summary

WWe will investigate the mechanism of action of a newly discovered natural rifamycin, B1, that has a novel mechanism of inhibition of bacterial RNA polymerase (RNAP), and through that bring new insights into mechanisms of transcription initiation. B1, unlike known rifamycins, inhibits synthesis of the first phosphodiester bond. In addition, B1 is up to ~1000 times more potent against rifampicin-resistant RNAPs than known rifamycins, and is active against rifampicin-resistant M. tuberculosis.
We will use a combination of organic chemistry, molecular biology and crystallography to understand the mode of action of B1 and how it manages to overcome rifampicin-resistance. We will apply semisynthesis and bioengineering to target the unique chemical groups of B1 in order to understand their functions. We will analyse B1 and derivatives by a large arsenal of in vitro transcription techniques to investigate unusual for rifamycins features of B1 action, proposed by preliminary data. Using crystallography and biochemistry we will solve the structures of B1 derivatives with promoter open complex and mutant RNAPs carrying the most frequent rifampicin-resistant mutations (which has already been achieved for B1 in preliminary work), and will use structure-based mutagenesis and in vitro assays to understand the structural principles of B1 ability to overcome rifampicin-resistant mutations. We will also investigate biochemically ADP-ribosylation (another rifampicin-resistance mechanism, which does not appear to affect B1) of rifampicin, B1 and its derivatives and the consequences for inhibition of transcription in in vitro assays.
The study will not only shed light on fundamental understanding of early stages of initiation of transcription and the plasticity of RNA polymerase active centre, but may highlight new targets for drug development and will be essential for the future development of B1 as an antimicrobial for the treatment of drug resistant TB.

Planned Impact

Translational research:
Description of a new inhibitor of RNAPs is of high importance for the field of biomedical sciences. The knowledge obtained in the proposed research will be of general interest to those, studying molecular mechanisms of transcription and their application for medicine and biotechnology. In addition, the research will obtain new information on functions of Arr proteins.

UK competitiveness (short-term):
The scientific disciplines of the proposal are very competitive internationally. The subject of the study is very novel (data concerning B1 is yet unpublished). Furthermore, our small consortium is currently has a monopoly on studying B1 and its derivatives, which will make the UK a leader in development of B1 and/or its derivatives as tools for probing RNAP function and as a potential drug lead against RIF-resistant infections, especially M. tuberculosis. The research will attract an exceptional European scientist (named in the proposal). These factors will strengthen the scientific competitiveness of the UK.

UK commercial sector (short-term):
Our fundamental work will provide vital information for future development, led by Industrial Partner Demuris Ltd, of B1 and/or its derivatives as new drugs against rifampicin-resistant bacteria, in particular, M. tuberculosis. Demuris has collaborations to test compounds against major pathogenic bacteria to support future drug development pathways. We have a good working relationship with Demuris and have regular meetings (see Pathways to Impact). Newcastle University and Demuris have filed a UK patent 1812078.2, covering rifamycin like antibiotics. Patent filings based on future development of B1 as a drug against rifampicin-resistant bacterial strains, building on the fundamental work in the application, are also anticipated.

Medicine (long-term):
The antibiotic resistance crisis is a serious global threat for human health and welfare. As a result, the development of new resistance-breaking antibiotics that are less susceptible to the rapid development of resistance has been identified as a top priority by the UK Government and the World Health Organization. 600,000 new rifampicin-resistant TB cases were reported in 2016, with, originating from them, MDR- and XDR-TB infections on the rise. B1 uniquely can overcome RIF-resistance, both, RNAP-based and Arr-based, and is active against MDR-M. tuberculosis. Therefore, in the longer-term, B1 and/or its derivatives will potentially be used as drugs against rifampicin-resistant diseases, including MDR- and XDR-M. tuberculosis, that are resistant to known rifamycins and other first and second line antibiotics.

Wider public:
In the short term, the proposed project will provide employment for two individuals at the postdoctoral level, thereby directly contributing to the national economy. The training of these PDRAs (and associated PhD, masters and undergraduate students) will benefit the UK biotechnology and pharmaceutical industries, as well as the academic base in the UK and its representation abroad, through the delivery of highly skilled individuals.
In the longer-term, potential beneficiaries will be health organisations and through them the wider public, both in developed and developing countries.
Additional benefits for the "wider public" will be in publicising the research via social media, press releases and interviews, which will continue to raise the awareness about the threat of antibiotic resistance, the value of antibiotic stewardship and the role of UK universities in combating these key global societal issues through fundamental biology research.

Capability, mechanisms for exploitation
The Impact activities will be managed by the PIs, RAs, and Demuris. It will be supported by Newcastle University Research and Enterprise Services (RES) and the press office. We will have regular formal meetings to promote exploitation every 3 months.

Publications

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Description Coupling of transcription with other cellular processes
Amount £2,086,031 (GBP)
Funding ID 217189/Z/19/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 08/2024