Construction of potent and specific inhibitors of M. tuberculosis redox enzymes using fragment screening methods

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

Mycobacterium tuberculosis (Mtb) is a human pathogen that causes the debilitating disease tuberculosis (TB). Recent reports from the World Health Organization (WHO) indicated that one third of the world's population is infected with TB, and the WHO declared a "global emergency" due to the worldwide spread of Mtb and since several Mtb strains are resistant to antibiotics that once formed the basis of effective TB treatments. For instance, there are now many Mtb strains that are resistant to the leading drugs rifampicin and isoniazid - referred to a multidrug resistant (MDR) TB. Other strains are resistant to many other drugs (extensively drug resistant, or XDR TB), and some are resistant to all major TB drugs (totally drug resistant, or TDR TB). The development of resistance to drugs by Mtb is a natural phenomenon caused by random mutations of bacterial DNA that result in changes to structures of proteins that prevent them being inactivated by particular antibiotics. The issues of Mtb drug resistance faced today arise from the gradual development and spread of resistance to several TB drugs over many years. In the same time period (~1960s to 1990s) there were very few new TB drugs developed, which resulted in a dearth of effective TB antibiotics. While new drugs are now coming through the pipeline, there is still a shortage of effective TB drugs and there are other complicating factors - among which is the issue of TB patients being more susceptible to infection by HIV/AIDS and vice versa. To address this issue, our plan is to progress a novel route to the development of new drugs that can inhibit the activities of a group of enzymes (called P450s) which are known to be essential to the survival of Mtb bacteria and to their ability to sustain infection in their human host. While previous TB drugs have usually been selected by extensive screening of large libraries of complex chemicals, we will use instead a relatively new approach to development of new and effective drugs against Mtb. The process of fragment screening involves using relatively small libraries of chemicals of generally quite small size. These libraries of chemicals are mixed with the proposed enzyme targets (the P450s) and in cases where different "fragments" bind to the P450s, this will be detected by methods including changes to the thermal stability of the proteins. With knowledge that selected fragment bind the P450s, the three dimensional structures of the proteins will be determined using the technique of X-ray crystallography, which will also reveal exactly where fragments bind within the P450s. This information is essential for the further development of the fragment screening process, since once the binding positions of a number of the chemical fragments inside the P450s are known a strategy can be developed to use chemistry approaches to link fragments bound in adjacent positions in order to make larger compounds that show much better selectivity for the particular P450 enzyme. Cycles of chemical "improvement" are usually needed, but these can lead to very effective antibiotic drugs. Our plan is to use this fragment screening and development programme on Mtb P450s essential for survival of Mtb (a P450 called CYP121A1), for its ability to survive in the lungs by using human cholesterol (CYPs 124A1, 125A and 142A1), and for its ability to infect and spread (called virulence, CYP128A1). The antibiotic drugs developed against each of these Mtb P450 proteins will be optimized for tight binding to their targets, and then their ability to terminate growth of Mtb/kill the bacterium will be verified by testing the inhibitors directly against the bacterial pathogen. Further important studies will look at the responses of Mtb to treatment with these inhibitors - in particular looking for changes in the profile of chemicals the bacteria produces in order to understand how the inhibitors affect the Mtb and to enable improvements in inhibitor effectiveness.

Technical Summary

Mycobacterium tuberculosis (Mtb) poses a grave threat to global health. A third of the world's human population is infected by Mtb, and recent years have seen increasing failure of existing TB drugs (developed in the 1940s to 60s) due to Mtb drug resistance by mechanisms including target protein mutations that diminish or abolish binding of inhibitor drugs. We are in an era where Mtb variants resistant to multiple drugs have spread around the world - including strains resistant to at least the two leading drugs (rifampicin and isoniazid, MDR TB) through to those also resistant to second line drugs (extensively drug resistant, XDR TB) or to all major drugs (totally drug resistant, TDR TB). New antibiotics and new approaches to TB drug development are desperately needed to replace failing antibiotics. The approach proposed here involves developing inhibitors of Mtb cytochrome P450 protein systems, where importance of the selected enzymes to bacterial viability, survival in the host and virulence are established. Building on extensive preliminary work underpinning this proposal, we will use fragment screening methods combined with structural biology, enzymology and bacterial MIC determination in order to produce and validate new drug leads. These will be developed against (i) the essential CYP121A1 P450, where we will build on existing tight binding scaffolds to make derivatives with improved affinity and cell penetration; (ii) the cholesterol degrading CYPs 124, 125 and 142 - where we will exploit structural similarity of the enzymes to produce inhibitors that inhibit all these P450s to prevent host cholesterol catabolism; and (iii) CYP128A1 and its partner Stf3, which influence Mtb virulence by hydroxylation and sulfation of menaquinone. Collectively, we will generate, optimize and validate new TB drug leads that attack crucial redox systems in Mtb, and use these reagents in antimicrobial studies to probe the Mtb metabolome/transcriptome to define modes of action.

Planned Impact

The proposal builds on substantial preliminary research done by the applicants to demonstrate that fragment screening technology can be used to produce potent inhibitors of M. tuberculosis (Mtb) cytochrome P450 (and partner) enzymes to provide new drug leads that are so desperately needed to treat infections with strains of Mtb that have become resistant to several antibiotics that once formed the basis of effective TB treatments. The emergence of drug resistant, multidrug resistant (MDR, resistant to at least rifampicin and isoniazid), extensively drug resistant (XDR, additionally resistant to a fluoroquinolone and to second line TB drugs) and totally drug resistant (TDR, resistant to all major TB drugs) strains of Mtb poses grave threats to human health worldwide. The appearance of these strains also brings focus on the lack of development of new anti-TB drugs in recent decades, with most of the "traditional" drugs effective against Mtb produced in the 1940s-60s. As resistance to these antibiotics emerged and spread, the lack of effective alternatives was exposed. While this has led to drug development in recent years (with two new drugs now available only for MDR TB treatment), there remains a dearth of new TB drugs at a time when these are most desperately needed. The research proposed in this programme is to progress a fragment screening approach to develop new types of anti-TB drugs that target P450 enzymes known to be crucial to bacterial viability, virulence and to ability to survive in the host through their ability to catabolise host cholesterol as a carbon source in the macrophage. Our previous research has proven the feasibility of this approach, particularly in developing potent inhibitors of the essential Mtb CYP121A1 enzyme. The current programme will extend this work and will also target the menaquinone hydroxylase CYP128A1 and its partner sulfotransferase Stf3, and the cholesterol hydroxylases (CYPs 124, 125 and 142) in order to produce novel compounds as effective new leads against a resilient human pathogen.

Beneficiaries from this research programme include researchers in the area of M. tuberculosis pathogenicity, compound screening and drug targeting. These include scientists in academia and industry who study the microbiology and biochemistry of Mtb with the aim of finding new reagents to treat drug-resistant forms of Mtb. In view of the current crisis in developing new TB drugs, this is an area of particular focus across the globe. Progress in this area has enormous potential to save lives and improve quality of life of millions of individuals. The success of a fragment-based strategy for TB drug development will also inspire researchers to adopt this relatively novel approach to produce drugs to address antibiotic resistance in other pathogenic microbes. This, in turn, should be transformative in the field and enhance the likelihood of identifying effective new antimicrobials through exploitation of new technological approaches. This should inspire further biomedical researchers to adopt the technology and biotechnologists/industrialists to make improvements to fragment screening strategies to streamline processes to enable faster development of potent drugs. Allied studies of the Mtb transcriptome and metabolome following treatment of Mtb with new compounds will also facilitate new insights into drug mechanisms and bacterial responses, and facilitate improvements in drug efficacy, highlighting to other researchers the value of these approaches in improving antibiotic potency. Researchers (PDRA and affiliated students) will be trained in areas including fragment screening, structural biology and transcriptomics, providing key skills for future employment in areas such as antimicrobial research and drug development in academic or industrial sectors. Collectively, this research study has potential to deliver important new drug molecules to provide a basis for new treatments for a deadly disease.
 
Description This grant is in its early stages and is a collaborative, 2-centre grant between Prof. Munro's group (PI) at the University of Manchester (MIB Building) and the group of Prof. Chris Abell at Cambridge. The subject of the grant is to use fragment screening and other chemical design methods in order to produc tight-binding inhibitors of cytochrome P450 enzymes and other key enzymes in the human pathogen in the bacterial pathogen Mycobacterium tuberculosis. In the early stages of this grant we have cloned, expressed and purified several cytochrome P450 enzymes and have solved the structures of a number of P450 enzymes bound to fragments, as well as larger inhibitor molecules. These studies provide a basis for the elaborations of these prototype molecules, ultimately to produce specific and tight-binding enzyme inhibitors.
Exploitation Route Tuberculosis is a deadly disease and is the largest killer among infectious diseases. The aim of this project is to develop new antibiotics/enzyme inhibitors for use in the treatment of Mycobacterium tuberculosis. Various routes forward include the further chemical elaboration of the compounds developed to date, including their testing for ability to kill M. tuberculosis cells. Once developed, potent inhibitors should be tested against drug- and multi-drug resistant strains of M. tuberculosis. At this stage, collaborations with industrial partners may be sought in order to enable further drug development and testing to identify the most effective drugs and to establish how effective these drugs are using in vitro and in vivo analysis.
Sectors Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Collaboration between the Andrew Munro (Manchester) and Chris Abell (Cambridge) groups for anti-tuberculosis drug compound development 
Organisation University of Cambridge
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution The BBSRC grant entitled "construction of potent and specific inhibitors of M. tuberculosis redox enzymes using fragment screening methods" is a 2-centre (University or Manchester and University of Cambridge) BBSRC-funded project - with the lead PI being Prof. Andrew Munro at Manchester and the PI at Cambridge being Prof. Chris Abell. The project is still in its early stages. Key aspects of the work done at Manchester include the expression and purification of cytochromes P450 and other enzymes from the pathogen Mycobacterium tuberculosis, the characterization of their interactions with substrates and inhibitors (including compounds developed by the Cambridge team) and structural studies (using X-ray crystallography) to define the binding modes of inhibitors, leading to the generation (using fragment screening technology) of tight-binding inhibitors designed to be effective in inactivating key enzymes in M. tuberculosis.
Collaborator Contribution The partners in this 2-centre project are Chris Abell's team from the University of Cambridge. The Cambridge team have expertise in fragment screening methods - where small compounds ("fragments") that bind to target enzymes are identified using high-throughput NMR and/or thermal shift assays. These initial "hits" are then sent to Manchester to assess whether they bind efficiently to the target enzyme(s) and (if successful) to guide further studies to produce derivatives that bind more tightly to the active site. Fragment growing, merging and elaborating strategies should then enable the production of tighter binding inhibitors, some of which (for the P450s) should bind efficiently to the P450 heme iron. Other classes of potential inhibitor compounds are also being produced at Cambridge. Compounds shown to bind tightly to the target enzymes will then be tested for their ability to prevent M. tuberculosis growth (or kill the bacteria outright) to determine MIC values in collaboration with researchers at the Crick Institute.
Impact The project is in its early stages, but ongoing work has identified a number of good inhibitors for M. tuberculosis P450 enzymes important to the viability of M. tuberculosis. Inhibitors have been synthesised and were shown to be effective in binding target P450 enzymes, and crystal structures defining the inhibitor binding modes in M. tuberculosis P450 enzymes have been solved. Ongoing work is focusing on the elaboration of these compounds to produce more effective compounds that can kill the M. tuberculosis bacterium.
Start Year 2018
 
Description Participation in an open day or visit at my research institution - Manchester Institute of Biotechnology Open Day - annual event from 2012 onwards 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact The Manchester Institute of Biotechnology holds an annual open day at which PhD students, Postdocs and Academics engage with the visiting secondary school students and display scientific experiments. The main aims are to engage with potential undergraduates of the future who have interests in science as a career (e.g. Chemistry, Biochemistry, Biotechnology etc).
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016