A fragment based screening approach to rationalizing M. tuberculosis P450 molecular selectivity

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

The development of methods for the determination of entire DNA sequences of humans and other organisms has revealed huge amounts of new information in terms of identification of novel proteins and enzymes and unusual and unexpected chemical processes that take place within cells. From perspectives such as antibiotic therapy and biotechnological applications, there are obviously numerous opportunities once the substrates and reactions catalyzed by these novel enzymes are established. However, considerable efforts are often required to establish the function of a newly identified enzyme if there is no initial knowledge of the physiological role it plays. In addition, identification of specific inhibitors of its function can also be laborious and involve screening of enormous libraries of chemicals. However, recent years have seen the advent of a novel type of approach to defining molecules that bind to purified enzymes. This fragment based screening (FBS) technology involves analysis of binding of much more limited libraries of small molecules than would be used in typical compound screens as used, for example, in the Pharmaceutical sector. Initial 'hits' from the FBS approach may bind the target enzyme only quite weakly, but with the help of protein structural methods their position of binding can be determined accurately. Once a number of such hits are obtained, the combined information on their binding locations and the nature of the chemistry in these sites and the surrounding environment can provide a basis for 'elaborating' the structure of the molecules such that tighter binding is achieved, or even chemically 'merging' fragments bound at adjacent sites to enable construction of much tighter binding molecules as potent inhibitors of the target enzyme. This FBS method is now increasingly used in industry (in areas such as drug development) as a complementary method to the more typical 'high throughput screening' techniques that involved huge chemical libraries. In the current proposal, we will exploit and develop the FBS technology in studies of a class of enzyme called cytochromes P450 (P450s) from the TB-causing bacterium Mycobacterium tuberculosis (Mtb). The P450s are enzymes that bind oxygen to an iron atom in a heme group bound to the protein, and then 'activate' the oxygen to enable the insertion of an oxygen atom into their substrates, which are typically lipid molecules that bind in the active site of the enzyme, close to the heme group. A large number (20) of P450s are found in Mtb, and it is recognized that many of these are essential enzymes for bacterial survival and for their infection of the host. However, there is a paucity of information on the substrates and physiological functions of the majority of the P450s. This project will use FBS methods to define fragments that bind to selected Mtb P450s involved in cholesterol metabolism and in other unusual biochemistry, and then define their binding mode using structural biology methods. The identities and positions of binding of the fragments will then be used in further work to chemically join fragments and to add functionalities to fragments in order to create much tighter binding molecules as specific inhibitors of catalytic functions of the P450s, and as probes of their mechanism. Further, we will make new types of libraries that contain fragments of lipids, steroids and other molecules that are typical substrates for P450s, and use FBS in a novel way to identify substrate molecules for 'orphan' Mtb P450s with unknown function - again using a combination of chemical synthesis and structural biology to 'home in' on true substrates for these P450s, and so provide new knowledge on Mtb's complex lipid biochemistry. This application will thus provide important new information on the physiological chemistry of an important bacterium, and drive new applications of FBS in enzyme substrate identification through technical developments in the methodologies used.

Technical Summary

Cytochrome P450 (P450) enzymes are a superfamily of oxygenases that perform an array of physiologically important reactions in organisms from bacteria through to man, including steroid and bioactive lipid syntheses, and xenobiotic transformations. With increasing numbers of P450 (CYP) genes identified from genome sequences, it is apparent that there is a large untapped resource of uncharacterized oxidase enzymes of unknown specificity. In Mycobacterium tuberculosis (Mtb) there are 20 P450s, with substrates definitively identified for ~6. The purpose of this proposal is to exploit and develop fragment based screening (FBS) technology to identify small molecule ligands that bind the active site of a panel of key Mtb P450 enzymes (using X-ray crystallography to define the binding modes), and then to (i) use a combination of chemical elaboration and 'merging' of fragments binding at adjacent positions, to iteratively improve their efficiency of binding and potency as inhibitors and as probes of structural/catalytic features of targeted P450s, and (ii) to generate novel libraries containing fragments representing both likely substrates classes for P450s (fatty acids, steroids, polyketides) and unusual lipids prevalent in and/or peculiar to Mtb, and then to exploit FBS with these libraries to generate/identify physiologically relevant substrate-like molecules, with reference to active site structure of the target P450s and knowledge of the metabolomics of Mtb. Studies on effects of inhibitory and substrate-like molecules on Mtb growth and transcriptional responses will also be done. The project will exploit FBS, structural biology, synthetic chemistry and various spectroscopic methods to generate novel inhibitors for Mtb P450s involved in cholesterol oxidation and secondary metabolite synthesis, and (in a development of the technological applications) to identify substrates for 'orphan' P450s to enhance knowledge of the biochemistry of a biomedically important bacterium.

Planned Impact

The research proposal aims to exploit and develop fragment-based screening (FBS) technology to enable both (i) the generation of tight binding ligands as inhibitors and mechanistic probes for individual cytochrome P450 (P450) enzymes from Mycobacterium tuberculosis (Mtb); and (ii) the pinpointing and confirmation of substrate and substrate-like molecules using specially designed fragment libraries composed of fragments representative of both 'typical' P450 substrates (fatty acids, steroids, polyketides etc) and of lipid molecules peculiar to the metabolism of Mtb and related mycobacteria. The work proposed is multidisciplinary, involving protein expression and isolation, enzyme-ligand binding analysis using NMR, ITC and other techniques, structural biology and synthetic chemistry, and the ultimate aim is the exploitation and development of FBS methodology to provide routes to both specific inhibitors and substrate identification for a group of key enzymes in Mtb, ultimately enhancing the knowledge base on the biochemistry of Mtb and on the roles of P450 enzymes in its physiology. Beneficiaries from the research include scientists in industrial/academic sectors working on Mtb from perspectives of both (i) understanding its unusual lipid biochemistry, physiology and infectivity, and (ii) developing therapeutic strategies to combat the pathogen. TB is a global health problem and huge numbers of scientists worldwide are engaged in fundamental and applied research in the area. This extends from Pharma with major programmes on TB therapeutics through to University labs exploring Mtb's biological chemistry, metabolomics and genetics. The proposal features a combination of academic and applied research that will be of interest to scientists researching fundamental Mtb biochemistry, to technologists able to apply further the novel approaches and applications of FBS methodologies detailed in this application, and to biochemists/industrialists who can capitalise on novel information forthcoming on the roles of P450 enzymes in Mtb physiology and on tight-binding inhibitory ligands that can be exploited for mechanistic studies and to inhibit activities of P450s critical to viability and cholesterol metabolism for energy generation. From an even wider perspective, TB affects not only an increasing number of the UK and world population (with drug resistant strains spreading), but is also an important disease of cattle, with bovine TB and badgers as a vector of infection currently being a controversial scientific area. In this respect, research in the TB area is of major interest worldwide, with the disease endemic in numerous third world countries and with several antibiotics redundant as a consequence of the proliferation of drug-resistant and MDR strains of Mtb and other related mycobacterial pathogens. Thus, research proposed here (with direct impact on understanding of Mtb physiology and enzymology) could be of great relevance to national/international agencies as well as influencing Pharma by advancing technologies in the area and by identifying new therapeutic targets and strategies for intervention. The timescale for such impact is difficult to predict, but we expect that the developments in FBS approaches and concomitant work to identify/validate specific P450 inhibitor and substrate-like molecules will yield numerous compounds and novel insights into Mtb P450 molecular selectivity in 2-3 years. According to outcomes from this work and allied studies validating specificity and potency of molecules generated, we expect the research to inspire similarly directed therapeutic studies and adoption of related screening approaches in academia/industry soon after relevant publications emerge. We are also confident that the range of approaches (spectroscopic, structural, chemical and microbiological) will provide the PDRA appointees with a range of professional skills suited to career development in both academic and industrial settings.

Publications

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Guengerich FP (2013) Unusual cytochrome p450 enzymes and reactions. in The Journal of biological chemistry

 
Description The main purpose of this project was to use the new methods of fragment screening (also known as fragment based screening, FBS) to identify small chemicals that bind to enzymes known as cytochromes P450 (or P450s) that are found in the human pathogenic bacterium Mycobacterium tuberculosis. The P450s are enzymes that bind and activate oxygen - allowing them to catalyze the insertion of oxygen atoms into substrate molecules that bind in the active sites of these enzymes. The substrates bind close to a heme group that binds the oxygen and inserts it into the substrate. There are a large number of P450s in M. tuberculosis, and we (and others) have shown that a number of these enzymes are crucial for the survival of M. tuberculosis and/or for its ability to infect the host organism. By using FBS, we have (in collaboration with Chris Abell's team at the University of Cambridge) been able to identify small chemicals (fragments) that bind to a range of different M. tuberculosis P450 enzymes that have been produced in the Munro lab at Manchester. This process is done using spectroscopic and protein stability methods (NMR and thermal shift techniques), and once fragments found to bind to a particular P450 are identified, work proceeds to quantify their affinity for the target P450, and then to determine their mode of binding to the enzyme using protein crystallography and X-ray diffraction methods. With this work completed, the next phase is to identify fragments bound sufficiently close in space in the P450 protein such that they can be chemically linked or merged (or otherwise chemically extended) to enable the production of tighter binding inhibitors that become more specific for the target P450 enzyme. An iterative process of further improvement of the next-phase inhibitors (following the same steps as above) then occurs until potent inhibitors are generated. In this work, this outcome has been achieved very effectively for the M. tuberculosis P450 CYP121 - which we showed to be an essential gene for bacterial viability and that we know produces an unusual cyclic dipeptide that likely has a crucial role in bacterial survival in the host. Tight binding inhibitors were developed using FBS (including molecules that bind tighter than does its cyclic dipeptide substrate) and are now undergoing evaluation for their potency against the M. tuberculosis bacterium. Earlier stage molecules were evaluated in the same way. Fragments hits have already been obtained against a series of other M. tuberculosis P450s (including cholesterol hydroxylases such as CYP125; and novel P450 enzymes including CYP144 and CYP143). A novel method by which a fragment library of compounds based on fragments of known P450 enzyme substrates was also developed, and used to probe effectively the substrate specificity of the M. tuberculosis CYP126 P450. Substantial progress has been made, and work is continuing in this area to produce a range of specific inhibitors for different M. tuberculosis P450 enzymes that could be effective new antibiotics that are much needed against multidrug resistant strains of the pathogen. Further papers in this area have been submitted as of early 2016.
Exploitation Route The aim is to produce highly specific inhibitors of Mycobacterium tuberculosis P450 enzymes that are important for viability and infectivity of the pathogen. Inhibitors developed in this way might be used effectively as antibiotics against the bacterium. Cytochrome P450s from M. tuberculosis have proven highly amenable to fragment screening approaches and work has led to development of a number of inhibitor and active site probe molecules for several of the M. tuberculosis P450s. Extending these studies and further improving the affinity of specific fragment-derived inhibitors against key M. tuberculosis P450 enzymes has potential for identification of novel TB therapeutics.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description A number of publications from this joint Manchester/Cambridge collaboration have been produced that demonstrate strong progress in the development of M. tuberculosis (Mtb) cytochrome P450 isoform specific inhibitors and probes of substrate selectivity. The research combines the expertise of the Manchester group in P450 expression and structural/biochemical analysis with that of the Cambridge group in fragment screening and chemical synthesis. This combined approach has been applied to a number of Mtb P450 enzymes that are known to be important for M. tuberculosis bacterial viability and infectivity, and molecules produced have been evaluated for their potency against M. tuberculosis and their potential for antibiotic exploitation evaluated. The effectiveness of the novel fragment screening process for enzyme inhibitor development was demonstrated for a number of the Mtb P450s, including the cyclodipeptide oxidase CYP121 (the product of a gene essential to Mtb viability), CYP144 (which is expressed as both a full length protein and as a 30 amino acid "truncated" form from a leaderless transcript) and CYP126 (which was probed using both a "regular" fragment library and a "biofragment" library). Combined studies have led to the development of a number of 2nd and 3rd generation molecules that have high potency (low Kd values) and which are effective inhibitors of Mtb P450 function. A number of inhibitors exhibit cross-reactivity against a set of the important Mtb P450s, and thus have potential anti-Mtb activity through simultaneous inhibition of several P450s. Work is still ongoing with the aim of producing and developing new compounds as mechanistic probes for Mtb P450 enzymes and/or therapeutics against the pathogen. Further publications are currently in submission and/or in press.
First Year Of Impact 2014
Sector Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
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 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 Schools
Results and Impact Scientific demonstrations to senior secondary school students to enthuse them about a scientific career and to provide advice on career development and the courses on offer at the University of Manchester.

Annual event - such that lessons are learned from one year's activity and are carried forward to the following year's presentations.
Year(s) Of Engagement Activity 2012,2013,2014,2015
 
Description Schools visit (Wilmslow) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to primary school children in final year on general science/genetics - talk sparked questions and general discussion

Students registered interests in scientific career. Invite for further talk in following year obtained.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011