A Systems Approach for the Fragment-Based Development of Selective Chemical Probes of Bromodomain Function

A key cellular mechanism for regulating expression of the genetic information stored in DNA is by mean of protein 'factors' that gene transcription. One group of such proteins affects gene expression levels by 'reading' epigenetics marks, i.e. reversible chemical modifications that are installed on other proteins that associate with DNA to form the highly compacted structure known as chromatin. A widely occurring modification is acetylation of lysine amino acids, which is specifically recognized by proteins that contain between one and six 'reader' domains called bromodomains. The human genome encodes 42 bromodomain containing proteins, giving a total number of 57 unique sequences that make up the bromodomain protein family. There is increasing evidence that link bromodomain proteins in various diseases, including cancer, however specific functions of many bromodomain proteins are yet unknown. Potent, cell-permeable small molecules that perturb the function of a biological target in a dose-dependent fashion are a powerful way to 'probe' the role of the target in a particular biological process as well as its association to disease and thus its therapeutic potential. Small molecules have several advantages over more traditional approaches involving gene knock outs or RNAi, including allowing spatial and temporal controls on the effect within a cell. However, identification of probe compounds can be laborious and often involves screening of large compound libraries. It can be challenging to develop 'tool compounds' that are not only sufficiently potent against a target protein but also highly selective so they do not bind to other similar proteins. This often hampers the successful application of chemical probes to establish a relationship between a molecular target and the biological consequences of modulating the target. Developing new approaches and tools to make advances in these areas would have an immediate impact in the field of chemical biology and for target validation in drug discovery. Recent years have seen the establishment of a novel, powerful approach to identify high quality binders against proteins. This involves screening libraries of molecules, so-called 'fragments', that are much smaller than those usually tested e.g. in 'high-throughput screening'. The binding modes of 'hits' identified from a fragment screen are characterized using protein structural techniques so their interactions with the protein are determined in details. Once several fragment hits are identified, the combined information on their interactions, on the nature of the binding site and knowledge of their chemistry can provide a basis for 'elaborating' these structures into more potent chemical probes. In the current proposal, we will combine fragment-based approaches with protein engineering, a technique to generate specific mutations on a protein by changing amino acids from one type to another. First we will elaborate bromodomain-targeting fragments by 1) 'growing' them to pick additional interactions with the binding site; 2) 'merging' fragments bound at overlapping sites at the acetyl-lysine binding pocket. This will generate tight binding ligands for bromodomains. Second we will elaborate these molecules to accommodate functional groups that chemically complement the mutation introduced in the binding site, e.g. filling space created by engineering a pocket, and/or 'clicking' the ligand covalently onto a cysteine. Such modified chemical probes should be highly selective for the mutant against wild-type or indeed any other bromodomain. Since the mutation can be rapidly introduced into any bromodomain protein and in a cell, the methods and tools that will be developed in this programme would allow a general strategy to chemically interrogate the biological function of bromodomain proteins at the system level. This approach could then be extended to study other reader domain systems as well.

Bromodomains are a family of small modules found in histone acetyl transferases and other chromatin-associated proteins that play crucial roles in many cellular physiological pathways and are also implicated in disease. They specifically recognise acetyl-lysine post-translational modifications in histones amongst other proteins, thereby acting as readers of the epigenetic code. The human genome encodes 57 bromodomain sequences, however the biological function of many of these remains to be dissected. This proposal is centred on the applications of fragment-based chemical tools and engineering of bromodomain-histone interfaces to develop highly selective small molecule probes that can aid elucidation of the individual roles of bromodomains at the system level. The project will exploit fragment-based design, synthetic chemistry, protein X-ray crystallography and various biophysical techniques including AlphaScreen, NMR spectroscopy and isothermal titration calorimetry to identify and characterize small molecule fragments that bind at the acetyl-lysine binding site of bromodomains. Two interdependent lines of enquiry will then be followed. First, we will chemically elaborate the fragments using 'growing' and 'merging' approaches to improve their affinity as inhibitors of the bromodomain-histone interaction. Second, we will modify fragments and fragment-derived ligands to chemically complement mutations in the bromodomain-histone interface in ways that would allow chemical probes to bind preferentially to engineered bromodomains against wild-type bromodomains. The selectivity of the chemical probes will be tested against a panel of bromodomains in vitro, and studies on their cellular activities will also be conducted. Collectively these studies will advance our knowledge and understanding of the molecular and structural basis for selectivity of chemical probes targeting this conserved protein-protein interaction family.

The research proposal aims to develop and apply fragment-based screening to the design of inhibitors of the human bromodomain-histone interaction, and to enable the development of such small molecules into highly selective chemical probes for individual bromodomains by combining the fragment approach with directed protein engineering at a system level. The work proposed is multidisciplinary, involving protein expression, purification and characterization, protein-peptide and protein-small molecule binding studies using biophysical techniques including NMR spectroscopy, ITC, and AlphaScreen, structural biology and organic synthesis. The ultimate aim of the project is to provide a novel, generally applicable approach to pin-point the individual roles of bromodomains and of their specific recognition of lysine acetylation, thus aiding elucidation of the function of bromodomain-containing transcription factors and chromatin remodelers in the human genome. Beneficiaries from the research include scientists in both industry and academia who are interested broadly in the fields of chemical and structural biology and drug design. It will impact biologists interested in epigenetics, chromatin structure, function and modifications, and gene regulation in physiology and disease. By stimulating wider use of multidisciplinary, systems and predictive approaches to bioscience, the work will impact a growing national and international community of synthetic and system biologists. These studies will also have strong impact on epigenetics target validation for drug discovery in many disease areas including inflammation, metabolism and cancer. This will be of interest to members within the Pharmaceutical Industry, within charities concerned with the threat of diseases and will influence policy-makers within government, national and international agencies in terms of showing that investment is being channelled into key areas that will underpin future drug discovery. By providing new tools to advance fundamental understanding in biological processes relevant to critical disease areas the research has therefore the potential to impact on the nation's health and economic competitiveness. Many beneficiaries will be direct 'users' of the research outputs, both immediately and in the longer term. Crystal structures of bromodomain-ligand complexes will be deposited in the PDB and in the SGC databases and will thus provide useful information to structural biologists, bioinformaticians and drug designers. Structures of chemical probes will be released and the compounds made available to the community upon request, meaning that many biologists studying chromatin structure, function and epigenetic modifications will be able to use these chemical tools to carry out experiments. The timescale for such impact is difficult to predict, but we expect that the developments in fragment-based screening approaches and concomitant work on manipulating bromodomain-histone interface to identify highly selective small molecule chemical probes will yield several compounds and new insights into the structural basis of bromodomain-histone target selectivity in the next 2-3 years. According to outcomes from studies in this work and allied programmes at validating potency and selectivity of molecules generated, we expect the research to inspire similarly directed targeting studies and embracing of related approaches and strategies in academia/industry soon after relevant publications emerge, also extending to other families of related reader domains e.g. chromodomains, Tudor and PHD fingers. We are also confident that the range of approaches that will be undertaken will provide the PDRA and RA appointees with a range of professional skills suited to career development in both academic and industrial settings, thereby maintaining and fostering national strengths in core underpinning disciplines such as molecular, chemical, cellular and structural biology.