Human SNM1A - a novel DNA repair role and the development of inhibitors.

Lead Research Organisation: University of Oxford
Department Name: Oncology

Abstract

Our genetic blueprint is contained within long, chromosomal complexes containing DNA present in the nucleus of our cells. However, cellular DNA is constantly under attack from reactive chemicals generated as a by-product of normal cellular function, and this damage must be repaired. Failure to repair DNA in an accurate or timely fashion is associated with a number of diseases, most prominently cancer. The DNA damage sustained by cells can, fortunately, be repaired by several complex cellular pathways, minimising changes in our DNA and thereby suppressing tumour formation.

Perhaps surprisingly, however, DNA damage is induced by many anticancer treatments (chemotherapy and radiotherapy) and it is this DNA damage that is effective in killing cancer cells. In this proposal we aim to define how a specific DNA repair factor, named SNM1A, acts to repair damaged DNA. We have recently discovered that SNM1A plays an important role in the repair of breaks in the DNA double-helix, which is the clinically important form of DNA damage produced by radiotherapy and certain anticancer drugs. We will define how SNM1A promotes the repair of these double-strand breaks in both whole cells and also in biochemical studies of purified SNM1A protein. Moreover, we have recently identified several chemical compounds that can inhibit SNM1A activity in the test-tube, and aim to develop these into molecules that can enter cancer cells and inhibit SNM1A. These will be very powerful tools for further studies if SNM1A, and also might ultimately allow us to develop SNM1A inhibitor drugs that could improve patients' response to DNA damaging chemotherapy agents and radiotherapy.

Technical Summary

Here we propose a combined biological and chemistry-based approach to characterise the role of the human SNM1A in DNA double-strand break repair, validating it as a therapeutic target for radio and chemo-sensitisation in cancer. We have recently discovered that, in addition to its established role in interstrand cross-link repair, SNM1A plays a role in the repair of DNA double-strand breaks (DSBs) induced by radiation and radiomimetic drugs. In one arm of this work, we will define the relationship of SNM1A to the other major DSB repair pathways, and discover how it is recruited to sites of damage, a process mediated, at least in part, through interactions with poly-ADP-ribose chains. We wil also undertake detailed biochemical and structural studies to define at the molecular level how SNM1A digests damaged DNA, including DNA containing oxidised bases, relevant to its role in the repair of DSBs induced by chemo- and radio-therapeutics.

We will combine this analysis with further development of small molecule inhibitors of SNM1A. We have recently discovered that several beta-lactam antibiotics are able to competitively inhibit DNA hydrolysis by SNM1A in vitro, within the low micromolar range, indicating that beta-lactam family molecules might provide an excellent starting point for the rational development of SNM1A inhibitors. We will employ focussed screens employing existing libraries of several thousand beta-lactam compounds and their structural analogues to inhibit SNM1A. In parallel with this we will further develop a cell-based assays that allow us to test the ability of these compounds to induce cellular sensitisation to DNA damage. Cellular specificity will be defined by the ability of the inhibitory compounds to selectively increase damage sensitisation and the hallmarks of repair defects in human cells, but not in isogenic lines disrupted for SNM1A.

Planned Impact

The work proposed here has broad implications for human health, which could bring significant benefit to the public and also the UK economy. The work will characterise a key molecular target required to maintain genetic stability by combating DNA cross-links and double-strand breaks, potentially suggesting strategies to ameliorate the symptoms of developmental disorders associated with genomic instability (including ageing) and, most critically, improve our ability to treat cancer. Therefore, outside of the more immediate academic field, the following groups should ultimately benefit: (i) Pharmaceutical and Biotechnology Industries, where there is a strong interest in targeting factors that influence sensitivity to cytotoxic cancer therapies. Exploitation of our research in this fashion would both benefit human health and be of economic benefit to the UK. Moreover, SNM1A is a metallo-beta-lactamase (MBL) fold enzyme, and is ancestrally related to bacterial MBL enzymes that combat antibiotics, which are a major emerging source of antibiotic resistance. There is intense current interest in bacterial MBL inhibition in both academia and industry, and it will be critical that any such bacterial MBL inhibitors do not also inhibit human MBL-domain proteins since the side effects (particularly long-term) are likely to be severe. Our work will provide an exemplar of how discrimination between bacterial and human MBLs can be achieved. (ii) The NHS and related Public Sector, who also have a strong interest in disease prevention, might ultimately be able employ the SNM1A and related genome stability factors as biomarkers to screen for those in danger of prematurely developing degenerative diseases or malignancies, allowing interventions or modification of lifestyle to counteract this. Our work also has the potential to improve prediction of therapeutic response since SNM1A status is likely to be indicative of tumour response to a variety of cancer therapies. (iii) Policy makers and Government. Those involved in policy making are required to make recommendations and laws that to help protect the population from health risks, established and emerging (a recent example is nano-particles). Here, an understanding of the pathways required to combat environmental DNA damage is important as it will help in the assessment of threats that could produce acute or longer-term damage to human cells and tissue. Policy could be modified to reduce human risk from exposure to a minimum. (iv) The UK employment sector - the two Post-Doctoral Fellows working on this project will gain a wealth of experience in biochemistry, genetics, cell biology and structural biology and medicinal chemistry, respectively which is directly transferable within the sectors described herein. (v) The general public will benefit as our groups, academic departments and the WIMM are all heavily committed to public engagement. The applicants and their groups regularly communicate their findings to the public at events held within the Oncology and Chemistry Departments and WIMM, including at Oxford Science week Public Lecture evenings which are strongly angled towards local schools. Naturally, we maintain an up-to-date websites detailing our work and its outputs, and where appropriate the University utilises its very effective media outlets to publicise findings nationally and internationally. Finally, we will publish our work in leading academic journals and disseminate findings at the appropriate academic conferences in a timely manner.

Publications


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Brem J (2015) Structural Basis of Metallo-ß-Lactamase Inhibition by Captopril Stereoisomers. in Antimicrobial agents and chemotherapy
Cahill ST (2016) Use of ferrous iron by metallo-ß-lactamases. in Journal of inorganic biochemistry
Pettinati I (2016) The Chemical Biology of Human Metallo-ß-Lactamase Fold Proteins. in Trends in biochemical sciences
Rendeková J (2016) Mgm101: A double-duty Rad52-like protein. in Cell cycle (Georgetown, Tex.)
 
Description Collaboration with European Lead Factory/Innovative Medicines Initiative to discover small molecule inhibitors of SNM1-family nucleases. 
Organisation European Commission (EC)
Department Innovative Medicines Initiative (IMI)
Country European Union (EU) 
Sector Multiple 
PI Contribution We have fully validated an assay for SNM1-family nuclease inhibition to the standard required for high-throughput screens. This enabled a screen of a library of over 300,000 compounds selected for their novelty, diversity potential, innovative design and synthetic tractability. The initial screen is completed, and further validation of potential 'hits' is ongoing.
Collaborator Contribution The European Lead Factory/Innovative Medicines Initiative performed the screen using our validated reagents and assay.
Impact We have a list of potential inhibitor 'hits', which will require careful validation in a multidisciplinary manner utilising the biochemical, cell biological and structural biology expertise of the grant awardees. Moreover, these compounds will provide leads for further medicinal chemistry efforts supported by this MRC grant.
Start Year 2014
 
Description BACK FROM THE DEAD: Demystifying Antibiotics, University of Oxford Museum of the History of Science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This exhibition marks penicillin's 75th anniversary as a life-saving drug. Discovered as an antibacterial substance produced by the penicillium notatum mould in 1928 by Alexander Fleming, it was turned into a medicine in Oxford ten years later and first tested on humans here in 1941. The success of penicillin was extraordinary; it seemed to bring patients back from the dead.
Penicillin became the most iconic drug of a new era of antibiotics. While vital to modern medicine, penicillin and other antibiotics have not eradicated infectious disease. Bacteria develop resistance to antibiotics so the work of preventing and fighting infection continues not just in Oxford but around the world.

The exhibition brings together dramatic stories and surviving material from the original Oxford research, displayed in parallel with today's antibiotic issues, allowing past and present to illuminate each other.
Year(s) Of Engagement Activity 2016,2017
URL http://www.mhs.ox.ac.uk/backfromthedead/
 
Description Professor Chris Schofield lab tours and lay talk. 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Lots of interest in talk and discussion of what chemistry involves, especially its application to drug discovery.

Increase in enquiries regarding studying chemistry here.
Year(s) Of Engagement Activity 2014
URL http://www.chem.ox.ac.uk/iycr2014.html