Dynamic modulators: Novel effectors of NF-kappaB signalling

Lead Research Organisation: University of Liverpool
Department Name: Sch of Biological Sciences

Abstract

The NFkappaB pathway is widely recognised as crucial to a variety of important cellular processes including both proliferation and apopotosis. It is correspondingly implicated in a variety of pathologies such as cancer and inflammatory disease. In recent work (e.g. Nelson, D. E., Ihekwaba, A. E. C., Elliott, M., Gibney, C. A., Foreman, B. E., Nelson, G., See, V., Horton, C. A., Spiller, D. G., Edwards, S. W., McDowell, H. P., Unitt, J. F., Sullivan, E., Grimley, R., Benson, N., Broomhead, D. S., Kell, D. B. & White, M. R. H. (2004). Oscillations in NF-kappaB signalling control the dynamics of target gene expression. Science 306, 704-708), we have shown that signalling in the NFkappaB likely operates not by modulations in the amplitude of NFkappaB per se but in its frequency or dynamics. We therefore need tools that can modulate these differentially, and chemical genomics provides an exciting prospect for acquiring them. An important set of such tools is represented by small (drug-like) molecules, and a useful strategy is to screen libraries of them for molecules with the desired activities. As we obtain 'hits' that are active in the way we require we shall study their structures and properties in a computer so as to screen 'virtually' much larger libraries of available molecules for substances that have appropriate chemical similarities, and then screen these too. This will lead to molecules with higher potency and thus higher specificity. These will be improved still further by making variants synthetically and again testing them. The most potent molecules (those with the highest affinity) will be immobilised on a column and used to fish out the target(s) to which they bind, thus allowing the identification of the target(s). Attractive features of this strategy are that (i) we do not need to know the 'target' that is necessary to affect the dynamics in the first place, (ii) targets may come from any pathway, (iii) we may find targets in quite 'distant' areas of the cellular network that were not even known or considered to interact functionally with the NFkappaB pathway, (iv) we thereby acquire a series of reagents that can modulate the dynamics of biological signalling pathways.

Technical Summary

Most cellular signalling systems are arranged in a 'bowtie' configuration, in which multiple extracellular stimuli can cause multiple downstream responses, but they do so by using only a comparatively small number of signalling intermediates. It is not obvious a priori how crosstalk can therefore be avoided. Using single-cell time lapse video imaging together with numerical modelling, we have shown that individual steps in the NF-kappaB signalling pathway can differentially affect both the amplitude and the frequency of the oscillatory behaviour of the nuclear NFkappaB concentration. This has led us to suggest that it is the DYNAMICS, rather than the amplitude, of such signals that control downstream events (a phenomenon that in principle can deal with the crosstalk problem entirely). What we need to help develop this new idea, therefore, are experimental methods that can conveniently manipulate the two aspects of these dynamics independently. Chemical genomics provides a novel approach by which we can acquire reagents with the necessary specificities and activities. These candidate reagents will be screened in our single-cell assays using cellular arrays, and their important properties assessed in silico by the use of QSAR methods, which will allow us to 'cherry pick' from other libraries and thus improve their likely potency. The most potent molecules will be improved further by the methods of synthetic and medicinal chemistry. By using whole cell assays we shall automatically be able to interrogate all pathways that are functionally linked to the NFkappaB pathway, and by immobilising the most potent molecules we shall be able to purify the proteins with which they interact. The deliverable will be a series of reagents that can differentially affect the amplitudes and dynamics of components of the NFkappaB signalling pathway.

Publications

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Mullassery D (2008) Single live-cell imaging for systems biology. in Essays in biochemistry

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Spiller DG (2010) Measurement of single-cell dynamics. in Nature

 
Description Chemiinformatic analysis and classification of a broad set of NF-kB modulators

Identification of the drugs aspirin and diclofenac as modulators of NF-kB oscillation period.. We have subsequently shown that these drugs affect the timing of A20 transcription and interestingly aspirin negates the fynamic effects of temperature changes in the fever range
Exploitation Route New screens planned with pharmaceutical companies to benefit from this early screen.

The results with aspirin and diclofenac are very interesting and suggested future utility. This remains ongoing
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Ongoing interest in the work from CRT, GSK and AZ.
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Appointed to Pool of Experts for BBSRC Panels, 2012-2013.
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description BBSRC Bioscience for Industry Committee (single meeting for presentation)
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description BBSRC Panel: Enabling Theme 2, the Exploiting New Ways of Working Strategy Panel (ENWW)
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description BBSRC Strategic Tools and Resources Development Fund (sTRDF) Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description External Reviewer for FRISYS-ZBSA (Zentrum fur biosystemanalyse), Freiberg, Germany, 26th May 2011.
Geographic Reach Asia 
Policy Influence Type Membership of a guidance committee
 
Description German Virtual Liver Scientific Advisory Board (42m euros programme )
Geographic Reach Asia 
Policy Influence Type Participation in a advisory committee
URL http://www.virtual-liver.de/wordpress/en/media/handling-biological-complexity-using-systems-approach...
 
Description Invited to attend Research Sponsors' Consultation Meeting (BBSRC &MRC) on Imaging for the biological and biomedical sciences, 14 May 2012.
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
 
Description MRC Microbiome
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description MRC Systems Immunology
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description MRC/BBSRC New Microscopy Initiative
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Member of BBSRC Research Equipment Initiative (REI) Review Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC Site visit to Imperial Centre for Systems Biology
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC Site visit to Oxford Centre for Systems Biology
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC Tools and Resources Development Fund Panel, Feb 2012.
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of BBSRC/EPSRC TDRI Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of MRC Doctoral Training Panel
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Member of MRC Population and System Medicine Board, 1 April 2012 - 1 April 2016
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Membership of BBSRC Interdisciplinary Systems Biology Strategy Committee
Geographic Reach Asia 
Policy Influence Type Participation in advisory committee
 
Description Wellcome Trust/Wolfson Capital Awards in Biomedical Sciences Committee
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Dynamics and function of the NF-?B signalling system
Amount £5,072,010 (GBP)
Funding ID BB/F005938/2 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2008 
End 03/2013
 
Description Japanese Partnering Award
Amount £31,026 (GBP)
Funding ID BB/G530276/2 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2009 
End 03/2014
 
Description Strategic Lola
Amount £4,160,524 (GBP)
Funding ID BB/K003097/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2013 
End 04/2018
 
Description Carl Zeiss 
Organisation Carl Zeiss AG
Country Germany 
Sector Private 
PI Contribution We have advised Zeiss on trends in bioimaging since 1996. We have provided new data and tested prototype equipment. We have spoken at Zeiss organised meetings. We have given them an opportunity to display Zeiss equipment at our training courses. We have organised symposia that have been supported by Zeiss. We have held expert discussion meetings to review microscopy trends that have involved senior Zeiss staff
Collaborator Contribution Zeiss have made a cash contribution to training courses (received) of £16,250. Zeiss estimate of total value of in-kind staff time for collaboration, training courses and other meetings (including visits of teams from Germany) ?25,000. In addition, Zeiss have also committed over £30,000 in cash and ~£80000 in in kind staff for future training meetings and collaborative visits. Zeiss helped to design the new Systems Microscopy Centre in Manchester and made a 45% discount (value ?350k) for the purchase of equipment in 2011. Zeiss are a formal MICA partner on both Liverpool and Manchester awards from the MRC/BBSRC New Microscopy Initiative. In the award to Manchester they have made a contribution of £614,314 in staff time, development costs and equipment contribution. This involves FCS (developed during this project), luminescence fluorescence imaging, light sheet microscopy and SOFI super-resolution imaging. More recently Zeiss have made a further contribution to our new clinical single cell centre. This includes over £400k in equipment discounts and £25k in cash contribution to training and symposia. Over the years the Zeiss contributions have included them helping us with public understanding of science exhibitions where they loaned equipment, provided support for professional poster preparation and used their delivery services to transport our exhibit materials and equipment to the exhibition venues. This included an exhibition in Buckingham Palace in 2006. Zeiss have sponsored 2-3 meetings per year in Manchester. In 2016 this included a session on light sheet imaging and a session on new confocal imaging technologies. In 2017 they have sponsored an image analysis daya and will sponsor a single cell biology workshop.
Impact Annual training courses MICA collaborative MRC grant MICA collaboration on new single cell centre The relationship with Zeiss has been two way. We have been given the opportunity to be early adopters f new technology and to feedback idease for improvement. We receive very favourable deals on microscope purchases and maintenance contracts. Specific areas of successful collaboration lie in improvements to higher throughput live cell imaging using the confocal microscopes; optimisation of truly dark microscopes for quantitative luminescence imaging and the development of FCS. Multiple workshops organised (2-3 per yeat)
 
Description School Visit, Altrincham Girls School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Dean Jackson - Advice and practice interviews for students
Year(s) Of Engagement Activity 2016
 
Description School visit (Liverpool Life Sciences, University Technical College) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Liverpool Life Sciences UTC is the first school in the UK specialising in Science and Health Care for 14 to 19 year olds. Talk at Liverpool Life Sciences UTC conference on "How Organisms Age"
Year(s) Of Engagement Activity 2016