Dynamic modulators: Novel effectors of NF-kappaB signalling

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.

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.