As the population ages, the incidence of psychiatric disorders, neurodegenerative diseases, and pain will increase. In terms of therapeutic intervention, this is self-evidently an area that requires the identification of novel pathways and targets. The CB1 and CB2 cannabinoid receptors are being investigated as potential therapeutic targets, however there are also likely to be considerable therapeutic opportunities based on modulating the synthesis of the most abundant endocannabinoid in the adult brain, 2-arachidonlyglycerol (2-AG). The molecule is generated from diacylglycerol by the sn1-specific DAG lipases (DAGL alpha and DAGL beta) that we recently cloned (1). We have recently established that these enzymes regulate the level of both 2-AG and arachidonic acid throughout the body, and that they are responsible for endocannabinoid mediated synaptic plasticity and neurogenesis in the adult brain (2). To fully exploit the DAGLs as a targets, we need to understand how their activity is regulated. In this context we have identified a number of conserved serine (S) and threonine (T) containing motifs within the catalytic domains of the enzymes. We have also generated a unique cell line that allows us to readily measure DAGL activity in cells. This has involved modification of a commercially available CB1-TANGO cell line. This line contains a chimeric CB1 receptor with a protease sensitive site - in brief, when the CB1 receptor is activated a transcription factor is released and this drives expression of a beta-lactamase reporter gene (for full details see (3)). We have used a lentivirus approach to stably express human DAGL alpha in these cells, and identified conditions where DAGL is inactive in the cells, and conditions where it is active (e.g. following stimulation with calcium). Activity can be monitored over several hours and is reflected in a DAGL/CB1 dependent increase in expression of the beta lactamase reporter. We have used a panel of selective antagonists in pharmacological screen to identify several kinases that are required for DAGL alpha activation by calcium (including PKA, PKC and Cdk5), and used bioinformatics to identify the sites within the catalytic domains that these kinases are most likely to phosphorylate. The project will build upon and extent the pharmacological approach to identify the kinases and phosphatases that regulate DAGL activity. siRNA will be used to further test the importance of candidate kinases/phosphatases by knocking them down. We will use proteomics to directly map the sites that are phosphorylated on DAGL by the candidate kinases in cells, as well as following treatment of purified DAGL protein with the candidate kinases. We will screen a number of commercially available antibodies that recognise 'generic' phosphorylated epitopes in order to identify tools that directly report on the activation status of DAGL. We will also raise phospo-specific antibodies to the key activation sites. Finally, we will mutate key phosphorylation sites and determine the effects of this on enzyme activity following expression of the constructs in the CB1-TANGO cells. 1. Bisogno, T., Howell, F., Williams, G., Minassi, A., Cascio, M. G., Ligresti, A., Matias, I., Schiano-Moriello, A., Paul, P., Williams, E. J., Gangadharan, U., Hobbs, C., Di Marzo, V., and Doherty, P. (2003) J Cell Biol 163, 463-468 2. Gao, Y., Vasilyev, D. V., Goncalves, M. B., Howell, F. V., Hobbs, C., Reisenberg, M., Shen, R., Zhang, M. Y., Strassle, B. W., Lu, P., Mark, L., Piesla, M. J., Deng, K., Kouranova, E. V., Ring, R. H., Whiteside, G. T., Bates, B., Walsh, F. S., Williams, G., Pangalos, M. N., Samad, T. A., and Doherty, P. (2010) J Neurosci 30, 2017-2024 3. van der Lee, M. M., Blomenrohr, M., van der Doelen, A. A., Wat, J. W., Smits, N., Hanson, B. J., van Koppen, C. J., and Zaman, G. J. (2009) J Biomol Screen

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Multi-cellular organisms rely on a large array of different transmitter substances to allow certain cells to control the behavior of others. The more sophisticated the organism the more complex the cell to cell communication. In mammals this language probably involves hundreds of fundamentally different types of transmitter. Clearly such systems need a large collection of specialized receptor molecules that can detect the individual presence of any particular transmitter. Further, these receptors, typically found on the outer surface of the cell's limiting membrane, have to signal their specific stimulation by passing a molecular message into the cells interior, effectively informing the cell that the receptor has been activated. Clearly, if a cell has many different types of receptors on its surface the molecular signal generated inside the cell by each different receptor (often called an intracellular message) must identify and distinguish which specific receptor has been stimulated. Otherwise the cell could not discriminate between the transmitters present on the outside of the cell and could not respond correctly. Hence, mammalian cells have vastly complex intracellular signalling mechanisms continuously informing the cell of what is happening in other parts of the organism or its environment. One such intracellular signalling molecule or 'message' is PIP3. It is a phospholipid molecule found on the inside surface of the cell's limiting membrane. Levels of PIP3 rise rapidly on activation of a large number of receptors. This is surprising given the problems the cell faces in knowing precisely which receptor has been activated when it detects an intracellular signal. This grant application is to understand how it is possible that rises in PIP3 can encode specific messages from so many different receptors. We have performed some experiments that have, in fact, shown that PIP3 in cells is not a single type of molecule. At least four tiny variants of PIP3 can be detected, called molecular species of PIP3. Interestingly, we find that these different molecular species of PIP3 do not respond equivalently to different ways of activating the cells we work with. We and others have also found that the different receptors can make the levels of PIP3 rise for different times and to different maximum levels. We propose that these small differences are very important inside the cell for discriminating whether a certain receptor has been stimulated. This is a 'clever' economy or efficiency on the part of the cell and allows it to use similar mechanisms to perform many different jobs. Although on the surface these might appear trivial details in the business of understanding biology, it has recently been discovered that many different cancers are caused by mutations in genes that regulate PIP3 levels in cells. Mutations that by chance cause the production of PIP3 to be increased without any need for receptor stimulation make cancers much more likely to occur. Mutations that by chance stop the enzymes that normally break down PIP3 from working also make cancer more likely to occur. As a result it is clear that understanding how PIP3 is made and then interpreted by cells is crucial for us to better understand how cancer occurs and how to treat it. Many companies are already trying to design drugs that will reduce PIP3 levels to fight cancer. This work will help us understand how to make better drugs of that type.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Organolithium reagents (i.e., compounds that contain a direct bond between a lithium and a carbon atom) are extremely important reagents in chemical synthesis. It has been estimated that 95% of all pharmaceuticals rely upon the use of these cornerstone reagents at some point in their preparation. In general, organolithium compounds are very reactive; however, this is sometimes coupled with the compounds exhibiting a lack of selectivity even when the reactions are carried out at very low, cryogenic temperatures. This is a massive hurdle to the synthetic chemist! To overcome this situation, less reactive, but more selective compounds [such as organomagnesium (e.g., Grignard) or organozinc (e.g., Reformatsky) reagents] are often used; however, these reagents are often too inert. Recent research has shown that by combining a lithium reagent with a magnesium (or zinc) one, a whole new and in many cases surprising chemistry can be produced. Fascinatingly, in these cases the reactivity cannot be replicated using the monometallic compounds on their own! Another important theme of this work is the generation of organic molecules (i.e., molecules which contain no metal atoms) which can be used as building blocks for key pharmaceuticals. Just as human beings have a left and a right hand, certain organic molecules (known as chiral compounds) can also be considered left- or right-handed. In medicine, it is common that only one handed form of an organic molecule has the required therapeutic effect; it is also usual for the other handed form to induce nasty side effects. Therefore it is critical that the synthetic chemist can easily produce only one handed form of a specific organic compound. In chemistry this is known as enantioselective synthesis. This research will systematically investigate the two aforementioned topics and combine them for the first time - that is enantioselective synthesis using alkali metal-magnesium or alkali metal-zinc complexes. During the first part of this research many new mixed-metal compounds which contain chiral molecules will be prepared. Various analytical techniques will be used to determine their structure, both in solution and in the solid state. Then a systematic study of how these compounds react with organic molecules will be conducted. It is envisaged that in the near future these new "bimetallics" will be used to complement the well known organolithium reagents that presently corner the market in the pharmaceutical industry. Another area which will be explored is the chemistry of alkali metal amides. Despite their widespread usage, the structural chemistry of alkali metal amides and their complexes continues to spring many fascinating surprises. We have recently observed that molecular rings of various sizes - consisting only of alkali metal cations and amide anions - can capture anions (such as hydroxide and chloride) in the presence of chiral diamine ligands. This new direction in s-block macrocyclic chemistry turns conventional crown ether chemistry on its head and opens the door for simple alkali metal amide/diamine compositions to be utilised in anion recognition chemistry. Results from this new direction within synergic chemistry will undoubtedly appeal to a broad spectrum of academics, including inorganic, organometallic and organic chemists, as well as to supramolecular chemists due to the strong structural and coordination chemistry nature of the area. This new methodology in enantioselective synthesis will also be of considerable interest to researchers in the chemical industry (fine chemicals, pharmaceutical, agrochemical etc.) who strive to produce chiral molecules (new and old!) in a facile manner.