MICA: Defining G protein- and arrestin-dependent signalling pathways of biased DOPr agonists and the relevance to their in vivo effects

A recent survey revealed that almost 20% of the European population suffers pain persisting for more than 6 months. The present ability to treat chronic pain is, however, limited, as the currently available medicines can provide no cure and give limited relief. Chronic pain from diseases such as osteoarthritis and diabetic neuropathy can have a devastating effect on the sufferers and their families, with accompanying high rates of depression and sleep loss. Chronic pain also constitutes a huge economic problem with a considerable loss of working days. Thus, the search for a chronic pain killer is pivotal in order to help individual sufferers and improve quality of life.

The delta opioid receptor (DOPr) is a nerve cell drug receptor whose activation leads to a number of beneficial outcomes in the patient including relief of chronic pain and the production of antidepressant and anxiety-reducing activity. DOPr activation may also in some cases lead to undesirable effects including epileptic behaviour, which detracts from their potential as medicines.

Drug activation of DOPr in neurons is likely to lead to the activation or inhibition of a large number of intracellular signalling pathways, which contribute to the therapeutic and/or undesirable effects of DOPr activation. The development of "biased" ligands at GPCRs, which are new types of drugs which can selectively activate particular cell signalling pathways, opens the way for novel ligands to be developed that potentially produce the desired therapeutic effects in the patient but which avoid undesirable effects. The drug discovery company Pharmnovo, the industrial partner in this application, has made a number of DOPr activating drugs with "biased" signalling which activate intracellular signalling pathways in different ways. These drugs provide pain relief in experimental models of chronic pain.

A problem with current studies is that the bias of a ligand is primarily determined using overexpressed receptors in clonal cell lines, which may not be reflect the global signalling response that occurs in a physiological system. It follows that the best way to determine bias and assess its functional significance must be to monitor as many signalling outputs as possible following activation of the endogenous receptor in the organism or relevant part of an organism.

Therefore if the signalling pattern (phosphoproteome) generated by DOPr drugs in a neuron can be determined, then by comparing this signalling profile with functional effects produced by the ligands, such as the ability to induce pain relief, then it should be possible to identify signalling pathways associated with desirable effects and those associated with undesirable effects of DOPr drugs. This approach will assist the design of new DOPr drugs with the best neuronal signalling profile possible to benefit the patient and avoid side effects. With this in mind the objectives of this present proposal are to:

- apply phosphoproteomics to DOPr in neurons to compare the global signalling response to biased signalling drugs at this receptor

- correlate the neuronal signalling profiles identified from the phosphoproteome with the functional profiles of the tested DOPr agonists (e.g. their relative abilities to induce pain relief, and to potentially promote epileptic-like activity etc)

- use these data to identify the very best neuronal signalling profile for the development of DOPr activating drugs for the treatment of pain, depression and anxiety

The drug discovery company Pharmnovo AB and Pharmnovo UK Ltd, the industrial partner in this application, has synthesised and developed a number of very selective DOPr agonists with "biased signalling" which can couple efficiently to G protein but partially or not at all to arrestins. These ligands provide analgesia in experimental models of chronic pain.

The best way to determine bias and assess its functional significance must be to monitor as many signalling outputs as possible following activation of the endogenous receptor in the organism or relevant part of an organism, and then correlate the pattern of signalling with the observed in vivo effects of the biased ligand. Hence if the global signalling fingerprint (phosphoproteome) in a neuron following DOPr activation can be determined, then by comparing functional effects, such as the ability of DOPr agonists to induce analgesia or an antidepressant effect, with the neuronal phosphoproteome produced by each ligand, we can begin to identify signalling pathways associated with desirable effects and those associated with undesirable effects of DOPr ligands.

Consequently the objectives of this present proposal are to:

- determine precisely the G protein versus arrestin bias of novel DOPr agonist ligands

- apply advanced phosphoproteomics and bioinformatics to native DOPr in neurons to determine and compare the global signalling response of novel DOPr agonists with variable G protein/arrestin bias

- correlate the neuronal signalling profiles identified from the phosphoproteome with the in vivo functional profiles of the tested DOPr agonists (e.g. their relative abilities to induce analgesia, and to potentially promote seizure activity, tolerance and dependence)

- use these data to identify the optimal neuronal signalling profile for future DOPr agonist ligands for the treatment of pain, depression and anxiety

There are a number of beneficiaries for whom this research could be helpful in the longer term:

1. Academia - the immediate grouping. The PDRA, who will be equipped with cutting edge skills in phosphoproteomics and bioinformatics, will in the future be able to provide essential skills for research or related jobs in academia, education, healthcare, or industry. The immediate beneficiaries will thus be the wider UK and international academic communities, public and private education, the healthcare sector and industry. The Investigators on this grant will also be beneficiaries, with for example EK and KH bringing their experience of GPCR biology and Proteomics together to ask new questions of fundamental importance. Each are very keen to expand their skills into Proteomics and GPCR biology, respectively.

2. Academia - Bristol. The bioinformatics training and advice provided by Barcelona will benefit other proteomics/phosphoproteomics projects undertaken here in the future. This is particularly significant as the level of bioinformatics expertise in Bristol is at present fairly limited.

3. Academia - the wider picture. The Kelly laboratory has a number of other collaborations with researchers interested in understanding the molecular details of MOPr function (e.g. Schulz/Jena; Bailey/Bath; Benovic/Philadelphia; Gurevich/Vanderbilt Nashville). It is likely that the research that these groups undertake will be directly affected by the knowledge and understanding obtained from the present application.

4. Economic and societal impact - public health and wealth creation. The findings of this work will be of great interest to the Pharmaceutical industry, a very important component of the UK economy. On the one hand the industry will be interested in the basic ideas about GPCR biology, how receptors work, and the functional consequences of biased agonism. This can inform them in their search for new drug targets as well as new ways in which to analyse drug action. In particular in recent years there has been a move to develop new types for drugs for GPCRs, including ones that show bias, or act at allosteric sites on the receptor. Our proposed work is in direct line with these interests and for example the idea of using the phosphoproteome to characterize bias, and to find new biased ligands will be of very great interest to the pharmaceutical sector, keen to exploit new paradigms for the >800 GPCRs in the mammalian genome. Secondly, opioid drugs are extremely important in the treatment of pain in animals and humans, whilst the abuse of opioid drugs has major health, welfare and economic consequences for the UK. Greater knowledge about these drugs and the way in which they interact with their receptor will also help to inform future public policy.

5. Economic and societal impact - evidence based policy making. GH is an invited member of the Independent Scientific Committee on Drugs (ISCD) a body set up to provide advice on drug harm to government and all other interested parties. ISCD submits responses to government calls, publishes reviews, posts information on its web site, informs the press and holds symposia on important current issues on drug use. GH's membership of this organisation allows him to provide research-driven advice that can potentially change Government policy in relation to the social and health problems associated with opioid drug therapy and opioid drug abuse.

6. Economic and societal impact - the public. In more general terms our work is of benefit to the UK in terms of our commitment to public engagement. This includes speaking in schools and hosting regular work experience visits to our laboratories by sixth-form students interested in studying science at university. Participation in these activities helps to ensure our knowledge and influence passes beyond the laboratories and walls of the University.