New tools for acute spatiotemporal control of GPCR signalling in vivo

The G protein coupled receptor (GPCR) family consists of approximately 800 members. They are proteins found on the surface of cells and they play important roles in virtually every process in the body. These proteins are also the target of approximately one third of all medicines used in the clinic. Unfortunately, even though these receptors have proved to be good drug targets, GPCR drug discovery is still associated with a high rate of failure, predominantly because many developed drugs do not display a sufficient therapeutic effect and/or cause unwanted side effects. This attrition is due, to a large part, to our incomplete understanding of how individual members of this receptor family control processes in the body. This is made even more complicated as many of these receptors are expressed in different cell types in different tissues. This means that a drug that acts at one of these receptors in one part of the body to have a therapeutic effect, might act at the same receptors in another part of the body to cause an adverse effect.

In order to develop better medicines, we need to understand how GPCRs in different tissues control the different processes in the body. Up until now, progress in this regard has been limited because we did not have the tools with which to selectively interrogate these processes in a cell specific manner. We aim to develop two synergistic approaches that will enable us to do this. The first approach will be to develop a strategy to restrict the action of a drug to a specific cell type by tethering it to the outside of that specific cell. The second method will allow us to switch off processes inside the cell that are initiated by GPCRs in a cell type-specific manner. We will use these methods to understand how drugs act at a GPCR that is the target of all opioid pain killers, the mu opioid receptor.

Opioids, like morphine, are the most effective drugs to treat severe pain. Unfortunately, opioids cause adverse effects such as addiction, constipation and suppression of breathing (the cause of fatality from overdose). The mu opioid receptor, a member of the GPCR family, controls both the therapeutic effects and the adverse side effects of opioid pain killers. It has been suggested opioid receptors activate two different types of proteins in nerve cells, one (G protein) that mediates pain relief, while another (arrestin) mediates adverse effects. However, recent research disputes this finding. This highlights the importance of understanding how opioid receptors activate processes in neurons that control pain relief or unwanted side effects. We propose to generate new tools that will allow us to block mu opioid receptor activation and signalling within the cell at a precise time and location in the brain. We will use this approach to understand the cell populations and cellular signalling processes that are responsible for causing the effect of opioids on breathing rate. These approaches will be widely applicable and will allow us to understand the physiological effects of a medicine when it acts at its target GPCR in an unprecedented cell specific manner with temporal precision. By understanding the role of specific cell signals in determining the therapeutic and side effects of medicines we can use this information to facilitate the discovery and development of newer safer medicines.

We hypothesise that the high attrition rate in GPCR drug discovery reflects our limited understanding of how GPCR signalling controls physiological effects. This is especially relevant for receptors that have complex patterns of expression throughout the body and for the exploitation of biased agonism. Within this context, our aim is to develop two synergistic and complementary approaches to interrogate the physiological consequences of GPCR signalling in discrete cellular populations using the Mu opioid receptor as a model. Specifically, these approaches are:
1. A MOR DART-antagonist to understand the role of MOR signalling in distinct neuronal populations. The anatomical distribution of MORs is complex. Efforts to understand the physiological role of MORs in distinct neuronal populations has been hampered by the lack of tools with which to do so. In this regard, the use of Drugs Acutely Restricted by a Tether (DARTs) can address this knowledge gap by localising drug action to a specific cell population. We will develop a DART MOR antagonist and target it to neurons of the pre-Bötzinger complex. We hypothesise that this blockade will reduce morphine-induced respiratory depression but not its antinociceptive effect.
2. A strategy for cell selective and acute inhibition of G proteins in vivo. Over the last decade, considerable efforts have been directed towards the development of G protein biased ligands as safer opioid analgesics. These studies have highlighted our limited understanding of how different signalling pathways might dictate opioid responses. Work to date, using genetic knockdown models, has focused on the role of arrestin signalling only. The role of G proteins has been indirectly inferred from these studies. We have developed an approach that allows us to interrogate G protein signalling in an acute and cell specific manner. We will use it to interrogate the role of MOR G protein signalling in the pre-Bötzinger complex to control respiration.

The basic fundamental research in this project will have relevance for basic scientists and industrial researchers and eventually for the general public. The success of the proposed research programme will have direct benefits for our collaborators as well as academic GPCR pharmacologists, neuroscientists, physiologists and drug discovery researchers. The longer-term benefits of the work have the potential to spread out more widely to companies in the pharmaceutical sector and to society at large.

1. Short term direct impact.
1.1 The collaborations established and consolidated with this programme between the University of Nottingham (JRL and MC), the University of Bath (CPB), the National Institute of Drug Addiction (AHN) and Columbia University (JAJ) will benefit from knowledge exchange throughout the project which will provide state-of-the-art methods in the control GPCR signalling in defined neuronal populations that are relevant to their research in aspects of opioid neuropharmacology (JRL, MC and CPB), addiction (AHN) and neuropsychiatry (JAJ).

1.2. GPCR pharmacologists, neuroscientists, physiologists and drug discovery researchers. The underpinning knowledge of GPCR biology and signalling can be used by the GPCR research community at wide. The results from this project will have immediate impact for researchers in the opioid field. Our novel MOR DARTs approach will allow researchers to deconvolute the complexities of opioid receptor signalling in distinct cellular populations. These studies will pave the way for future more comprehensive studies aimed at unlocking the complex role of MOR in distinct physiological roles such as pain sensation, respiration, gut motility, reward and emotion. Our work will provide an invaluable tool for the future study of MOR neurocircuitry. The broad applicability of our methods to other GPCR targets will enable similar mechanistic insights for other GPCRs in areas of high clinical need.

1.3 Junior researchers (the PDRAs) will benefit from training during the project in cutting-edge methods for the manipulation of GPCR signalling in vivo and in vitro. This will build research capacity and provide researchers for the future equipped with the relevant knowledge and expertise for roles in academia, industry, or policy.

2. Longer term indirect impact.
2.1 Pharmaceutical companies. Humans have around 800 GPCRs and it is thought that 400 have the potential to be targeted therapeutically. Fully understanding biased agonism of GPCRs may enable the development of novel drugs with greater tissue specificity and reduced side effects. Our research will also refine experimental design in biased agonism drug discovery, providing major improvements within the principles of the 3Rs and reducing the risk of failure of drugs at early pre-clinical stages. Together, our approaches will contribute to reducing the attrition rate associated with GPCR drug discovery and development.

2.2 Ensuring UK leadership in GPCR biology. The training of junior researchers and planned dissemination and outreach activities will ensure that awareness of the project is spread to the widest possible audience, both nationally and internationally. The training gained during the project will accelerate the career development and trajectory of the PDRAs, putting them on track to apply for independent fellowships so that they can lead their own groups to build capacity.

2.3 Benefits for quality of life and public health: The general public will benefit in the long term through improved knowledge of GPCR drug action and improved medicines. For example, pain has a significant economic and social impact whilst also reducing quality of life. The generation of novel analgesics will improve health and wellbeing whilst also reducing economic burden from sick leave.