Defining physiological and pathophysiological roles of the Free Fatty Acid Receptor2 by analysis of novel transgenic mouse models

Development of new medicines is a long term ambition and challenge. Initial efforts are often based on observations and analysis of effects in rodents of either activating or blocking a specific protein or a signal transduction pathway that is believed to contribute to or influence the development of a disease in humans. Means to do this include elimination of expression of specific genes in mouse or treatment of animals with synthetic drug-like molecules with the hope that responses produced will inform or mimic those that might occur in humans. Although it is true that many important physiological process are controlled and regulated in equivalent ways between mice and humans there are also many differences that reflect the 90 million years of evolution separating the species. One common aspect of this is that there are often differences in sequence between the equivalent proteins from different species and in a number of cases this is known to markedly effect the ability of a synthetic drug to produce effects. This is the situation for the receptor FFA2 that is activated by short chain fatty acids produced by the bacteria that are present in the intestine. Antagonists of this receptor that were identified by finding ligands that block the human form simply don't work at rodent orthologues and, because of this, it has been difficult to perform preliminary studies in mice that might predict their effects in humans. To overcome this we have generated mice in which we replaced the mouse version of FFA2 with the human form, and have shown that in tissues from these animals the identified antagonists are now able to block FFA2-mediated effects. We have also taken this one step further. As there are at least two receptors in both mouse and human that are activated by short chain fatty acids we further adapted human FFA2 such that it NOT activated by short chain fatty acids but is activated instead by a set of synthetic molecules that don't activate the normal forms of FFA2. We have also generated mice in which this modified human receptor is expressed instead of the mouse form.
With this underpinning knowledge we now plan to fully investigate the roles that are controlled by FFA2 in mouse. We will pay particular attention to actions in both the intestine where our preliminary studies have shown that FFA2 is a key regulator of release of a hormone called GLP-1 that after its release plays a central role in promoting insulin production from the pancreas when blood glucose levels rise after a meal. There have also been conflicting reports from other groups as to whether activating or blocking FFA2 might be an effective approach to either prevent the development of, or to treat, diseases such as ulcerative colitis that are based on inflammation of the lower intestine. We will now be able to address these questions in mouse models of this and related diseases, something that has hitherto been impossible.
Another key area where there has been debate and contradictory views is whether direct activation or blockade of FFA2 would be able to enhance or limit release of insulin in humans, and specifically in type II diabetics. There are reasons to believe the control mechanisms in mouse are different from in humans and now, using both the animal models described above and detailed molecular studies on both mouse and human forms of the receptor protein we plan to assess this question. This is likely to influence thinking with the pharmaceutical industry as to the most appropriate strategy to consider as a therapeutic treatment and whether studies that have been performed in wild type mice might actually be pointing in the wrong direction for therapy in humans.

Cross-species variation in pharmacological responsiveness of tool compounds and potential clinical candidate drugs frequently challenges both understanding of basic biological processes and the suitability of potential drug targets for therapeutic development. In a number of cases humanised mouse lines have been developed to address this challenge. In the case of the G protein-coupled receptor (GPCR) FFA2, all antagonists reported to date in either the primary or patent literature are 'human specific' and unable to bind to and block rodent FFA2 orthologues. To overcome this issue we have generated a knock-in mouse line in which a humanised form of FFA2 replaces the mouse orthologue. The endogenous activators of FFA2 are short chain fatty acids (SCFAs) produced largely by the microbiota. However, a closely related receptor, FFA3, is also activated by the same group of SCFAs, and no high affinity blockers of FFA3 are available. Inspired by the development and use of 'Designer Receptors Exclusively Activated by Designer Drugs' (DREADD) variants of muscarinic acetylcholine receptors that have allowed identification of effects mediated specifically by each of the 5 members of this group of GPCRs that all respond to acetylcholine, we developed a DREADD variant of human FFA2. This is unresponsive to SCFAs but instead is activated by a number of ligands we have identified, including sorbate. We have also generated a transgenic mouse line expressing this variant. In preliminary studies we have confirmed that the anticipated pharmacology is generated in tissues expressing hFFA2 or hFFA2-DREADD. These mice will now be studied to fully define and characterise effects of SCFAs that truly reflect activation of FFA2, with specific focus on regulation of entero-endocrine function, regulation of insulin release and the role of FFA2 in either the development or potential treatment of colitis and related inflammatory diseases of the lower gut.

There is widespread interest in each of the biological effects of metabolites produced by the microbiota, how this may influence cell and tissue homeostasis and how manipulation of the production of such metabolites might affect health across the lifecourse. There is also considerable interest in understanding the specific roles of receptors that respond to such metabolites and how targeting these receptors with synthetic ligands might provide therapeutic opportunity.

Who might benefit from this research?
Based on the above the outcomes of the proposed research will benefit a broad range of researchers spanning those interested directly in health and nutrition, via those interested in the molecular details of G protein-coupled receptor function and those involved in considering the validity and usefulness of mouse models of physiology and disease, to translational scientists based in both academia and industry that are tasked with therapeutic target validation.

How might they benefit from this research?
Reviews on the biology of the receptors for short chain fatty acids have highlighted a number of potential areas of therapeutic utility in targeting these receptors. However, although it is broadly clear that Free Fatty Acid receptor 2 (FFA2) is more widespread in distribution and has been implicated in a broader range of roles than FFA3, in many of these areas it remains surprisingly uncertain if activation or blockade of this receptor would be the most effective strategy, or indeed if a strategy of blocking this receptor would be effective at all. In substantial part this reflects that no antagonists able to block rodent orthologues of FFA2 have been described. As such, although antagonists with affinity at the human receptor are known, these cannot be used in wild type rodents. This was in large part the basis for production of the 'humanised' knock-in lines of mice as we describe in detail in 'Case for Support'. The detailed characterisation and study of these animals now proposed will address such questions specifically and define unambiguously the roles of this receptor. The detailed analysis we will perform on the molecular basis of G protein subtype selection by specific orthologues will also benefit greatly researchers who have been making predictions on these topics based on in silico evaluation but without testing their hypotheses directly. Finally, translational scientists who are interested to know if mouse models of physiology and function are pertinent to human pathophysiology will find the outcomes of particular interest as the proposed research will inform on the potential therapeutic validation (or otherwise) of FFA2 as a disease modifier in areas of inflammation and metabolic dysfunction. As such the pharmaceutical industry will follow the outcome of these studies closely.

The project also has substantial potential in terms of staff training. Although the named PDRA has generated much of the preliminary data and has gained experience of maintaining the transgenic mouse colonies Dr Bolognini will benefit further as he extends his ex vivo and in vivo skills set and from opportunities to perform further cutting edge research in a broad swathe of areas relevant to modern pharmacological and physiological studies. This training will ensure he has the greatest range of subsequent career opportunities.