Developing a non-animal model system to investigate bitter tastants as new treatments for asthma

The recent observation that bitter tasting compounds cause the opening of airways in the lung suggest a role for these compounds in the treatment of asthma. This is unexpected, since bitter taste detection was thought act to protect animals (including humans) from ingesting harmful, often bitter, compounds. Bitter tasting compounds are detected by a family of receptors called TAS2Rs that are found on the tongue. Intriguingly, however, it remains unclear if these TAS2Rs are involved in asthma treatment, or if this effect occurs through unidentified proteins. Increasing evidence suggests that these compounds may act through TAS2R independent targets, but these targets are still to be discovered.

Research into bitter compounds and the way they work in asthma has focused on using lung tissue obtained from guinea pigs and mice, but also from humans. This research involves using this animal tissue to identify the process that bitter compounds trigger to cause muscle relaxation (related to relieving asthma induction). Using this approach, it was expected that the effect of each bitter compound could be mapped, and resulting information used to develop improved treatments for chronic asthma. Unfortunately, to date, bitter tasting compounds have been shown to mediate their dilatory effects in multiple ways, some acting independently of known TAS2Rs-dependent processes within these animal tissues. Identifying these mechanisms is thus highly important.

Developing alternate non-animal systems to investigate the mechanisms of bitter tasting compounds will significantly reduce the use of animals in this research area. This research will also provide experimental approaches unavailable in animal models leading to breakthroughs in the treatment of asthma. We propose to develop a two stage system bitter tastant research, initially using the social amoeba Dictyostelium to identify how these tastants work, and then using human cells to confirm our discoveries are relevant to asthma treatment.

We have shown that Dictyostelium can be successfully used to identify new mechanisms of action for compound relevant to human health. The effective use of this model is beyond doubt, as it has been used to find the mechanism of bipolar disorder drugs (Williams et al (2002) Nature, 417, 292-5; Williams (2005) Prog. in Neuro-Psychopharmacol. and Biol. Psychiatry 29, 1029-37), the mechanism of the most wildly used epilepsy treatment, valproic acid (Xu et al (2007) Euk Cell, 6, 899-90; Chang et al (2012) Disease Models and Mechanism, 5, 115-124; Chang et al (2013) Neurobiology of Disease, 62C, 296-306) and the underlying therapeutic mechanism of the MCT ketogenic diet (Chang et al (2013) Neuropharmacology, 69,105-14; Williams and Walker (2013) Biochem. Soc. Trans, 41,1625-8). It has also been used to identify novel molecular targets for two structurally independent bitter tastants been (Robery et al (2013) Journal of Cell Science, 126, 5465-76; Waheed et al (2014) British Journal of Pharmacology, 171, 2659-70). Hence therapeutically relevant targets for bitter compounds in the treatment of asthma will be identified in the project using Dictyostelium as an animal replacement model.

Ultimately, our goal is to develop Dictyostelium as a non-animal system to uncover the mechanisms whereby bitter tasting compounds mediate their effects, to identify novel molecular targets for the treatment of asthma.

We will initially screen a variety of 'reference' bitter tastants associated with bronchodilation and asthma relief effects using Dictyostelium growth as a model. Sensitivity of Dictyostelium to these compounds will enable a genetic screen to identify novel gene products controlling sensitivity to bitter compounds, as we have shown in regards to three other bitter tastants; phenylthiourea, naringenin and valproic acid. A mutant library used in the screen will be provided by Prof Thompson (Manchester)(support letter provided to Royal Holloway), and two bitter tastants will be used to isolate new bitter tastant targets. Recapitulation of identified mutants in wild type cell lines will confirm a role for each target in sensitivity. These mutants will also be rescued by expression of the endogenous gene and by expression of the human homologue (as we have previously published), and the characterisation of bitter tastant signal transduction. Cross-specificity for these bitter tastants against common molecular targets will be investigated. Resistance will be monitored using sensitivity to growth inhibition, movement, and development (as we have previously published). This approach will thus enable the analysis of these new targets without animal loss. We have demonstrated the successful use of all these approaches in multiple projects and we foresee no technical problems in this research.

We will continue to develop the system by the inclusion of a human research model. In these studies we will employ both primary human airway smooth muscle cells and human bronchi and vessels. Both known and newly identified bitter tastant targets will be ablated by established RNAi techniques and calcium imagine will be used to monitor tastant effects. We have well established expertise in these approaches and we foresee no technical problems in this research.

Research on bitter tastants is a rapidly emerging field with potential for new drug development. At this stage, there are no "gold-standard" models for rational design of new chemical entities to interfere with bitter tastants. As also discussed in the introduction of the research plan, one conventional path for development of the field would involve setting up high-throughput screening experiments using animal tissues. The calculation of how many animals that would be necessary for such studies depends upon a number of currently unknown factors, it may be sufficient to conclude at this early stage in this new field of research that the number of animals required would be overwhelming. We would rather like to focus on the opportunity that our proposal offers, namely to pro-actively introduce a non-mammalian test system even before animal models have been attempted to any greater extent. Together with the build-in validation of findings in the Dictyostelium model in the human cells and tissues, this project has the potential to completely pre-empt the need for animal models in research on bitter tastants. We therefore argue that the project is highly relevant for all 3R aspects. In addition, the PIs of the application belong to strong international networks and may therefore be able to effectively implement the dissemination of new non-animal methods in their respective scientific communities.

The ultimate impact of this project, in addition to developing a world leading non-animal model approach for bitter tastant function in asthma, is patient benefit. The societal importance of asthma and related chronic obstructive pulmonary disease is huge, since these conditions affect more than 15% or the European population, with an estimated cost of over Euro 210 billion in 2011. This project will impact on this population through the ultimate improved understanding of mechanisms of action of potential new treatments. To indicate and underline this, we have received two letters of support from UK based Asthma Charities, demonstrating their support for the project due to potential impact on those living with these diseases. This clearly shows the potential long-term and beneficial output from this project.