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

Aims

This project aims to develop a novel Dictyostelium model to investigate the potential for bitter tastants as new treatments for asthma, and replace the mammalian models currently used for these studies.

Background

Bitter tasting compounds have been demonstrated to open the airways in the lung, suggesting a role for these compounds in the treatment of asthma. 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 ex vivo tissue to elucidate the mechanism by which bitter compounds trigger muscle relaxation (related to relieving asthma induction), and uses large numbers of animals.

Developing alternative non-animal systems to investigate the mechanisms of bitter tasting compounds will reduce the use of animals in this research area, and provide experimental approaches unavailable in animal models. This project proposes to develop a two stage system, initially using the social amoeba Dictyostelium to identify the mechanism of action of bitter tastants as bronchodilators, and subsequently using human cells to confirm discoveries are relevant to asthma treatment.

Research details and methods

A variety of 'reference' bitter tastants associated with bronchodilation will be screened 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. A mutant library used in the screen 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. Resistance will be monitored using sensitivity to growth inhibition, movement, and development. This approach will enable the analysis of these new targets without using animals.

Bitter compounds and identified signalling pathways from the Dictyostelium studies will be investigated in human-derived tissues to illustrate functional mechanism(s) for these compounds in human tissues.

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.

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Strategic grant

Status:

Closed

Principal investigator

Professor Robin Williams

Institution

Royal Holloway University of London

Co-Investigator

Professor Sven-Erik Dahlen

Grant reference number

NC/M001504/1

Award date:

Jan 2015 - Jan 2016

Grant amount

£98,894