The UFAW 3Rs Liaison Group Research Studentship
Steven Robery, Royal Holloway College, University of London
Employing the social amoeba, Dictyostelium, as a first pass screen in drug development
A major problem when developing new drugs for human (or animal) use is that potential drugs can cause nausea or vomiting (emesis) as a side effect. In fact, emesis is reported as a possible side effect in approximately one third of medicines. Whilst the physiological mechanisms behind the emetic response are well characterized, the diverse range of stimuli that can generate the response indicate the molecular mechanisms involved are poorly understood.
Rats, mice, ferrets, dogs, house musk shrews and non-human primates are commonly used as models in emetic research. In clinical (human) studies nausea and vomiting are perceived as unpleasant and it is very likely that animals experience similarly unpleasant feelings when undergoing emetic testing.
In an effort to reduce the number of animals required for testing emetic drugs, in 2009 the UFAW 3Rs Liaison Group supported a project by Mr Steven Robery (under the supervision of Robin Williams and Paul Andrews) at Royal Holloway, University of London. Steven examined the possibility of using the soil-living amoeba Dictyostelium discoideum as a preliminary screen for emetic liability in drug development in order to reduce or replace animal use in this area.
A diverse range of emetic compounds were investigated and the effect on Dictyostelium discoideum behaviour was monitored. Steven found that a small number of known emetic compounds strongly inhibited cell migration in a concentration-dependent and reversible manner, and thus provided specific families of emetic/aversive compounds that were suitable for more detailed investigation. These active compounds included a range of bitter compounds, including phenylthiourea and naringenin, and the hot tastant capsaicin.
Figure 1: Analysis of Dictyostelium cell behaviour.
Cells moving under a chemotactic gradient were analysed using ImagePro software to determine cell velocity (μm/sec); cell aspect (shape measured as a ratio between the length of cells across each axis); cell angle (degrees-where cell migration was measured in comparison to the y-axis); and cell tracking (where the coordinates of cell pathways were illustrated following normalisation to (0,0) at 5 minutes) in order to illustrate changes in migration before and after compound addition.
Analysis of the bitter tastant phenylthiourea, identified a poorly characterised receptor for this compound in Dictyostelium. This receptor was analysed in Dictyostelium as a potential cellular mechanism for bitter tastant receptor activity. This study then translated the discovery of this receptor to a human context, by identifying a poorly characterised human receptor (part of the inhibitory neurotransmitter receptor for GABA) that is responsible for bitter tastant function. Analysis of another bitter tastant, naringenin (a flavonoid), also identified a poorly characterised ion channel responsible for naringenin function using Dictyostelium as an animal replacement model. The discovery of this channel as a target for naringenin was then characterised in mammalian cell lines, to confirm the functionality of this interaction in mammalian models.
Overall, Steven concluded that because only seven of the twenty-nine compounds with known emetic effects in mammals affected Dictyostelium behaviour, its use as a general model for predicting emetic liability is limited. However, his research identified four different sub-groups of compounds that blocked chemotaxis, including a range of bitter tastants. He concluded that Dictyostelium may therefore provide a useful model in the analysis of these compounds in structure-activity relationships or translational studies to identify novel active vanilloids or bitter tastants. Using Dictyostelium, compounds structurally related to bitter tastants such as denatonium benzoate may also help to identify other bitter and non-bitter analogues, which may target specific bitter taste receptors, and in the characterisation of bitter tastants potency in translational studies. The project led to further industry collaboration to potentially reduce the use of sentient animals (ferrets, dogs and non-human primates) in procedures classified as of moderate severity under the Animals (Scientific Procedures) Act 1986.
Dr Robery was awarded his PhD in 2013
Published papers arising from Steven’s work supported by UFAW:
Waheed A, Ludtmann MHR, Pakes N, Robery S, Kuspa A, Dinh C, Baines D, Williams RSB and Carew MA. 2014. Naringenin inhibits the growth of Dictyostelium and MDCK-derived cysts in a TRPP2 (polycystin-2)-dependent manner. British Journal of Pharmacology. 171(10): 2659-2670. https://dx.doi.org/10.1111/bph.12443.
Robery S, Tyson R, Dinh C, Kuspa A, Noegel AA, Bretschneider T, Andrews PL & Williams RS. 2013. A novel human receptor involved in bitter tastant detection identified using the model organism Dictyostelium discoideum. Journal of Cell Science. 126: 5465-5476http://dx.doi.org/10.1242/jcs.136440
Robery S, Mukanowa J, Percie du Sert N, Andrews PLR. & Williams RSB. 2011. Investigating the effect of emetic compounds on chemotaxis in Dictyostelium identifies a non-sentient model for bitter and hot tastant research. PLoS One 6(9): e24439. http://dx.doi.org/10.1371/journal.pone.0024439
