An integrated strategy for control of animal and plant parasitic nematodes through targeting a 5-HT-gated chloride channel MOD-1

Lead Research Organisation: University of Leeds
Department Name: Sch of Biology


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Technical Summary

We will characterise a 5-HT-gated chloride channel MOD-1 as a new target for chemical control of parasitic nematodes: Its potential is demonstrated as 5-HT acting as an agonist on MOD-1 causes paralysis and the antagonist methiothepin protects plants from invasion. MOD-1 is not found in arthropods and therefore has potential for low environmental impact. We will resolve MOD-1 orthosteric and allosteric binding sites through identification of conserved residues using an evolutionary approach, homology modelling and virtual screening. This will be facilitated by identification of agonists and antagonists in a novel assay in which selective expression of parasite MOD-1 in the M4 neuron of C. elegans confers lethality in the presence of an agonist such as 5-HT, and this is blocked by the orthosteric ligand methiothepin. It incorporates bioavailability and off-target effects. This M4 assay will be used to express MOD-1 from parasites of economic importance and select MOD-1 agonists and antagonists from biased compound libraries. MOD-1 expression in Xenopus oocyte will permit pharmacological definition of compound efficacy. Orthosteric agonists will be distinguished from positive allosteric modulators (PAMS) by methiothepin antagonism. In silico docking of MOD-1 compounds will refine homology modelling using available crystal structures of cys-loop ligand-gated chloride channels to resolve un-explored chemical space including both orthosteric and allosteric sites and guide synthesis of chemicals targeted at MOD-1. These will be validated in the M4 assay. Validated lead compounds will be screened for efficacy against parasites and tested for off target toxicity against a panel of organisms including beneficial insects.

Planned Impact

A: Beneficiaries A1: Commercial private sector The Animal Health industry and Agrochemical sectors are a mainstay of the economy e.g. livestock generates 41% of the EU agricultural output and the British Potato Council (BPC) estimates UK potato production has at c. £3 billion pa. Recent nematicide bans and anthelmintic resistance, drive a need for improved control methods underpinned by basic pre-competitive research. This project will demonstrate that an integrated approach to control for crops and animals can support agronomic and animal health needs. A2. Environmental organisations There are increasing concerns on the use of chemical in the environment reflecting growing public awareness of the damage they can cause. This project will show this can be addressed by a sound scientific rationale that constrains the impact of control methods to pests and parasites. A3. Farmers and growers Nematicides are the largest variable cost of the growers. This project will address concerns on the lack of basic biological knowledge of the parasitic nematodes e.g. UK Potato Council, Agr & Hort Develop Board, Research Strategy 2012-15). A4. Livestock producers. Anthelmintics account for c. Euros 2 billion of the veterinary pharmaceutical market but resistance limits their effectiveness. Our project will demonstrate a new approach to control. A5. Companion animals Dogs will benefit from the investigation of new heart worm treatments (c. 1 million dogs in the US). A.6 International growers: Nematodes cause losses of c. $ 157 billion pa; this will increase without new controls. The project will underpin global efforts to improve crop protection strategies and in the longer term will benefit growers. A7. Supermarkets: Some supermarkets see value in removing even the theoretical risk of pesticide residues from crops including potatoes. The outputs of this project, by identifying new mechanisms for plant parasitic nematode control, may in the longer term support that policy. A8: Policy-makers: DEFRA and the Scottish Government (SG) must implement the EU's Directive 91/414/EEC withdrawing nematicides. DEFRA and SG must also support an EU Directive for potato cyst nematodes (2007/33/EC; 2010) and need evidence of alternative controls. A9: General Public: There is a need to maintain efficient and inexpensive staple foods for the UK population. This work will support that need in the long term. B: Nature of benefits from this research B1: Benefits for UK economic competitiveness: LHD/VOC/PEU filed patent no. PCT/GB2018/051770 (25/06/2018) for exploitation of 5-HT signalling, including MOD-1. The project will provide patents as and when appropriate. Together this will create dialogue with industry for commercial exploitation of intellectual property: Thus it will support industry in tackling the challenges they face in developing new products with low environmental toxicity. B2. Benefits for UK producers: This work will build a platform to address the problem of nematode control that faces many UK producers i.e. how to sustain yields in the face of an increasing threat from parasitic nematodes whilst sparing beneficial ecosystems. B3: Increasing the effectiveness of public services and policy: It is relevant for those who seek to implement change in policies such as EU regulation EC 1107/2009 and implementation of 2007/33/EC to be aware that the UK science base supports its agricultural sector and is working to develop measures to address new legislation. B4: Enhancing cultural enrichment, quality of life and health i) Reducing reliance on hazardous pesticides benefits UK biodiversity and food production ii) Researchers will be trained in an important area alongside a new Enterprise Unit at Southampton, primed by a BBSRC Follow-on-Fund, that will translate basic science to food security and facilitate movement of trained personnel to industry iii) Links with schools will raise awareness of the of role science in sustainable food production.


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Description This project developed and used a novel screening platform (PhaGeM4) to identify active compounds that target a nematode-specific neuronal receptor as an effective mechanism for nematode control. A specific benefit of this approach is potential control chemicals with lower environmental impact. At the centre of our approach was the MOD-1 serotonin-gated chloride channel. This neuronal receptor is essential for appropriate nematode behaviour and, crucially, is largely restricted to nematodes. It is not widely found in insects e.g. bees, and not at all in higher animals including mammals. Therefore chemicals that act on MOD-1 would have an excellent profile in terms of their selective toxicity.
We first identified and cloned orthologues of the C. elegans mod-1 gene from representative plant (4 species) and animal parasitic nematodes. MOD-1 function was confirmed for 2 of the orthologues by heterologous expression and complementation studies in C. elegans. This highlighted that MOD-1 is highly conserved in the nematode phylum. Localising the sites of endogenous mod-1 expression in G. pallida proved difficult due to low expression levels however fluorescent in situ hybridization generated promising results and we were able to localise serotonin-producing neurons in the nematode using a fluorescent antibody.
A versatile screening platform, the PhaGeM4 assay, has been developed to identify novel compounds acting on the MOD-1 channel. In this assay, compounds can be assessed in an in vivo setting on whole C. elegans worms expressing a mod-1 gene in the essential neuron M4. If the compound activates the MOD-1 receptor this will result in C. elegans developmental arrest which can be easily scored. A chemical library collection of diverse drug-like molecules that span a wide range of pharmacologically active molecules, the Pathogen Box, was used to screen for MOD-1 selective modulators. From 400 compounds tested ten induced a significant developmental arrest in C. elegans worms expressing MOD-1 in the M4 neuron. Furthermore, these lead compounds displayed a similar potency when assessed on C. elegans strains expressing G. pallida MOD-1 in the M4 neuron. This supports the idea that bioactivity of drugs against C. elegans MOD-1 has a high chance of being conserved in parasitic species.
Of the ten lead compounds identified, one compound was known to possess anthelmintic properties and another was previously shown to be a growth inhibitor of C. elegans. None of the other eight compounds have been associated with any anthelmintic activity, and as such they represent new compounds of high interest, discovered by our new assay platform. The lead compounds have been further characterized for their ability to offer protection against plant parasitic nematodes. A root invasion assay revealed that 40% of the lead compounds effectively impaired nematode invasion at the concentrations tested. This result highlights the value of the PhaGeM4 assay as a robust platform capable to screen and identify relevant compounds.
Exploitation Route The methodology of the PhaGeM4 assay platform has been made available to the scientific community in the following publication: Calahorro F. et. al. PharmacoGenetic targeting of a C. elegans essential neuron provides an in vivo screening for novel modulators of nematode ion channel function. Pestic Biochem Physiol. 2022 Aug; 186:105152. doi: 10.1016/j.pestbp.2022.105152. The underpinning rationale of the M4 screening platform can be extended to other targets of interest if their ectopic expression in M4 neuron disrupts its function in a quantifiable way following external activation.
The lead compounds identified in this work could be tested against a wide range of plant and animal parasitic nematodes.
Sectors Agriculture, Food and Drink,Environment,Pharmaceuticals and Medical Biotechnology

Title PhaGeM4 platform 
Description The PhaGeM4 assay platform is a screening platform in which test compounds can be assessed in an in vivo setting using C. elegans worms expressing a target receptor specifically in the essential neuron M4. Any compound that activates the target receptor in the M4 neuron will lead to developmental arrest of the nematodes. This phenotype can be readily scored in a 96-well plate assay. One advantage of this method is that is can be used for heterologous screening, where a target receptor from a parasitic nematode species can be expressed in C. elegans. Furthermore, it uses whole organism screening and thus intrinsic to the screen is the ability to optimise bioavailability in vivo. It differentially selects chemicals that act on target receptor-expressing C. elegans compared to non-transgenic controls and so will eliminate chemicals that act off-target in C. elegans. Given the evolutionary conservation of many core cell signalling pathways this can predict toxicity in other animals. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? Yes  
Impact The PhaGeM4 assay platform was used in this project to identify a number of new lead compounds that activate the MOD-1 serotonin-gated chloride channel receptor of the potato cyst nematode Globodera pallida. Some of these compounds were then successful in significantly reducing the invasion the invasion of potato roots by the nematode.