An integrated strategy for control of animal and plant parasitic nematodes through targeting MOD-1

Nematodes are simple roundworms that share a similar worm-like shape yet range in size from the smallest microscopic species to a gigantic 3 metre long parasitic worm of sperm whale. They inhabit a correspondingly diverse range of habitats and include free-living and parasitic species. Free-living species are beneficial to the ecosystem. In contrast parasitic species cause major losses in food production and also 'neglected' human tropical diseases. The control of these nematodes is a particularly timely problem now for two reasons: The chemicals used to protect crops from plant parasitic nematodes are extremely toxic to users and the environment and are being withdrawn from use whilst at the same time drugs used to treat animal parasitic worm infections are losing their efficacy because nematodes are becoming resistant. Clearly, an approach is needed which will deliver chemicals that have low environmental impact and drugs that break resistance by acting in a completely new way. Arguably, the most successful nematicidal compounds to date have been those that have effects on the neuromuscular system of the worm. This means the worms can't move, feed or lay eggs and ultimately die. Of these, the most outstanding compound is ivermectin. Indeed, its discoverers won the Nobel Prize for the extraordinary benefit this compound has delivered to veterinary and human medicine. It revolutionised the treatment of human river blindness. An ivermectin-like chemical is also used in crop protection as a seed treatment. A key reason to the success of ivermectin is its selective toxicity i.e. it kills the parasite but has no detrimental effect on the mammalian host. It achieves this because it acts on a discrete signalling molecule, called a GluCl receptor that is only found in invertebrates. Therefore, the nematode worm has this receptor, and is killed by ivermectin, while the vertebrate mammalian host is unharmed. Unfortunately, due to more than three decades of use resistance to ivermectin has emerged and is a severe and increasing problem in the treatment of parasitic worms. Moreover, ivermectin kills a wide range of invertebrates including beneficial organisms and thus is not without environmental impact. We have found a new target that not only would deliver resistance-breaking chemicals but would also have lower toxicity to beneficial ecosystems. This target is the receptor MOD-1, first discovered in the nervous system of the nematode C. elegans that is widely used in laboratories around the world as a 'model' organism for parasitology. Importantly, activating the MOD-1 channel paralyses C. elegans showing that chemicals that target MOD-1 would be nematicidal. In addition, blocking MOD-1 disables plant parasitic nematodes and they can no longer enter roots. Notably, MOD-1 is largely restricted to the nematodes and is not widely found in insects e.g. in bees, and not at all in higher animals including mammals: This provides a compelling argument that chemicals that act on MOD-1 would have an excellent profile in terms of their selective toxicity. They could be deployed in veterinary medicine to kill the parasite whilst being well-tolerated by the animal receiving treatment and in crop protection to prevent plant disease but leave pollinating insects safe. The biology of this channel is intrinsically fascinating: We will characterise it using genetics, pharmacology and molecular modelling and in doing so find chemicals that interact with MOD-1. The latter will address the urgent need by both the agrochemical sector and the animal health sector for new environmentally friendly approaches to parasite control.

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.

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.