The matrix associated astacin enzymes: novel targets in the control of key GI nematodes of ruminants.

The parasitic nematodes are important pathogens of man and his domestic animals that cause chronic debilitating diseases. The trichostrongylid nematodes represent widespread and economically important pathogens of grazing livestock and have a worldwide prevalence. The infective stages are taken up on pasture and pathogenic stages are found in the gastro-intestinal tract. These parasites are common throughout the United Kingdom and they are estimated to cost the sheep farming industry in excess of £84 million per annum. The numbers of reported outbreaks and the geographic range of these parasites are increasing, with an increasingly warmer and wetter climate prolonging the survival of the free-living stages being a contributing factor. A limited number of drugs are available to control these infections with resistance being commonplace due to their over-application. Multiple resistance to different classes of drugs has now also been reported and there is an urgent requirement to develop new classes of drugs. All nematodes are protected by a tough exoskeleton called the cuticle, a matrix that is shed and re-synthesised repeatedly through a process called moulting. The cuticle structure and the moulting process are specific to nematodes and we will target key enzymes that are involved in the construction and shedding of this structure. We have used a laboratory model system called Caenorhabditis elegans to study this structure and we have identified the key enzymes that are shared with the parasitic nematodes. We now propose to study these enzymes in the important nematode species that infect sheep and will use a combination of structural biology, genetics and biochemistry to validate these targets. In collaboration with experts in drug discovery we will identify small molecules that are effective against these enzymes and in collaboration with experts in veterinary parasitology we will test the compounds in the parasites themselves. This work will lay the important groundwork in developing new drugs to control these economically important parasites and it is envisaged that these compounds will be effective against a wider range of infective nematode parasites of cattle, domestic animals and man.

Trichostrongylid nematodes of grazing livestock represent a significant economic burden to the global livestock farming industry. Control measures depend upon the administration of a limited number of anthelmintics that against the backdrop of widespread resistance mean an uncertain future for this sector. We propose to examine the feasibility of targeting the nematode specific cuticle and the moulting process to develop novel classes of anthelmintics. My recent research in the experimental model nematode C. elegans has focused on key astacin metalloprotease enzymes that are involved in cuticle collagen biosynthesis and cuticle ecdysis. This proposal aims to translate this research to examine the function of these enzymes in the key parasite species of small ruminants, namely Haemonchus contortus and Teladersagia circumcincta. H. contortus is predominantly a tropical species that is taking hold in the UK whereas T. circumcincta is the most significant nematode in the UK sheep flock. Characterisation is underway for the H. contortus enzymes and work on the T. circumcincta genes and enzymes is now feasible due to the ongoing BBSRC-funded sequencing project. We will apply recently adapted trichostrongylid RNA interference methods to functionally characterise these genes and will use well established transgenic techniques to examined their ability to complement the corresponding C. elegans mutants. Biochemical and cell biological techniques will be applied to characterize the parasite enzymes in detail. Our preliminary molecular modeling experiments based on available astacin structures have identified a series of inhibitors that are predicted to inhibit both subsets of enzymes. These compounds can replicate the mutant phenotype of a corresponding mutant in C. elegans. We will examine the inhibitory potential of the identified compounds in the trichostrongylid parasites using a range of in vitro and in vivo assays and all positive leads will be actively pursued.

The approach outlined here, which is built on a very significant and broad scientific base, provides the UK with an opportunity to take a lead in the understanding of the enzymology of collagen biogenesis and ECM formation and develop a novel class of anti-nematode compounds. This proposal will increase our basic knowledge of parasitic nematodes that are a major cause of serious economic losses in the livestock farming industries worldwide. Treating nematode infections in livestock will also provide economically sustainable agriculture in the UK that will help maintain the UK's food security. The parasitic gastro-intestinal (GI) nematodes cause a significant economic impact on the global livestock industry. These common parasites induce widespread debilitating infections in small ruminants and cattle resulting in poor weight gain and significant production losses. The estimated cost of GI nematode infections to the UK sheep farming industry alone has be calculated to be in excess of £84 million per annum and is likely to increase in the future due to the combined effects of climate change and anthemintic resistance. Strong evidence exists to support the recent change in the UK climate that has resulted in milder, wetter winters with an extended herbage growing season that will affect the free-living stages of the trichostrongylid ruminant parasites. In the UK this has impacted on both the range and number of outbreaks reported for Haemonchus contortus, Nematodirus battus and Telodersagia circumcincta in sheep. The GI trichostrongylid infections can be treated with three specific classes of anthelmintics, but widespread prophylactic use has led to the emergence of serious resistance in both sheep and cattle populations, and more worrying triple resistance has also been reported on sheep farms in Scotland. The outlook for sheep farming in the UK is increasingly bleak, and it may ultimately fall below the level of economic viability. With respect to cattle farming the widespread advance of anthelmintic resistance has now also began to cause serious concern in the USA. Therefore there is an urgent need to develop new drugs to target the GI nematodes of ruminants. As a consequence of single and multiple drug resistance the real need is to identify and target novel pathways that will allow the control of the existing anthelmintic-resistant strains. The primary causes of concern to the UK sheep farming industry are H. contortus and T. circumcincta and these parasites will be the focus of this proposal. We plan to target the cuticle biosynthetic and cuticle moulting astacin enzymes that play essential nematode-specific roles and we envisage that compounds identified in this study may have related efficacy against the other major trichostrongylid nematodes and will feasibly be effective against a wide range of parasitic nematodes. This pathway has not previously been targeted in drug development schemes, and if successful, will provide an effective means of treating the existing anthelmintic resistant strains. In addition, by targeting the cuticle synthesis and moulting processes we may also effectively control the arrested L4s, a stage normally refractive to current treatments. This will help break the season- season transmission and will reduce or remove the pathogenic stages associated with the common trichostrongylid species.