Parasitic nematodes infect hundreds of millions of people and farmed livestock. Further, plant parasitic nematodes result in major crop damage. The pipeline of therapeutic compounds is limited and parasite resistance to the existing anthelmintic compounds is a global threat. We have developed an INVertebrate Automated Phenotyping Platform (INVAPP) for high-throughput, plate-based chemical screening, and an algorithm (Paragon) which allows screening for compounds that have an effect on motility and development of parasitic worms. We have validated its utility by determining the efficacy of a panel of known anthelmintics against model and parasitic nematodes: Caenorhabditis elegans, Haemonchus contortus, Teladorsagia circumcincta, and Trichuris muris. We then applied the system to screen the Pathogen Box chemical library in a blinded fashion and identified compounds already known to have anthelmintic or anti-parasitic activity, including tolfenpyrad, auranofin, and mebendazole; and 14 compounds previously undescribed as anthelmintics, including benzoxaborole and isoxazole chemotypes. This system offers an effective, high-throughput system for the discovery of novel anthelmintics.

The human whipworm Trichuris trichiura is a parasite that infects around 500 million people globally, with consequences including damage to physical growth and educational performance. Current drugs such as mebendazole have a notable lack of efficacy against whipworm, compared to other soil-transmitted helminths. Mass drug administration programs are therefore unlikely to achieve eradication and new treatments for trichuriasis are desperately needed. All current drug control strategies focus on post-infection eradication, targeting the parasite in vivo. Here we propose developing novel anthelmintics which target the egg stage of the parasite in the soil as an adjunct environmental strategy. As evidence in support of such an approach we describe the actions of a new class of anthelmintic compounds, the 2,4-diaminothieno[3,2-d]pyrimidines (DATPs). This compound class has found broad utility in medicinal chemistry, but has not previously been described as having anthelmintic activity. Importantly, these compounds show efficacy against not only the adult parasite, but also both the embryonated and unembryonated egg stages and thereby may enable a break in the parasite lifecycle.

2,4-Diaminothieno[3,2-d]pyrimidines, a new class of anthelmintic 1 with activity against adult and egg stages of whipworm 2 Short title: Diaminothienopyrimidines, a new chemotype for the control of 3 whipworm 4 35The human whipworm, Trichuris trichiura, infects around 500 million people globally, impacting on 36 their physical growth and educational performance. There are currently huge mass drug 37 administration (MDA) programs aiming to control whipworm, along with the other major soil 38 transmitted helminths, Ascaris and hookworm. However single doses of albendazole and 39 mebendazole, which are used in MDA, have particularly poor effectiveness against whipworm, with 40 cure rates less than 40%. This means that MDA may not be able to control and eliminate whipworm 41 infection, and risks the spread of resistance to albendazole and mebendazole in the parasite 42 population. 43We are attempting to develop new treatments for parasitic worm infection, particularly focused on 44 whipworm. Herein we report the identification of a class of compounds, diaminothienopyrimidines 45 (DATPs), which have not previously been described as anthelmintics. These compounds are effective 46 against adult stages of whipworm, and also block the development of the model nematode C. elegans. 47Our DATP compounds reduce the ability of treated eggs to successfully establish infection in a mouse 48 model of human whipworm. These results support a potential environmental spray to control 49 whipworm by targeting the infectious egg stage in environmental hotspots. 50 Current anthelmintics 52The benzimidazole anthelmintics albendazole and mebendazole are typically used to treat human 53 whipworm infection but are compromised by lack of single-dose efficacy and the risk of resistance. 54Thus, existing drugs lack sufficient efficacy in mass drug administration (MDA) programs to 55 adequately control or potentially eradicate whipworm. This is a major stumbling block in the WHO 56 target to eliminate morbidity from soil transmitted helminthiases in children by 2020. The current 57 approach for controlling soil-transmitted helminths such as Trichuris is mass drug administration of a 58 single-dose of albendazole or mebendazole, typically repeated annually [1]. However for infection 59 with T. trichiura, single doses of benzimidazoles lead to low cure rates, only 28% and 36% for 60 albendazole and mebendazole respectively[2]. These cure rates are much lower than those of other 61 major human soil-transmitted helminths, Ascaris lumbricoides and hookworm, demonstrating the 62 need for improvements to therapy specifically targeting Trichuris. Indeed modelling studies have 63 demonstrated that, due to these low cure rates, MDA with benzimidazoles does not interrupt 64 whipworm transmission and thus cannot achieve eradication in many settings [3]. 65 Furthermore, the experience from studies on veterinary parasites is that widespread usage of 66 anthelmintics can lead to rapid development of resistance. The discovery of isolates of two species of 67 g...

anthelmintics, including tolfenpyrad, auranofin, and mebendazole and 14 compounds previously 31 undescribed as anthelmintics, including benzoxaborole and isoxazole chemotypes. This system offers 32 an effective, high-throughput system for the discovery of novel anthelmintics. 33. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a

Hundreds of millions of people worldwide are infected with the whipwormTrichuris trichiura. Novel treatments are urgently needed as current drugs, such as albendazole, have relatively low efficacy. We have investigated whether drugs approved for other human diseases could be repurposed as novel anti-whipworm drugs. In a previous comparative genomics analysis, we identified 409 drugs approved for human use that we predicted to target parasitic worm proteins. Here we tested theseex vivoby assessing motility of adult worms ofTrichuris muris, the murine whipworm, an established model for human whipworm research. We identified 14 compounds with EC50 values of≤50μM againstT. muris ex vivo, and selected nine for testingin vivo. However, the best worm burden reduction seen in mice was just 19%. The high number ofex vivohits againstT. murisshows that we were successful at predicting parasite proteins that could be targeted by approved drugs. In contrast, the low efficacy of these compounds in mice suggest challenges due to their chemical properties (e.g. lipophilicity, polarity, molecular weight) and pharmacokinetics (e.g. absorption, distribution, metabolism, and excretion) that may (i) promote absorption by the host gastrointestinal tract, thereby reducing availability to the worms embedded in the large intestine, and/or (ii) restrict drug uptake by the worms. This indicates that identifying structural analogues that have reduced absorption by the host, and increased uptake by worms, may be necessary for successful drug repurposing against whipworms. Therefore, we recommend that prior toin vivostudies, future researchers first assess drug absorption by the host, for example, using human intestinal organoids or cell lines, and drug uptake by whipworms using intestinal organoids infected withT. muris.