An automated high-throughput system for phenotypic screening of chemical libraries on C. elegans and parasitic nematodes

Abstract

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.

Repurposing of an old drug: In vitro and in vivo efficacies of buparvaquone against Echinococcus multilocularis

“…So far, the Pathogen Box has been screened against the nematode H. contortus ( Preston et al, 2016 ), the fungi Candida albicans and Cryptococcus neoformans ( Vila and Lopez-Ribot, 2016 ; Mayer and Kronstad, 2017 ), Plasmodium and the kinetoplastids Leishmania and Trypanosoma ( Calit et al, 2018 ; Dennis et al, 2018 ; Duffy et al, 2017 ), Neospora caninum ( Müller et al, 2017 ) , Mycobacterium abscessus and M. avium ( Jeong et al, 2018 ; Low et al, 2017 ), Toxoplasma gondii ( Spalenka et al, 2017 ), C. elegans ( Partridge et al, 2018 ), Entamoeba histolytica ( Mi-Ichi et al, 2018 ), and Giardia lamblia and Cryptosporidium parvum ( Hennessey et al, 2018 ). Interestingly, all compounds that exhibited activity against E. multilocularis were also active against at least one more pathogen other than the one it was selected for by MMV (with the exception of nitazoxanide), thus underlining the importance and potential of the concept of drug repurposing.…”

2,4-Diaminothieno[3,2-d]pyrimidines, a new class of anthelmintic with activity against adult and egg stages of whipworm

“…An earlier version of this system has been previously described [ 13 , 14 ]. Two hundred frame movies of the whole plate were recorded at 10 frames per second and then motility determined by an algorithm based on thresholding pixel variance over time [ 15 ]. For the hit confirmation and expansion assays, library material was used at a final concentration of 100μM.…”

“…Worm movement was stimulated by inserting and removing a 96-well PCR plate into/from the wells of the assay plate, and then whole plate 200 frame movies were recorded at 30 frames per second. Growth was quantified as a correlate of movement using the same automated system described earlier, which estimates movement for each well by categorising pixels as imaging movement if their variance is greater the mean plus one standard deviation of the variances of all the pixels on the plate [ 15 ].…”

Plant-Based Natural Products for the Discovery and Development of Novel Anthelmintics against Nematodes

“…Most studies use only a single target organism, which in many cases is not a parasite (i.e., C. elegans). In vitro evidence of activity on a suitable parasite is probably essential for progressing a compound for further development, although well-documented anthelmintic traditional use, in conjunction with C. elegans activity, gives more confidence that the activity will persist on parasites, compared to compounds emerging from a screen of a chemical library [114,115].…”