Screening of the 'Pathogen Box' identifies an approved pesticide with major anthelmintic activity against the barber's pole worm

Abstract

There is a substantial need to develop new medicines against parasitic diseases via public-private partnerships. Based on high throughput phenotypic screens of largely protozoal pathogens and bacteria, the Medicines for Malaria Venture (MMV) has recently assembled an open-access 'Pathogen Box' containing 400 well-curated chemical compounds. In the present study, we tested these compounds for activity against parasitic stages of the nematode Haemonchus contortus (barber's pole worm). In an optimised, whole-organism screening assay, using exsheathed third-stage (xL3) and fourth-stage (L4) larvae, we measured the inhibition of larval motility, growth and development of H. contortus. We also studied the effect of the 'hit' compound on mitochondrial function by measuring oxygen consumption. Among the 400 Pathogen Box compounds, we identified one chemical, called tolfenpyrad (compound identification code: MMV688934) that reproducibly inhibits xL3 motility as well as L4 motility, growth and development, with IC₅₀ values ranging between 0.02 and 3 μM. An assessment of mitochondrial function showed that xL3s treated with tolfenpyrad consumed significantly less oxygen than untreated xL3s, which was consistent with specific inhibition of complex I of the respiratory electron transport chain in arthropods. Given that tolfenpyrad was developed as a pesticide and has already been tested for absorption, distribution, excretion, biotransformation, toxicity and metabolism, it shows considerable promise for hit-to-lead optimisation and/or repurposing for use against H. contortus and other parasitic nematodes. Future work should assess its activity against hookworms and other pathogens that cause neglected tropical diseases.

Keywords: Anthelmintic screening; Haemonchus contortus; Mitochondrial respiratory chain; Pathogen Box; Tolfenpyrad.

Graphical abstract

Fig. 1

The ‘Pathogen Box’ from the Medicines for Malaria Venture (MMV) (Panel A) contains 400 diverse drug-like molecules with confirmed activity against one or more key pathogens that cause some of the most socioeconomically important diseases worldwide, including malaria, toxoplasmosis, cryptosporidiosis, trypanosomiasis, leishmaniasis, hookworm disease, trichuriasis, schistosomiasis; numbers of chemicals active against different pathogen/pathogen groups are indicated in parentheses. The graphs (Panels B to D) show the activity of tolfenpyrad on the motility of exsheathed third-stage (xL3) and fourth-stage (L4) larvae of Haemonchus contortus (after 24 h, 48 h and 72 h of exposure) and on the development of the L4 stage (after 7 days of exposure), respectively.

Fig. 2

Panel A. Images of fourth-stage larvae (L4) exposed for 48 h to LB* containing either 1% dimethyl sulfoxide (untreated, negative control), 100 μM of each tolfenpyrad, moxidectin (positive-control) or monepantel (positive-control). Displayed are whole worm (L4) or anterior, mid-body and posterior regions (20–100× magnification; white scale bar: 20 μm). Solid, black arrows show structural damage to the cuticle and mouth of L4s. Panel B. Graph showing the mean widths of L4s under the same experimental conditions. The data points represent the mean width of 30 individual L4s ± the standard error of the mean (SEM). A representative graph of two separate experiments is shown. Significance between values (mean ± SEM) was determined using a non-parametric (Kruskal-Wallis) one-way ANOVA and Dunn's multiple comparison test. *^/** indicate which values are significantly different from one another (P < 0.05 and P < 0.01, respectively).

Fig. 3

Panel A. Motility of exsheathed third-stage larvae (xL3s) of Haemonchus contortus incubated in culture medium (LB* + 1% DMSO) (untreated), and in the presence of 100 μM of either tolfenpyrad (‘hit’ compound) or moxidectin (control) at three distinct time points; at each data point, motility was normalised to time (0 min). Panel B. Based on results in panel A, oxygen consumption was measured seven times (8 min each) over a period of 1 h in untreated xL3s, and xL3s treated with the same concentration (100 μM) of tolfenpyrad (‘hit’ compound) or moxidectin (control) using a flux analyser. The mean values from 10 replicates (motility) and 8 replicates (oxygen consumption) from two separate experiments are shown, with variation between measurements displayed as a standard error of the mean (SEM). *^/** indicate values (mean ± SEM) which are significantly different from one another (P < 0.05 and P < 0.01, respectively), determined using a two-way ANOVA and Dunnett's multiple comparison test.