A phenotypic screen of the Global Health Priority Box identifies an insecticide with anthelmintic activity

Abstract

Background: Infection with parasitic nematodes (helminths), particularly those of the order Strongylida (such as Haemonchus contortus), can cause significant and burdensome diseases in humans and animals. Widespread drug (anthelmintic) resistance in livestock parasites, the absence of vaccines against most of these nematodes, and a lack of new and effective chemical entities on the commercial market demands the discovery of new anthelmintics. In the present study, we searched the Global Health Priority Box (Medicines for Malaria Venture) for new candidates for anthelmintic development.

Methods: We employed a whole-organism, motility-based phenotypic screening assay to identify compounds from the Global Health Priority Box with activity against larvae of the model parasite H. contortus, and the free-living comparator nematode Caenorhabditis elegans. Hit compounds were further validated via dose-response assays, with lead candidates then assessed for nematocidal activity against H. contortus adult worms, and additionally, for cytotoxic and mitotoxic effects on human hepatoma (HepG2) cells.

Results: The primary screen against H. contortus and C. elegans revealed or reidentified 16 hit compounds; further validation established MMV1794206, otherwise known as 'flufenerim', as a significant inhibitor of H. contortus larval motility (half-maximal inhibitory concentration [IC₅₀] = 18 μM) and development (IC₅₀ = 1.2 μM), H. contortus adult female motility (100% after 12 h of incubation) and C. elegans larval motility (IC₅₀ = 0.22 μM). Further testing on a mammalian cell line (human hepatoma HepG2 cells), however, identified flufenerim to be both cytotoxic (half-maximal cytotoxic concentration [CC₅₀] < 0.7 μM) and mitotoxic (half-maximal mitotoxic concentration [MC₅₀] < 0.7 μM).

Conclusions: The in vitro efficacy of MMV1794206 against the most pathogenic stages of H. contortus, as well as the free-living C. elegans, suggests the potential for development as a broad-spectrum anthelmintic compound; however, the high toxicity towards mammalian cells presents a significant hindrance. Further work should seek to establish the protein-drug interactions of MMV1794206 in a nematode model, to unravel the mechanism of action, in addition to an advanced structure-activity relationship investigation to optimise anthelmintic activity and eliminate mammalian cell toxicity.

Keywords: Caenorhabditis elegans; Haemonchus contortus; Anthelmintics; Antiparasitics; Drug discovery; Flufenerim; Global Health Priority Box; Nematodes; Phenotypic screening.

Fig. 1

Results of the primary screen of the Medicines for Malaria Venture (MMV) Global Health Priority Box (n = 240) against (A) exsheathed third-stage larvae (xL3s) of Haemonchus contortus and (B) young adults of Caenorhabditis elegans with reference to four distinct control compounds (monepantel, monepantel/abamectin, M-666 and moxidectin) and a negative (LB* + 1% DMSO) control. All test and positive-control compounds were tested at 20 μM. Each grey dot represents an individual test compound. Mean ± standard error of the mean (SEM) indicated for positive-control compounds (four data points) and negative controls (32 data points for LB* + 1% DMSO). For all screens, the Z′ factor ranged between 0.76 and 0.92

Fig. 2

The potency of four active test compounds (MMV1794214, MMV1794206, MMV1577463 and MMV027339) against exsheathed third-stage larvae (xL3s) of Haemonchus contortus, the potency of two active test compounds (MMV1794214 and MMV1794206) on adult females of H. contortus, and the potency of one active test compound (MMV1794206) on young adults of Caenorhabditis elegans, with reference to monepantel and/or moxidectin (positive controls). Each curve shows (A) the inhibition of H. contortus larval motility at 90 h, (B) the inhibition of H. contortus larval development at 7 days, (C) the in vitro motility inhibition (%) of H. contortus adult females over a period of 24 h (motility scores assessed at 3-, 6-, 12- and 24 h time points; cf. Taki et al. [59]) and (D) the reduction of C. elegans motility at 40 h. Data points represent either one (C) or three (A, B and D) experiments conducted in triplicate; the mean ± standard deviation (SD, C) or standard error the mean (SEM, A, B and D). Statistical significance was evaluated with reference to a negative control (C); **** denotes P ≤ 0.0001

Fig. 3

Toxicity assessment of MMV1794206, monepantel and moxidectin on human hepatoma (HepG2) cells, with reference to two positive controls, doxorubicin (Dox; cytotoxic) and M-666 (mitotoxic). For each compound, the (A) half-maximal cytotoxic concentration (CC₅₀) and (B) half-maximal mitotoxic concentration (MC₅₀) were established via a cell viability assay after 48 h of incubation. Crystal violet staining was used to measure the absorbance (595 nm) of treated cells, which was negative- (blank) and baseline- (100% cell viability) corrected. Data points represent triplicates and are presented as a mean ± standard deviation (SD)

Fig. 4

The chemical structure of MMV1794206 (flufenerim)