Thermal proteome profiling reveals Haemonchus orphan protein HCO_011565 as a target of the nematocidal small molecule UMW-868

Abstract

Parasitic roundworms (nematodes) cause destructive diseases, and immense suffering in humans and other animals around the world. The control of these parasites relies heavily on anthelmintic therapy, but treatment failures and resistance to these drugs are widespread. As efforts to develop vaccines against parasitic nematodes have been largely unsuccessful, there is an increased focus on discovering new anthelmintic entities to combat drug resistant worms. Here, we employed thermal proteome profiling (TPP) to explore hit pharmacology and to support optimisation of a hit compound (UMW-868), identified in a high-throughput whole-worm, phenotypic screen. Using advanced structural prediction and docking tools, we inferred an entirely novel, parasite-specific target (HCO_011565) of this anthelmintic small molecule in the highly pathogenic, blood-feeding barber's pole worm, and in other socioeconomically important parasitic nematodes. The "hit-to-target" workflow constructed here provides a unique prospect of accelerating the simultaneous discovery of novel anthelmintics and associated parasite-specific targets.

Keywords: anthelmintic discovery; in silico docking; structure modelling; target identification; thermal proteome profiling.

FIGURE 1

Workflow used for the selection of the candidate nematocidal compound UMW-868. (A) All compounds (diverse chemical space) in the HitFinder library (Maybridge) library were screened in an established whole-organism phenotypic assay (Taki et al., 2021a) using exsheathed third-stage (xL3) larvae of Haemonchus contortus. The screen of this library identified 11 definitive hits with favourable drug-like features. (B) Secondary screen identified six compounds with strong activity, one of which (designated UMW-868) had favourable structural characteristics for further investigation. (C) Potency assessments of UMW-868 on different developmental stages of H. contortus—i.e., xL3 (motility), L4 (= fourth larval stage; development), female adult (motility) and egg (hatching). (D) UMW-868 is linked to the chemical space of the HitFinder library, based on the physicochemical properties (molecular weight and cLogP).

FIGURE 2

Bioassay characterisation of compound UMW-868. (A) Structures of UMW-868 (active) and its analog A-3155 (inactive). (B) Dose-response assessment of the in vitro lethality of UMW-868 for inducing cell death in exsheathed third-stage larvae (xL3s) of Haemonchus contortus over 90 h, with reference to xL3s exposed to control compounds, M-666 (lethal) and A-3155 (non-lethal). (C) Dead cells detected microscopically of UMW-868-treated xL3s stained with Sytox Green nucleic acid stain, observed under 40-times magnification, with reference to xL3s exposed to A-3155 (non-lethal) and no-compound control (containing 0.2% (v/v) DMSO). (D) Cellular and mitochondrial toxicities of UMW-868 and two non-toxic compounds, monepantel (MON) and moxidectin (MOX) on HepG2 human hepatoma cells, using control compounds (doxorubicin and M-666) for respective toxicities.

FIGURE 3

Thermal proteome profiling. (A) Proteins from exsheathed third-stage larvae (xL3s) of Haemonchus contortus were released by cell lysis in phosphate-buffered saline (PBS) containing 0.4% NP-40 (detergent). Prior to heat treatment, aliquots were incubated with UMW-868 or PBS, respectively. Subsequently, remaining soluble proteins were collected by centrifugation and analysed by tandem mass tag (TMT)-based quantitative proteomics. Melting curves of each quantified protein were plotted using the R package. (B) Heat map of quantified soluble proteins showing relative protein abundances upon treatment with UMW-868 or PBS with temperature gradient from 37°C to 67°C. Normalised protein abundance is shown as a grey to white scale, depicting high to low relative protein abundance. (C) Boxplot of overall quantified soluble proteins showing relative fold-change in abundance upon treatment with UMW-868 or PBS in a temperature gradient from 37°C to 67°C. (D) Thermal shift plot showing the melting curves of two potential protein targets for UMW-868: HCO_011565 and HCO_014287. Data from two replicates.

FIGURE 4

Molecular characterisation of HCO_011565 and docking with UMW-868. (A) Structure of HCO_011565 protein predicted in silico using the Alphafold2 algorithm. The per-residue confidence scores (pLDDT; left to right) for individual sections of the protein are indicated. (B) Transcription and expression profiles for the gene encoding HCO_011565 in different developmental stages/sexes of Haemonchus contortus larvae: eggs, first-, second- and third-stage larvae (L1, L2 and L3, respectively), exsheathed third-stage larvae (xL3s), fourth-stage larvae (L4s), female and male L4s (L4f and L4m, respectively), and female and male adults (Af and Am, respectively). Heatmaps indicate levels of transcription/expression: red indicates moderate to high transcription/expression; green is low expression; and grey indicates that expression was not detected. (C) Three-dimensional structure of the protein HCO_011565 and its interaction with compound UMW-868; the inset shows the positioning of UMW-868 in the pocket predicted within HCO_011565. (D) Phylogenetic relationship of protein HCO_011565 with its orthologs in five other species of nematode (left) and the structural models of these orthologs predicted using Alphafold (right).

FIGURE 5

UMW-868 and its analogs evaluated for activity on Haemonchus contortus. Sequential modifications of UMW-868 analogs with substitutions of the aryl ring (bottom) and n-propyl (top). Potencies (IC₅₀ in µM) of individual compounds on the motility of exsheathed third-stage larvae (xL3s) of H. contortus are indicated.