Rooney, J., Rivera-de-Torre, E., Li, R., Mclean, K., Price, D.R.G., Nisbet, A.J., Laustsen, A.H., Jenkins, T.P., Hofmann, A., Bakshi, S., Zarkan, A., Cantacessi, C. (2024) Comp. Struct. Biotech. J., in press

The complex relationships between gastrointestinal (GI) nematodes and the host gut microbiota have been implicated in key aspects of helminth disease and infection outcomes. Nevertheless, the direct and indirect mechanisms governing these interactions are, thus far, largely unknown. Here, we demonstrate that the excretory-secretory products (ESPs) and extracellular vesicles (EVs) of key GI nematodes contain peptides that, when recombinantly expressed, exert antimicrobial activity in vitro against Bacillus subtilis. In particular, using time-lapse microfluidics microscopy, we demonstrate that exposure of B. subtilis to a recombinant saposin-domain containing peptide from the ‘brown stomach worm’, Teladorsagia circumcincta, and a metridin-like ShK toxin from the ‘barber’s pole worm’, Haemonchus contortus, results in substantial membrane damage, membrane blebbing, and cell lysis. This study provides key evidence of the occurrence of direct mechanisms of worm-microbiota interactions and paves the way for future investigations aimed at deciphering the impact of parasite-associated changes in host gut microbiota composition and function on the pathophysiology of GI helminth infection.

Shanley, H.T., Taki, A.C., Nguyen, N., Wang, T., Byrne, J.J., Ang, C.-S., Leeming, M.G., Nie, S., Williamson, N., Zheng, Y., Young, N.D., Korhonen, P.K., Hofmann, A., Chang, B.C.H., Wells, T.N.C., Häberli, C., Keiser, J., Jabbar, A., Sleebs, B.E., Gasser, R.B. (2024) Int. J. Parasitol. Drugs Drug Resist. 24, 100522

Within the context of our anthelmintic discovery program, we recently identified and evaluated a quinoline derivative, called ABX464 or obefazimod, as a nematocidal candidate; synthesised a series of analogues which were assessed for activity against the free-living nematode Caenorhabditis elegans; and predicted compound-target relationships by thermal proteome profiling (TPP) and in silico docking. Here, we logically extended this work and critically evaluated the anthelmintic activity of ABX464 analogues on Haemonchus contortus (barber’s pole worm) – a highly pathogenic nematode of ruminant livestock. First, we tested a series of 44 analogues on H. contortus (larvae and adults) to investigate the nematocidal pharmacophore of ABX464, and identified one compound with greater potency than the parent compound and showed moderate activity against a select number of other parasitic nematodes (including Ancylostoma, Heligmosomoides and Strongyloides species). Using TPP and in silico modelling studies, we predicted protein HCON_00074590 (a predicted aldo-keto reductase) as a target candidate for ABX464 in H. contortus. Future work aims to optimise this compound as a nematocidal candidate and investigate its pharmacokinetic properties. Overall, this study presents the first steps toward the development of a new anthelmintic with a novel, distinct mechanism of action.

Shanley, H.T., Taki, A.C., Nguyen, N., Wang, T., Byrne, J.J., Ang, C.-S., Leeming, M.G., Nie, S., Williamson, N., Zheng, Y., Young, N.D., Korhonen, P.K., Hofmann, A., Wells, T.N.C., Jabbar, A., Sleebs, B.E., Gasser, R.B. (2024) Bioorg. Med. Chem. 98, 117540

Global challenges with treatment failures and/or widespread resistance in parasitic worms against commercially available anthelmintics lend impetus to the development of new anthelmintics with novel mechanism(s) of action. The free-living nematode Caenorhabditis elegans is an important model organism used for drug discovery, including the screening and structure-activity investigation of new compounds, and target deconvolution. Previously, we conducted a whole-organism phenotypic screen of the ‘Pandemic Response Box’ (from Medicines for Malaria Venture, MMV) and identified a hit compound, called ABX464, with activity against C. elegans, and a related, parasitic nematode, Haemonchus contortus. Here, we tested a series of 44 synthesized analogues to explore the pharmacophore of activity on C. elegans and revealed five compounds whose potency was similar or greater than that of ABX464, but which were not toxic to human hepatoma (HepG2) cells. Subsequently, we employed thermal proteome profiling (TPP), protein structure prediction and an in silico-docking algorithm to predict ABX464-target candidates. Taken together, the findings from this study contribute significantly to the early-stage drug discovery of a new nematocide based on ABX464. Future work is aimed at validating the ABX464-protein interactions identified here, and at assessing ABX464 and associated analogues against a panel of parasitic nematodes, towards developing a new anthelmintic with a mechanism of action that is distinct from any of the compounds currently-available commercially.

Labus, J., Tang, K., Henklein, P., Krüger, U., Hofmann, A., Hondke, S., Wöltje, K., Freund, C., Lucka, L., Danker, K. (2024) Biochim. Biophys. Acta - Biomembranes 1866, 184257

Integrin α1β1 is an adhesion receptor that binds to collagen and laminin. It regulates cell adhesion, cytoskeletal organization, and migration. The cytoplasmic tail of the α1 subunit consists of 15 amino acids and contains six positively charged lysine residues. It resembles those of polybasic protein structures known to interact with acidic phosphoinositides of the plasma membrane. Using different biochemical methods, we present evidence that the α1 integrin cytoplasmic tail directly associates with phosphoinositides, preferentially with phosphatidylinositol 3,4,5- trisphosphate (PI(3,4,5)P₃). Since the association was disrupted by calcium, magnesium and phosphate ions, this interaction seems to be of ionic nature. Testing peptides with different α1 C-terminally derived amino acid sequences revealed that the peptide-lipid interaction is driven by the conserved KIGFFKR motif. Further experiments using point mutations in the cytoplasmic α1 tail highlight the importance of the two potential phospholipid-binding lysines in the KIGFFKR motif for α1β1 integrin function. The exchange of lysine with glycine residues in this motif (positions 1166 and 1171) increase α1β1 integrin-specific adhesion and F-actin cytoskeleton formation compared to cells expressing the unmodified α1 subunit. Cells with the mutation at position 1171 have increased levels of active β1 integrins, form more pronounced focal adhesions and show increased phosphorylation of focal adhesion kinase. In contrast, phosphorylation of AKT is dramatically inhibited in these cells. We conclude that the KIGFFKR motif, and in particular lysine1171 of α1 integrin is involved in PI3K recruitment and subsequently recruitment of PI(3,4,5)P₃-binding molecules and their activation, which is necessary for the dynamic regulation of α1β1 integrin activity.