The narrow-spectrum anthelmintic oxantel is a potent agonist of a novel acetylcholine receptor subtype in whipworms

Abstract

In the absence of efficient alternative strategies, the control of parasitic nematodes, impacting human and animal health, mainly relies on the use of broad-spectrum anthelmintic compounds. Unfortunately, most of these drugs have a limited single-dose efficacy against infections caused by the whipworm, Trichuris. These infections are of both human and veterinary importance. However, in contrast to a wide range of parasitic nematode species, the narrow-spectrum anthelmintic oxantel has a high efficacy on Trichuris spp. Despite this knowledge, the molecular target(s) of oxantel within Trichuris is still unknown. In the distantly related pig roundworm, Ascaris suum, oxantel has a small, but significant effect on the recombinant homomeric Nicotine-sensitive ionotropic acetylcholine receptor (N-AChR) made up of five ACR-16 subunits. Therefore, we hypothesized that in whipworms, a putative homolog of an ACR-16 subunit, can form a functional oxantel-sensitive receptor. Using the pig whipworm T. suis as a model, we identified and cloned a novel ACR-16-like subunit and successfully expressed the corresponding homomeric channel in Xenopus laevis oocytes. Electrophysiological experiments revealed this receptor to have distinctive pharmacological properties with oxantel acting as a full agonist, hence we refer to the receptor as an O-AChR subtype. Pyrantel activated this novel O-AChR subtype moderately, whereas classic nicotinic agonists surprisingly resulted in only minor responses. We observed that the expression of the ACR-16-like subunit in the free-living nematode Caenorhabditis elegans conferred an increased sensitivity to oxantel of recombinant worms. We demonstrated that the novel Tsu-ACR-16-like receptor is indeed a target for oxantel, although other receptors may be involved. These finding brings new insight into the understanding of the high sensitivity of whipworms to oxantel, and highlights the importance of the discovery of additional distinct receptor subunit types within Trichuris that can be used as screening tools to evaluate the effect of new synthetic or natural anthelmintic compounds.

Functional Characterization of the Oxantel-Sensitive Acetylcholine Receptor from <em>Trichuris muris</em>

“…Here, we found that oxantel acted as a partial agonist with similar potency as ACh, epibatidine acted as a partial agonist with a higher potency than ACh, whereas pyrantel induced only a minor response on the Tmu-ACR-16-like receptor. These findings emphasize species-specific differences at the receptor level, since oxantel acted as a full-, pyrantel as a partial-, and epibatidine as a weak agonist on the Tsu-ACR-16-like receptor [30]. Oxantel was predicted to bind to the ACR-16 of T. muris (Tmu-ACR-16-like) with high affinity in a recent in silico ligand binding analysis of the extracellular domains of nematode ACR-16 nAChRs [36].…”

“…Trichuris muris is, like T. suis, used as a model for T. trichiura. Oxantel was previously shown to act on the nAChR made of ACR-16-like subunits from T. suis [30]. In order to investigate the ACR-16-like nAChR from T. muris, we identified and cloned the full-length coding sequence of the T. muris acr-16-like subunit cDNA (Tmu-acr-16-like).…”

“…The cloned T. muris acr-16-like sequence differed from the putative full-length Tmu-acr-16-like cDNA sequence available in WormBase (accession number WBGene00290200) by two synonymous nucleotide substitutions (CCG → CCA at codon 327 and AGT → AGC at codon 427), and was therefore deposited in GenBank under the accession number MZ322663. In order to explore all the members of the ACR-16 group of Trichuris spp, we performed searches in genomic databanks that led to the identification of the C. elegans acr-19 gene orthologue in T. muris [30]. Consequently, the full-length Tmu-acr-19 cDNA sequence was amplified by PCR and subsequently cloned and sequenced.…”

“…In order to investigate potential functional nAChRs made of T. muris ACR-16-like and ACR-19 subunits, we microinjected their corresponding cRNAs in X. laevis oocytes and responses to agonists were subsequently monitored using two-electrode voltageclamp (TEVC) electrophysiology. The cRNA of the X. laevis chaperone protein Resistance to Inhibitors of Cholinesterase ric-3 was also added to the respective subunit cRNAs as it is required to promote the expression of nAChRs [30,[36][37][38][39][40]. We found that injections of the Tmu-acr-16-like cRNA led to the robust expression of a functional homomeric receptor, which elicited a response in the μA range when exposed to 100 μM acetylcholine (ACh) (Fig.…”

“…Recently, we identified a preferential drug target for oxantel from the pig whipworm T. suis, and achieved its functional characterization in Xenopus laevis oocytes and the free-living nematode Caenorhabditis elegans. This drug target is a homomeric acetylcholine receptor (AChR), constituted of five ACR-16-like subunits that are specific to Clade I nematodes [30]. Due to the high oxantel sensitivity of this receptor, and in reference to the previously reported L-AChR, N-AChR, B-AChR and M-AChR (respectively for Levamisole-sensitive, Nicotine-sensitive, Bephenium-sensitive and Morantel-sensitive) [31,32], we referred to this Tsu-ACR-16-like receptor as an O-AChR subtype.…”