Identification of the Binding Site for the Anthelmintic Drug Ivermectin in Cys-Loop Receptors

Abstract

Ivermectin (IVM) is an antiparasitic drug widely used in veterinary medicine, mainly in cattle, to kill intestinal worms. Additionally, it is used to treat human parasitic diseases like Onchocerciasis (river blindness). The major target of IVM is a heteropentameric glutamate-gated chloride channel (GluClα/β receptor), which is unique to invertebrates and belongs to the Cys-loop receptor superfamily. Nanomolar concentrations of IVM irreversibly activate the native GluClα/βR, thereby causing sustained hyperpolarization, suppression of nervous impulses, paralysis and death of the nematode. Little is known about the IVM binding site(s). We first studied a chimeric α7-GluClβ receptor that consists of the ligand-binding domain (LigBD) of the α7 nicotinic acetylcholine receptor (α7-nAChR) and the channel domain of a homopentameric GluClβR. IVM was expected to increase the chimera's activity since the activity of the α7-nAChR is enhanced by IVM and the homopentameric GluClβR is irresponsive to IVM. Surprisingly, IVM strongly inhibited the chimeric α7-GluClβR suggesting that IVM does not bind exclusively to either of the domains, but binds somewhere at the interface between the LigBD and the pore domain. Next, we explored the natural target of IVM, the GluClα/βR. Since the α-subunit is known to be responsible for IVM binding by the native GluClα/βR, we replaced the β8β9 and Cys loops of the β-subunit by the homologous loops of the α-subunit. These two loops reach the interface between the LigBD and the pore domain. Our results show that the aforementioned loop swapping significantly enhances receptor activation by IVM. Furthermore, site-specific mutagenesis suggests that IVM binds at the interface between the LigBD and the pore domain, in a crevice between the β8β9 loop and the Cys loop of two adjacent subunits. Supported by the Wolfson Foundation and the Israel Science Foundation.