• The aim of project is to obtain data for the improvement and innovation of antinematodal pharmacotherapy. The main targets for anthelmintic drugs are found in the neuromuscular system of parasitic nematodes. Many anthelmintics target nematode cys-loop ligand-gated ion channels, which are membrane-bound channels facing the extracellular matrix. That makes them easily accessible to drug molecules and are thus, „druggable“ targets. We will investigate the possibility for potentiating the action of anthelmintics on the existing targets and indicate potentially new sites for drug action. The examinations are divided into 4 parts:

a) The main objectives of the project are to obtain data on the possibility of upgrading the efficacy of anthelmintic drugs which are agonists оr antagonists of GABA and nicotinic acetylcholine receptors (nAChRs) in parasitic nematodes. Piperazine is the only GABA receptor agonist in clinical use. Although initially this receptor was designated as the main site of action of avermectins, it was later proven that they act primarily on the glutamate-dependent chloride channels in the nematode pharynx. The potential for druggability of GABA receptor in nematodes remains incomplete. We are particularly interested in the interaction of benzodiazepine receptor agonists and antagonists with the nematode chloride channel and the possibility of their combination with GABA-ergic drugs. Our previous results with diazepam and flumazenil suggest this possibility (Stevanović and al., 2021). We will test isoxazolines the antagonist of GABA-ergic chloride channel receptor in insects and acarines and examine their interaction with nematode GABA receptors. Furthermore, there are at least three subtypes of parasitic nematode nACh receptors (N, L, and B). Based on recent studies, potentially the fourth subtype is the morantel-sensitive nACh receptor (Charvet et al., 2018). We will examine the antinematodal effects of combinations of anthelmintic drugs with agonistic action on different nematode nAChR subtypes. Targeting multiple nAChR subtypes is a rational way to neutralize resistance caused by changes in the binding site of single  nAChR subtype in the parasite.

b) The second part of the research will refer to the examination of new potential drug targets in the neuromuscular system of nematodes. We will focus our research on the pharmacological characteristics of the muscarinic acetylcholine receptor (mAChR) in parasitic nematodes. In the previous research, we found that mAChR agonists cause a slow depolarization of the A. suum muscle cell membrane (Trailović et al., 2007). We will examine druggability of agonists and antagonists of the mAChR, determine receptor localization in the neuromuscular system of nematodes and evaluate potential importance for pharmacotherapy. On the other side, one of the most recently discovered pharmacological receptors in the neuromuscular system of nematodes is MOD 1. These receptors regulate the opening of chloride channels and are related to the 5-HT group of receptors in vertebrates. The MOD 1 channel is not blocked by calcium ions or 5-HT3a-specifc antagonists but is inhibited by the miasetrin and methiothepin (tricyclic antidepressants), metabotropic 5-HT receptor antagonists (Rodriguez Araujo et al., 2022). This indicates an interesting pharmacology of the MOD 1 receptors in nematodes. The research that we are planning on the parasitic nematode A. suum will give us answers about how MOD 1 chloride channels are involved in movement and contractility of the parasites and whether it can be an attractive drug target. On the other hand, the data on the inhibitory effect of tricyclic antidepressants is directly related to our next part of the project: repurposing of existing medicines with potential anthelmintic properties.

c) The third part of project will refer to the repurposing of existing medicines with potential anthelmintic properties. In more detail, an alternative drug development approach is the screening of drugs that have already been approved for the treatment of other diseases and reviewing them for anthelmintic treatment. The advantage of this strategy, also called ‘drug repurposing’ or ‘therapeutic switching’, is the availability of preclinical and clinical data that can accelerate the drug development and reduce development costs. The screening of a small-molecule library of compounds used in human clinical trials against C. elegans uncovered the anthelmintic activity of the neuromodulatory drugs sertraline, paroxetine and chlorpromazine (Weeks et al., 2018). Based on our previous results, obtained using in silico prediction tools, we will examine the effects of the already mentioned agonists and antagonists of benzodiazepine receptors and GSK575594A (Stevanović et al., 2021) on the neuromuscular system of parasitic nematodes.

d) The last but significant fourth part of our project refers to the investigation of anthelmintic properties of active ingredients of essential plant oils. Plants produce natural or essential oils as organic products of secondary metabolism. Essential oils and their active ingredients, based on previous pharmacological studies, may be able to efficiently and securely replace (or act as adjuncts to) traditional antiparasitic drugs. The focus of research is terpenoid Active Ingredients (AIs) of plant essential oils. Our previous results show antinematodal effects with a mechanism of action for carvacrol (plant monoterpenoid) that involves inhibition of parasite muscle contractions. Specifically, carvacrol inhibits ACh induced depolarizations of muscle cells indicating a direct interaction with nAChRs in Ascaris suum (Trailović et al., 2015, Marjanović et al., 2020).

• After the first year of research on the project, we will select substances with the most pronounced anthelmintic effect and continue testing with them. The selection will be made on the basis of the presence or absence of antinematodal effect, the value of certain mean effective concentrations (EC₅₀) and the maximum effect (E_max). In the following research, we will test different agonists, antagonists and modulators of the tested receptors and analyze in more detail the mechanisms of the antinematodal action. We will continue to examine these substances on the already described experimental models, but we will also test the selectivity of their pharmacological actions on contractile models of mammalian neuromuscular preparations: contractile model of the isolated diaphragm for testing the effects on nAChR (muscle type), model of contractions of the isolated ileum and colon of rats for testing the effect on muscarinic, GABA and 5-HT receptors. The goal of this part of research is to determine the selectivity of the pharmacological effects of tested substances.