The analysis of the effect of ivermectin on phytopathogenic strains of
Fusarium graminearum
(F‑55644, F-55748) and
Fusarium oxysporum
f. sp.
lycopersici
(F-52897, F-55547) was carried out; as a ...result, its concentrations were established at which a fungistatic effect on the growth of colonies of the specified strains was observed (2 and 3 mg/mL). It was found that
F. oxysporum
strains were more susceptible in general to ivermectin than
F. graminearum
strains. Since it is known that ivermectin is able to interact with β-tubulin (causing a stabilization of microtubules), to explain the obtained results, a 3-dimensional model of the complex of this compound with
Fusarium
β-tubulin was developed and ivermectin-induced changes in the conformation of β-tubulin were determined, including, particularly, the stabilization and spiralization of the M‑loop of the β-tubulin molecule. This structural element of β-tubulin plays an important role in the lateral contacts between tubulin subunits of adjacent protofilaments within the microtubule. Since the M-loop stabilization reflects a very important feature of microtubule stabilizing agents' binding to the taxane site of β-tubulin, it can be supposed, that ivermectin possesses the same effect on
Fusarium
microtubules. The results obtained allow for considering ivermectin or its derivatives as potential compounds with fungicidal activity.
The drug repurposing, which is the search of new applications for known bioactive compounds, has apparent benefits (e.g., cost efficiency, development risks, and time reduction) as compared to the ...traditional de novo drug design making it common for different research fields including the biological drug discovery 1. The nematocide and insecticide ivermectin (IVM) is considered a wide spectrum agent, which makes it a perspective candidade for repurposing 2. Although the structure-activity relationships of IVM interactions with its traditional targets of Cys-loop receptors family are studied 3, their structural patterns have not been described yet. The knowledge of structural patterns, which are groups of residues related spatially and by physico-chemical properties, with high affinity for IVM functional groups can be used to find local similarities in active sites of IVM potential targets when the direct alignment of sites cannot be implemented due to the evolutionary distinction between known and potential targets 4. Aim. To determine the structural patterns of IVM allosteric interaction with residues of its binding site located in the transmembrane domain of a-homopentameric glutamate-gated chloride channel (GluCla) of Caenorhabditis elegans. Methods. To consider different conformational states of IVM binding site two complexes of IVM bound to C. elegans GluCla (each with five site conformations) with identifiers 3RHW (https://doi. org/10.2210/pdb3RHW/pdb) and 3RIF (https://doi.org/10.2210/pdb3RIF/pdb) were obtained from PDB 5. The structures were examined in Analyzer Mode of SeeSAR v.12.1.0, in which contributions of IVM atoms into the complex affinity and their interactions with site structural patterns were determined for each site conformation using the HYDE scoring function 6. The residues belonging to identified structural patterns were classified by their properties using the Taylor's classification of amino acids 7. Results and Discussions. The binding site of IVM on cys-loop receptors including GluCla is located in the interface between transmembrane domains of (+) and (-) subunits and is formed by M2, M3, and M2-M3 of (+) subunit and M1 of (-) subunit (Fig. 1, A). As it is demonstrated on the Fig. 1, B, IVM is composed of the 16-membered heteromacrocyclic ring fused with the spiroketal and benzofuran groups, and linked with the disaccharide group. According to the results, the benzofuran group is critical for IVM recognition and binding: it interacts with the T-A-S-N-D-I-L-Q-I-P pattern, which is formed by T257, A258, S260, and N264 of M2, D277 and I280 of M3 of (+) subunit and L218, Q219, I222, P223 of M1 of (-) subunit (Fig. 2, A). Due to the size and hydrophobicity of macrocycle, its different parts interact with residues of all site-forming structural elements mentioned above resulting in the V-I-G-A-M and I-V-D-L patterns demonstrated on the Fig. 2, B, C. While the V-I-G-A-M pattern is formed by the residues of (+) subunit (V278, I280, G281, A282, and M284 of M3), the I-V-D-L pattern contains residues of both subunits: I273 of M2-M3, D277 and V278 of M3 of (+) subunit and L218 of M1 of (-) subunit. Finally, the spiroketal group interacts with M-T-F-C-M-I of (+) subunit (M284, T285, and F288 of M3) and (-) subunit (С225, M226, and I229 of M1) (Fig. 2, C). As opposed to other functional groups, the disaccharide is located outside of the binding site pocket. It interacts with I273 of M2-M3 of (+) subunit and L217, L218, and I222 of Ml of (-) subunit; however, considering that these residues are not united spatially, no pattern for the disaccharide can be determined based on the structural information which was analyzed. The determined structural patterns of IVM allosteric interaction with GluCla can be used in search of IVM binding site on its potential targets, in the development of hypotheses of IVM binding to identified sites, and to rationalize the drug design of new GluCl ligands. Conclusions. The structural patterns with high affinity for IVM functional groups have been determined based on the results of HYDE assessment and visual analysis of IVM-GluCla complexes and the possible implementations of patterns knowledge have been described. The identified patterns can be further corrected and extended using the structural information of other IVM targets deposited in PDB.
Aim. Determination of the ivermectin influence on plant pathogenic species of Fusarium genus; particularly, F. graminearum and F. oxysporum. Methods. The susceptibility of studied strains (F. ...graminearum F-55756 and F. oxysporum F-54635) to ivermectin was measured in vitro with the agar diffusion method. Ivermectin in concentrations from 0 to 3 mg/ml was poured into the wells made in media for that purpose. Further, mycelial discs of F. graminearum and F. oxysporum strains were placed into the central regions of Petri dishes, which were then maintained in the dark at 25 °C. Ivermectin influence on growth and morphology of studied strains was estimated after the 7 days using the ImageJ software and methods of statistical analysis to determine the most effective concentrations. Results. As the result, it was found that at 1 mg/ml concentration and above the fungistatic effect is observed and the 3 mg/ml concentration appeared to be the most effective one. Thus, the percentage of mycelium area in comparison with control at this concentration was 83,91 % for F. graminearum F-55756 and 69,95 % for F. oxysporum F-54635. Conclusions. The ivermectin effective fungistatic action on the studied strains was observed giving the reason for further analysis of the ivermectin influence on other strains of Fusarium complex species and search of molecular targets of its action.
