Homology modeling and docking analysis of phytochemicals of Catharanthus roseus and Vernonia amygdalina against Ancylostoma caninum metalloprotease-2

Introduction

The economically important canine hookworm, Ancylostoma caninum Ercolani, 1859 is a parasite of veterinary and public health problems, especially in developing countries that causes ancylostomiasis in many mammals (Lee et al., 2022; Bowman, et al., 2010) [10,3] . The adult A. caninum also infects humans and causes eosinophilic enteritis (Tsuji, 2020; Prociv and Croese, 1996) [18,15] . The anthelmintic drugs Albendazole, fenbendazole, febantel, moxidectin, ivermectin, pyrantel pamoate, and milbemycin oxime are that have been approved for the treatment of A. caninum infection (Aziz et al, 2020; Reinemeyer, 2016) [1,16] . The occurrence of multiple drug resistance to benzimidazoles, macrocyclic lactones, and pyrantel in A. caninum was shown (Jimenez et al., 2019) [8] . The reports of multiple anthelmintic drugresistant are numerous (Castro et al. 2023; Leutenegger et al. 2023; Venkatesan, et al. 2023; Marsh and Lakritz, 2023) [4,11,13,19] . The direct life cycle having A. caninum secreted/excreted many proteins, during their establishment in the host. The excretory/secretory proteins of the helminths participate in a variety of functions including host penetration, tissue invasion, immune evasion, reproduction, and feeding (Williamson et al. 2006; Haffner et al. 1998; Karanu et al. 1993) [21,7,9] . These proteins are important antigens for the development of new therapeutic or vaccine targets. Adult A. caninum secrete a unique metalloprotease that resembles astacin called Ac-MTP-2 which is identified only in hookworms. Ac-MTP-2 is localized in the oesophageal gland and uterus of the parasite and is found involved in the parasite attachment and digestion of the mucosa of the gastrointestinal tract, nourishment, and reproduction. The role of the Ac-MTP-2 is suggestive of the possibility of them being therapeutic or vaccine candidate (Feng et al.

2007) [5] . Many drugs were developed either directly or indirectly from plants, making plants a key source of new pharmacologically active molecules (Veeresham, 2012) [12] . The various tools of computer-aided drug design (CADD) facilitate the identification of newer lead from a great number of phytochemicals available in commonly available plants. In the present study, the phytochemicals of Catharanthus roseus (Periwinkle) and Vernonia amygdalina (Bitter leaf) were explored for their in silico anthelmintic potential against Ancylostoma caninum by molecular docking.

Materials and Methods

Homology modelling and preparation of the three-dimensional structure of Ancylostoma caninum protein for docking: The amino acid sequence of A. caninum acstatinlike metalloprotease 2 (Ac-MTP-2) (Accession number: ABG49116.1) was obtained from NCBI. The threedimensional model of Ac-MTP-2 (Figure-01) was built in server SWISS-MODEL using Ancylostoma caninum metalloendopeptidase with a sequence identity of 77.68% as a template. The built model was refined in a web server GalaxyRefine. The refined model was then validated in the SAVESv6.0 server. The validated model was then prepared in AutoDock Tools 1.5.6 by the addition of polar hydrogens, Kollman charges, and AD4-type atoms. The protein models were visualized in UCSF Chimera 1.16.

Selection and preparation of ligands for molecular docking:

The phytochemicals of Catharanthus roseus and Vernonia amygdalina obtained through a literature search are given in Table 1. The evaluation of the physicochemical properties, druglikeness (Lipinski, Veber, and Egan rules), lipophilicity, solubility, gastrointestinal absorption, and bioavailability score of the ligands are important before it is selected as a drug molecule. The canonical SMILES of the obtained phytochemicals (ligands) were available in the PubChem server and were submitted to the Swiss ADME server to select the ligands for docking. In silico toxicity of the ligands was predicted in the ProTox-II server. The three-dimensional structure of the ligands was obtained from the PubChem server. The selected phytochemicals (ligands) were prepared in AutoDock Tools 1.5.6 for docking.

In silico molecular docking and analysis of the docking results

The docking of the prepared Ac-MTP-2 and ligands were carried out in AutoDock tools 1.5.6 to know the active site where the ligands are binding to the protein. Using AutoGrid4 and AutoDock4, the grid box was set around the entire protein to perform blind docking. The dimension of the grid box was 126 x 92 x 126 with a spacing of 0.414 angstroms. The docking was carried out using the Lamarckian genetic algorithm to find the best binding pose of the ligands within the protein. The pose with the lowest binding energy is considered the best pose. The docking results were analyzed in AutoDock Tools 1.5.6, and Biovia discovery studio to view the amino acids of the protein interacting with the ligands. The active site of the protein was predicted in the Computed Atlas of Surface Topography of Proteins (CASTp) server to compare the residues of the protein interacting with the ligands.

Results and Discussion

The quality of the modeled and refined three-dimensional Ac-MTP-2 protein model was validated in SAVES v6.0 server based on the ERRAT score and Ramachandran plot. The ERRAT score for good-quality protein should be more than 50. The overall quality factor (ERRAT score) of the Ac-MTP-2 protein model was 93.66 (Figure 2a). The Ramachandran plot was obtained from PROCHECK which showed that 91% of residues were present in the most favored region and none of the residues were present in the disallowed regions (Figure 2b). This shows the higher quality of the modeled protein. In the SwissADME web server, forty ligands from two commonly available plants, Catharanthus roseus, and Vernonia amygdalina were evaluated for their pharmacokinetic and pharmacodynamic properties. The evaluation of the drug likeness of the compounds before proceeding with the clinical trials would save money and time for the researchers. The properties of the newer ligands are compared with the already existing drugs in the webserver to predict the compounds that are likely to thrive in all phases of clinical trials. To know the drug likeness of the compounds, Lipinski, Veber, and Egan rules that involve molecular weight, lipophilicity, number of hydrogen bond acceptors and donors, number of rotatable bonds, and topological polar surface area are considered (Lipinski et al. 1997) [12] . A total of 24 ligands obeyed all three (Lipinski, Veber, and Egan) rules without any violations. All 24 ligands had high gastrointestinal absorption and a bioavailability score of 0.55. The lipophilicity of the compounds contributes to their absorption, distribution, metabolism, and excretion. Lipophilicity is given by LogP and should be between 0 and 5 (Berger et al. 2022) [2] , and in the present study lipophilicity ranged from 0.66. All these compounds were in moderately soluble, soluble, and very soluble classes. These 24 compounds were selected for further docking study. The ADME property of 24 ligands selected for the docking study is given in Table 3. The binding energy of the 24 ligands with Ac-MTP-2 protein in the docking study ranged from -3.62 to -9.4 kcal/mol. Twenty of the ligands had binding energy lesser than -7 kcal/mol whereas ajmalicine, serpentine, and raubasin had the lowest binding energy of -9.4, -9.2, and -9.12 kcal/mol, respectively. Hydroxyvernolide and vernolide are the two ligands with a binding energy of -8.04 and -8.09 kcal/mol, respectively. The selected ligands interacted with the predicted active site residues. The interaction of the selected five ligands with Ancylostoma caninum metalloprotease-2 analyzed in Biovia discovery studio is given in Figure 4. 4. Ajmalicine and serpentine had good enzyme inhibitory activity against the cysteine protease, cruzain of Trypanosoma cruzi with a binding energy of -9.59 and -9.28 kcal/mol, respectively (Souza et al. 2022) [17] and in the present study, ajmalicine and serpentine had -9.4 and -9.2 kcal/mol binding energy against Ac-MTP-2. Yohimbine was identified as a novel inhibitor of NS2B-NS3 protease with a binding energy of -7.18 kcal/mol by the in-silico docking study performed in a study (Girija et al. 2019) [6] . However, in our study, yohimbine had a binding energy of -7.81 kcal/mol against Ac-MTP-2 which was comparatively higher than the other few ligands. The present study demonstrates the in silico anthelmintic activity of ajmalicine, raubasin, serpentine, hydroxyvernolide, and vernolide against a metalloprotease of Ancylostoma caninum (Ac-MTP-2). The selected ligands should be tested in vitro and in vivo for their anthelmintic activity and toxic properties against A. caninum. The successful development of lead compounds into drugs treats infected dogs in addition to human infections.

Conclusion

Gastrointestinal parasites are responsible for various diseases and lead to tremendous economic losses in the livestock industry. In addition to the diseases caused by them, anthelmintic resistance is a severe problem. The parasites alter their genetic makeup to protect themselves from the available drugs. Therefore, there is a need for the identification of newer drug molecules against different drug targets. The present study identified a few phytochemicals with promising in silico activity against a metalloprotease,

Acknowledgement

Ac-MTP-2. We suggest further evaluation of these phytochemicals in vitro against Ancylostoma caninum for their anthelmintic property.