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Molecular docking elucidates the plausible mechanisms underlying the anticancer properties of acetyldigitoxigenin from Adenium obesum

Adenium obesum (Forssk.) Roem. & Schult. is a promising medicinal plant belonging to the Apocynaceae family. It is a rich source of various phytochemicals such as cardiac glycosides, flavonoids, terpeniods, pregnanes etc. which have different pharmacological properties such as anticancer, antibacterial, acaricidal etc. While previous reports showed the anticancer activity of the aerial parts of the plant extract of A. obesum, the mechanisms of action of its chemical constituents are not known. The present study is aimed at elucidation of plausible mechanisms of anticancer activity of the plant by evaluating the binding interaction of its nine major selected compounds with macromolecular receptors implicated in the initiation and progression of cancer using various in silico approaches. Molecular docking results showed that the compound Δ16-3-Acetyldigitoxigenin (16-anhydro-3-acetylgitoxigenin) scored the best binding energy scores with the majority of the target proteins. The molecular binding of the compound was stabilized through hydrogen bonds as well as hydrophobic interactions, and also possesses favorable drug-like properties without significant toxicities.


Introduction
Every year, approximately 6.7 million deaths take place around the world due to various types of cancer (ACS, 2020). A wide array of cytotoxic agents and radiotherapy used in the cancer treatment have limitations in their usage e.g. side effects, and efficacy (Kim et al., 2007;Stopeck and Thompson, 2012); therefore, the development of better effective therapeutics for the cancer treatment from natural products remain continues because of its minimal side effects (Da Rocha et al., 2001;Vermani and Garg, 2002;Gurib-Fakim, 2006).
Although Adenium obesum (Forssk.) Roem. & Schult. (family Apocynaceae, commonly known as 'Desert Rose') is primarily an ornamental poisonous plant, the whole plant including latex has been used in traditional system of medicines for the treatment of various aliments e.g. skin lumps, wound, ear ache, rhinitis, gonorrhea and infectious diseases. It contains nearly 50 major chemicals constituents belonging to the class of cardenolides, flavonoids, pregnanes and triterpenes (Versiani et al., 2014). The recent reports (Almehdar et al;Hossain et al., 2017;Ali et al., 2019a,b) established the anticancer activity of extract of aerial part of A. obesum but the modes of action of the chemical constituents have not been understood; therefore, the aim of the current study is to elucidate the plausible molecular mechanisms underlying the anticancer activity of A. obesum extract using in silico approaches.

Preparation of ligand and receptor
The structures of nine major compounds of A. obesum (Table 1) were modeled using Chemsketch. The three-dimensional structures of selected macromolecular receptors e.g.  (Halgren, 1996), and the receptors prepared following our previously described method (Gurung et al., 2016) were used to execute docking. The binding sites were defined by choosing grid boxes of suitable dimensions around the bound co-crystal ligands.

Molecular docking
The Lamarckian Genetic Algorithm was used for performing molecular docking using AutoDock4.2 (Morris et al., 2009) considering the docking parameters from our previously described method (Gurung et al., 2016). A total number of 50 independent docking runs were performed for each ligand. The conformations were grouped under clusters by considering a difference of less than 2.0 Å of root mean square deviation (RMSD). The lowest free energy of binding (DG) and the lowest inhibition constant (K i ) were considered for choosing the most favorable binding pose. The molecular interactions between the compounds and receptors were studied using LigPlot + v 1.4.5 (Laskowski and Swindells, 2011).

Validation of docking method
In order to check the suitability of molecular docking parameters and algorithm to reproduce the native binding poses, redocking experiment was performed using the co-crystal ligands.

Determination of physicochemical properties of the compounds
DataWarrior program version 4.6.1 was used for the determination of various physicochemical properties of the selected compounds such as drug likeness and toxicity (Sander et al., 2015).
The physicochemical properties of the docked compounds are tabulated in Table 3. The majority of the compounds obeyed the Lipinski's rule of five (ROF) (Lipinski, 2004) 6,],   also showed similar results except for their possible toxicity on reproductive health. The majority of the compounds showed a good drug likeness score except for compounds 1 [D 16 -3-Acetyldigitoxigenin (16-anhydro-3-acetylgitoxigenin)], 6 [Dihydroifflaionic acid] and 7 [Lup-20 (29)-ene-3,28-diol (betulin)] which exhibited negative drug likeness score. The other physicochemical properties such as topological surface area (TPSA) and number of rotatable bonds (RB) were also found to be within permissible limits (TPSA 140 Å 2 and RB 10).
Thus, the present molecular docking studies revealed structural insights into possible binding modes of major active compounds of A. obesum, and identified the best docked compound for each target. The compound 1 (16-anhydro-3-acetylgitoxigenin) was found to be best docked (showed a high binding affinity, good number of hydrogen bonds and hydrophobic interactions with their respective molecular targets which play a key role in the pathogenesis of cancer) to four targets CDK-2, Topoisomerase-II, VEGFR-2 and Topoisomerase-I whereas Compound 2 (12b-Hydro xypregna-4,6,16-triene-3,20-dione), Compound 7 (Lup-20(29)ene-3,28-diol) and Compound 8 (Quercetin 3,3 0 -dimethyl ether) were found to be best docked to CDK-6, BCL-2 and Telomere:Gquadruplex respectively with favorable drug-like properties; and thus, these compounds can be promising leads for the design of specific target inhibitors which would help with management of the disease.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.