From docking experiments, the vinca domain and maytansine binding site showed energy values higher than those observed in the colchicine and pironetin binding site. These results allow us to propose a hierarchy order to the possible stability of the xanthanolides in each of the MDAs binding sites in tubulin: colchicine binding site > pironetin binding site > maytansine binding site > vinca domain. At the end of the 100 ns of simulations, the previous results become stronger, the results obtained in the vinca domain and maytansine binding site showing high RMSD values (> 15 Å). This is related to the dissociation of the ligand from the receptor, situation corroborated visualizing the molecular dynamic trajectory with VMD. Because the vinca domain and the seventh site are equivalent, the results obtained for the former were assumed for both. However, in order to validate this hypothesis, further studies are required.
On the other hand, the result obtained in the CBS and pironetin binding site showed stability along the molecular dynamic trajectory. In the first case, the RMSD obtained, showed minimal changes in the interaction of the ligand with the receptor, obtaining always values close or lower than 5 Å and a fast saturation of the curve, suggesting ligand stability in this domain [49]. In contrast, in the pironetin binding site, some RMSD values were higher and the curve presented big fluctuations related to low stability in the interactions between the ligands and the receptor. All previous results and the energy obtained with the LIE-D methodology suggest that the xanthanolides could interact through both, the colchicine and pironetin binding site, with greater affinity for the former.
In a recently published work, was shown that the compound Cevipabulin binds indistinctly into the vinca domain and the seventh binding site, which are equivalents but located in different subunits [8]. In this sense, a superposition study based on the sequence identity homology shared by α- and β-tubulin, the 3D structures similarities, and the disposition of pironetin and colchicine binding sites in their respective subunits reveal that these are equivalent sites from different subunits. They share the same fold and their sequences are similar, with 34% of sequence identity and many of the observed amino acid changes are conservative (29%) [50]. All mentioned above, make stronger our previous suggestion of two possible binding sites in tubulin for xanthanolides.
The higher stability observed for xanthanolides in colchicine and pironetin binding sites, against the vinca domain and maytansine binding site, could be for several reasons. First, the structural characteristics of each domain, the colchicine and pironetin binding sites are well-defined deep hydrophobic pockets [18, 30, 51], while the other sites are wider and more superficial, providing few hydrophobic interactions for the stability of xanthanolides. This leads to the antimitotic drugs that bind into the colchicine and pironetin binding sites being hydrophobic low molecular weight compounds [10, 30], while those that bind at the vinca domain and maytansine binding site have more complexes structures and bigger sizes, being in many times small peptides [18, 52]. Thus, considering that xanthanolides are compounds with a hydrophobic core with few hydrogen acceptors, two at one end (O17 and O18) and one in the other (O16), and low molecular weight, they can establish more favorable interaction in a well-defined hydrophobic pocket as the colchicine and pironetin binding site.
Additionally, pharmacophoric models that can help to understand the higher selectivity of the xanthanolides for the colchicine binding site have been developed. For the colchicine binding site, two pharmacophoric models have been proposed [53, 54]. In one of them, exist seven pharmacophoric points: three hydrogen acceptors atoms (A1, A2, and A3), one hydrogen donor atom (D1), two hydrophobics centers (H1 and H2), and one planar group (R1). However, to make the molecule bioactive it only requires at least one hydrogen acceptor atom, two hydrophobic centers, and a planar group [53]. The other pharmacophore was proposed by Wang and coworkers, 2016, in which five pharmacophoric points are proposed: three hydrophobic centers (I, II, and III) and two hydrogen bond centers (IV and V, either hydrogen bond acceptor or donor). Also, they calculate the distance to which these pharmacophoric points should be found: I-II (4–9 Å), II-III (4–8 Å), III-IV (3–5 Å), V-II (4.5-7 Å), II-IV (4.5-7 Å) y IV-I (5–6 Å) [54].
For the maytansine binding site and vinca domain, pharmacophoric models have also been proposed. The first one was suggested by Prota and coworkers in 2014 for the maytansine binding site, it consists of three keys interaction points (I, II, and III) with the β-subunit of tubulin. The proposed distance between these pharmacophoric points was much higher than those raised for the colchicine binding site: I-II (10.7–12.1 Å), II-III (7.4–8.4 Å) y III-I (4.7-5 Å) [12]. The second one. was proposed by Wang and coworkers in 2015, for the vinca domain, it contains two hydrophobic groups (I and II), and three hydrogen bond forming atoms. The two hydrophobic groups I and II interact with hydrophobic pockets β and α respectively. The three-hydrogen bond forming atoms interact with Dβ179 (one N atom) and Nα329 (one N and one O atom). There are two areas (A and B) at the opposite sides of the pharmacophore where extra chemical groups can be added to make additional interactions to increase the affinity of the ligand. As for previous pharmacophores, the possible distance between the pharmacophoric points was recommended: I-II (8.5–11.2 Å), II-N (5.8-8 Å), I-N (9-10.2 Å) [52].
Xanthanolides, xanthatin and 8-epi-xanthatin, are hydrophobic molecules with a representative core constituted by a bicyclic γ-lactamic ring which contains a seven-member ring fused with a five-member ring [55]. The first ring has an aliphatic branch while the second one has a methylene group. Additionally, they contain three oxygen atoms acting as hydrogen acceptors, enabling the interaction stabilizations by hydrogen bonds. According to these structural features, we propose preliminary pharmacophore model composed by five points: three hydrophobics centers (I, II, and III) and two hydrogen acceptors (IV and V) (Fig. 5). Points I and II are represented by the methyl (C15) in the aliphatic chain and the methylene (C13) from the five-member fused ring, respectively. Point II is a bulk hydrophobic center represented by the seven-member ring. The hydrogen acceptor center is the O16 (point IV) and O18 (point V). Distance between these points was measured (I-II: 6.70 Å, II-III: 4.06 Å, III-IV: 2.96 Å, V-II: 4.99 Å, II-IV: 5.89 Å, IV-I: 2.40 Å), and show to be similar to those proposed by Wang and coworkers in the pharmacophore model for the colchicine binding site [54] (Fig. 5). In addition, these proposed pharmacophoric points in xanthanolides, are distributed in such a way that if the γ-lactamic ring is pointing toward either, α or β-subunit, these keep a similar location and distances justifying the issue that two models equally stable were obtained in this work in the colchicine binding site. On other hand, when its isomers are compared with the pharmacophore’s models proposed for the vinca domain, it does not match. This analysis of the potential pharmacophoric points of the xanthanolides has great importance for the pharmaceutical industry, leading to possible chemical modifications to improve the pharmaceutical efficiency as chemotherapeutics drugs.
Regarding the higher stability observed for the xanthanolides in the colchicine binding site than in the pironetin binding site, could be associated with several facts. The colchicine and pironetin binding sites are two well-defined hydrophobic pockets that bind small molecular weight molecules. However, despite the high similarity in their 3D structure, they present some differences in their amino acid composition [50]. These differences in the primary sequence could contribute to the lower stability observed for the xanthanolides inside the pironetin binding site. Also, the CBS is located at the interface of two unseparated subunits from the same heterodimer, providing amino acid residues from both subunits for xanthanolides interaction [10]. On the other hand, the pironetin binding site is located exclusively in the α subunit reducing the number of interactions for the stabilization of xanthanolides [56]. To date, Pironetin is the only known ligand that binds into the pironetin binding site. This pocket is usually in a closed conformation, Pironetin binding opens it with some residues shifting more than 10 Å [56]. The ethyl group of Pironetin seems to play an essential role in its action, due to the biological activity decrease in Pironetin variants where this group has been removed or replaced by other groups [57–60]. All these facts represent a challenge for binding the xanthanolides into the pironetin binding site. Also, it could explain that the xanthanolides are located at the exit of the pironetin binding site at the end of molecular dynamics simulations.
So far, our results point to the idea that the isomers xanthatin and 8-epi-xanthatin are more likely to exert their mechanism of action as MTs destabilizing agents by interacting with the colchicine binding site. Based on the analysis of Pérez-Pérez et al. [61], the colchicine domain ligands were classified into classical and non-classical colchicine binding site inhibitors (CBSIs) according to their spatial orientations. The superposition of these ligands with x-ray crystal structures indicated that classical CBSIs with more globular or butterfly-like shapes occupy zones 1 and 2, mimicking the colchicine binding mode. However, non-classical CBSIs with more planar structures tend to locate deeper into the β-subunit making use of zones 2 and 3 [18, 61]. The studied xanthanolides in this work share some structural similarities with several non-classical CBSIs leading to the idea that these isomers could exert their activity by zones 2 and 3 of the colchicine binding site. However, the interaction pattern obtained in most complexes with high stability showed interactions with amino acid residues from the three zones. To date, only two CBSIs can interact with the 3 zones, ABT- 751 [29] and 2-(6-Ethoxy-3-(3-ethoxyphenylamino)-1-methyl-1,4- dihydroindeno[1,2-c]pyrazol-7-yloxy)acetamide [62], which interact mainly with zones 1 and 2 but also establish a single hydrogen bond with the Tyrβ202 from zone 3. Nevertheless, a convergent binding pose for xanthanolides in the CBS was not obtained. Despite all point to the possibility that these isomers could be classified as novel non-classical CBSIs, further studies are required to propose an accurate binding conformation and classification for xanthanolides as CBSI.
Despite Colchicine being a cheap chemotherapeutic option, its clinical applications are limited by its toxicity. It strongly binds to tubulin in non-cancerous cells and causes impaired protein assembly, mitotic arrest, and multiorgan dysfunction [10]. Because of this, the search for new and more efficient CBS inhibitors continues to be an attractive open research field. In a previous study, it was observed that mice treated with the chemical fraction containing the xanthanolides did not show signs of toxicity, but a significant inhibitory effect in the proliferation of the tumor [21]. These results could be related to the fact that xanthanolides have lower binding energy (-6.79 A -5.21 kcal / mol) than Colchicine (-10 A -9 kcal / mol) [63] in the CBS. Xanthanolides could efficiently bind to tubulin in the cancer cells with an overexpression of these proteins associated with their exacerbated cell growth. It would increase the arrest of tumor cells in the G2 / M phase, which led to the apoptosis of these cells and the reduction of the tumor. However, in non-cancerous cells in a quiescent state, the union of the xanthanolides to tubulin will be lower, decreasing toxicity in patients.
In summary, xanthatin and 8-epi-xanthatin isomers could bind indistinctly into the pironetin and colchicine binding sites. However, based on our results and the features of the Pironetin binding into the pironetin binding site, these isomers seem to have a greater affinity for the CBS. Additionally, the molecular structure of xanthanolides matches with the pharmacophore model proposed for the colchicine binding domain, especially with the model suggested by Wang and coworkers in 2016. The studied xanthanolides in this work share some structural similarities with several non-classical CBSIs, which consist in a planar structure and tend to locate deeper into the β-subunit making use of zones 2 and 3 in the colchicine-binding domain.