1. Introduction
The transient receptor potential (TRP) channel family is a group of transmembrane nonselective cation channels which includes seven subfamilies: canonical (TRPC), vanilloid (TRPV), melastatin (TRPM), mucolipin (TRPML), polycystin (TRPP), TRPN (NOMPC-like, no mechanoreceptor potential C), and ankyrin (TRPA1) [
1]. All of the 28 distinct channels have been considered drug targets for multiple diseases such as chronic pain, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), and asthma [
2,
3]. Among them, TRPA1, the only member of the TRPA subfamily, is currently one of the most promising and studied targets in preclinical biological research, especially in the context of chronic and acute pain and respiratory diseases.
The TRPA1 ion channel is composed of four homomonomers forming a single pore, and is structurally characterized by an N-terminal fragment of 14–16 ankyrin repeats (30–34 amino-acid-long helix turn motifs), responsible for mediating protein–protein interactions with cytoskeletal proteins as well as the thermal and chemical sensitivity of the channel [
4,
5,
6,
7]. Each monomer contains six transmembrane domains (S1–S6), a reentrant pore loop in the transmembrane domain, and an intracellular coiled-coil structured C-terminal domain (
Figure 1).
TRPA1 is a ligand-gated ion channel that is modulated by voltage. It is activated in response to various endogenous and exogenous molecules. Among TRPA1 agonists, two groups can be distinguished: electrophilic and nonelectrophilic ligands. The electrophilic compounds, such as pungent natural products (allyl isothiocyanate, diallyl disulfide), environmental irritants (acrolein, vehicle exhaust components), and reactive metabolites (4-hydroxynonenal) (
Figure 2A), activate the channel by covalently modifying cysteine residues. This applies primarily to Cys-621, but also to Cys-641, and Cys-665, all of which are located in the region comprising ankyrin repeats, linker, and pre-S1-helix that is responsible for allosteric regulation of TRPA1. Moreover, Lys-710, located in the pre-S1 helix, seems to play an equally important role, especially in the case of the binding of thiocyanates and ketoaldehydes. Furthermore, mass spectrometric analysis has shown that other lysine residues (Lys-704, Lys-787) are also crucial targets for electrophilic activation of hTRPA1 [
8]. In 2020, Suo and co-authors presented cryogenic electron microscopy (cryo-EM) structures of TRPA1 in a complex with the covalent agonists JT010 and BITC, where they confirmed the location of the binding site between ankyrin repeats, pre-S1-helix, and the TRP-like domain as a coupling domain. Both compounds modify Cys-621 by reversible thiol-Michael adduct formation (BITC) and irreversible SN2 reaction (JT010). Additionally, the thiazole group of JT010 formed CH-π and sulfur-π interactions with Phe-612 and Tyr-680 (
Figure 2B,C) [
9]. The latest research conducted by Jianhua Zhao’s group indicates that electrophilic agonists act through a two-step process. The first phase is connected with Cys-621, which causes the reorientation of a cytoplasmatic loop to enhance nucleophilicity. Subsequently, in the second phase, Cys-665 is modified to stabilize the loop in an activated conformation [
10].
The binding site of noncovalent agonists has not been directly described. However, the literature presents some assumptions about the hydrophobic pocket located at the interface of the pore helix-1, S5, and S6. It is worth noting that this binding site is similar to that of a known antagonist A-967079 [
4]. Molecular modeling studies have indicated that piperidine carboxamides, a class of potent agonists described by Chernov-Rogan et al., and general anesthetics such as isoflurane, desflurane, and propofol interact right at that site. The lead compounds PIPC1 and PIPC2 were docked to homology models based on TRPV1/6, and interacted with Val-875, Ile-946, Ser-873, and Phe-909 (
Figure 3) [
11]. However, subsequent molecular modeling studies on the group of anesthetics suggested that isoflurane and propofol, while acting at the same binding site, have different binding modes focused on halogen bonds with Ser-873, Met-915, and Met-956 (isoflurane) or sulfur-aromatic interactions with Met-912 (propofol). Interactions with these amino acids are thought to be responsible for the agonism of volatile anesthetics at this target [
12]. Both the above-mentioned predictions were confirmed by mutagenesis studies and appropriate functional assays. Recently, another binding site for a new noncovalent agonist GNE551 was discovered and confirmed with the cryo-EM structure of the TRPA1-GNE551 complex by Chen’s group [
13]. The binding pocket was formed by the voltage-sensor-like domain (VSLD) of one subunit and the pore domain (S5–S6 helices) of the neighboring subunit and was characterized as a mostly hydrophobic pocket. Nevertheless, two hydrogen bonds with Tyr-840 and Ser-943 stabilized the complex, while Gln-940 was a critical residue for ion channel activation by GNE551 (
Figure 3).
In the treatment of pain and respiratory diseases, TRPA1 antagonists appear to be more pharmacologically relevant. A-967079 and HC-030031 are among the most widely studied TRPA1 antagonists discovered in recent years (
Figure 4). The binding site of the former compound is located in the pocket formed by S5, S6, and the first pore helix. This site was first determined using a mutagenesis approach and then confirmed by a density map obtained through cryo-electron microscopy studies [
1,
4,
10,
11,
14]. Paulsen et al. proposed that A-967079 forms hydrogen bonds with Ser-873 and/or Thr-874, both located at the bottom of the putative pocket, and π-π interactions with highly conserved Phe-909—a key residue, the mutation of which deprives A-967079 of its antagonistic effect [
4]. This binding site for TRPA1 antagonists was confirmed in the experimental structure (PDB: 6WJ5), which represented the interaction pattern of the GDC-0334 inhibitor in the study by Lorena Riol-Blanco’s group [
15]. Unfortunately, the binding site of HC-030031 could not be identified in these studies [
4,
16]. Moreover, it was proved that Phe-909 does not participate in the binding of the methylxanthine antagonist [
12]. Further efforts have been made to find a binding site for HC-030031 and two main hypotheses have been published. The site-directed mutagenesis studies described in the paper by Gupta et al. indicated that interaction with a single amino acid residue Asn-855 is responsible for the inhibitory activity of HC-030031. The results of molecular dynamics (MD) simulation showed that HC-030031 formed a hydrogen bond with Asn-855 and the hydrophobic moiety of the molecule was located in a nearby hydrophobic pocket [
17]. No other ligand–protein interactions were identified that would indicate the exact position of the compound at the binding site. On the other hand, Wenlei Ye and co-authors pointed out in their mutagenesis study that methylxanthine antagonists may bind in the pore in the vicinity of Asp-915, and its bioactive conformation is stabilized by the presence of acidic residues [
18]. In addition, molecular modeling studies recently presented in an article by Kravchenko et al. described this region as a potential binding site for the TRPA1/TRPV1 antagonist HSV-DKH-0450 [
19]. Given the lack of conclusive evidence, identification of a specific binding site and interactions formed by HC-030031 would be essential for the drug design of methylxanthine derivatives, which remain at the forefront of scientific interest. Considering the above, the aim of the present study was to determine the potential binding site of a representative of xanthine antagonists, compound HC-030031, and to illustrate its binding mode in order to support rational molecular design in this chemical space.
2. Results and Discussion
In the Sitemap program, 20 potential binding sites were identified and assessed (
Table 1). The possible ligand binding sites were as follows: the region of the ankyrin repeats, the channel pore, the transmembrane domain (region around S1, S2, and S3), as well as the area between pre-S1, TRP-like domain, and S4–S5 helices, and below the TRP-like domain. The highest values of the druggability assessment score (DScore) were assigned to a place in the transmembrane region (1.213) for the 3J9P-based model, and to sites near the pre-S1/S4/S5/TRP-like domain (1.137) for the 6PQQ-based model.
The analysis of one subunit of the TRPA1 channel only by the Deepsite web server indicated three likely binding sites in the 3J9P-based model, which included the area around the linker domain, the transmembrane domain (S1/S2/S3), and the ankyrin repeats. In the case of the 6PQQ-based model homomonomer, only two binding sites were predicted. The favorably scored site was localized around the pre-S1/S4/S5/TRP-like domain which contains Asn-855, among others. The second binding site was indicated around the ankyrin repeats (
Table 2).
As a result of TRPA1 structural analysis using the MetaPocket consensus program, three probable binding sites were predicted for each model (
Table 3). Interestingly, for each of the models, two designated places were located near the pre-S1/S4/S5/TRP-like domain—the only difference was the individual subunits that constructed these areas. However, in the case of the TRPA1 channel, which is built of four homomonomers, the selected pocket would apply analogically to all subunits. The other two different sites were identified around the ankyrin repeats and the transmembrane domain for the 3J9P-based model and the 6PQQ-based model, respectively.
Castp3.0 determined 18 probable binding sites for both models, which are presented in detail in
Table 4. In the case of the 3J9P-based model, the cavities were located mainly in the linker domain and ankyrin repeats, and, to a lesser extent, across the entire channel pore and in the transmembrane domain at the site formed by the helices S1, S2, and S3. On the other hand, the majority of sites in the 6PQQ-based model were found in the transmembrane area as well as in the entrance to the channel pore. Only a few of them were found at the intracellular channel pore. No binding sites were indicated near the pre-S1/S4/S5/TRP-like domain, contrary to the results from the previous algorithms.
It was observed that the potential binding sites were indicated across the entire TRPA1 structure, representing four main areas. The sites located within the ankyrin repeats and the transmembrane domain were similar to the already known binding sites of the TRPA1 ligands, particularly for the electrophilic agonists, which interact covalently with cysteine residues of the ankyrin repeats, but also for the nonelectrophilic agonists, and antagonists derived from A-967079 that bind in the region of the transmembrane domain. Due to the above-mentioned literature indications that HC-030031 does not interact with the TRPA1 ion channel in these areas [
4,
9,
16], they were excluded from further analysis. The remaining putative binding sites were the actual channel pore, a fragment below the TRP-like domain and the space formed by the TRP-like domain and pre-S1, and the S4 and S5 transmembrane helices. However, the TRP-like domain and the region of the channel pore around Asp-915 already have established functions in ion channel activity. The TRP-like domain is involved in the activation of TRPA1 by electrophilic irritants and subserves allosteric regulation [
4], and the channel pore in the vicinity of Asp-915 represents the site of TRPA1 pore constriction and is critical in controlling ion permeation [
20]. The most prevalent binding sites were found in the pre-S1/S4/S5/TRP-like domain area (
Table 5), and they were characterized by preferable values from the Sitemap (
Table 1), Deepsite (
Table 2), and MetaPocket2.0 (
Table 3) scoring functions. Moreover, these active sites contained Asn-855 residue, which was found to be significant for the inhibitory effect of HC-030031 in research presented in the paper by Gupta et al. [
17]. Therefore, this area was designated for a further step, which consisted of the docking of the TRPA1 antagonist HC-030031.
In the search for potential binding sites, we used two TRPA1 ion channel models. For further steps of HC-030031 binding mode determination, starting with docking, we chose only one of them—the more reliable 6PQQ-based model. This model was prepared based on the recently released TRPA1 cryo-EM structure, which is characterized by a higher resolution and accuracy of matching atoms to the electron density map compared to 3J9P, while the predictors indicated analogous binding sites (among those regarded as relevant) for these two models (seven sites in the pre-S1/S4/S5/ TRP-like domain). The docking step was performed as an induced-fit docking procedure, which allows for conformational changes of the protein side chains and minor relaxation of the protein backbone that further enable the receptor to alter its binding site to be more compatible with the shape and functional group arrangement of the ligand. HC-030031 was docked to the TRPA1 ion channel at each of the seven potential sites that represented the selected group of pre-S1/S4/S5/TRP-like domain sites (
Figure 5). Out of the docking results, one HC-030031-TRPA1 complex was selected for each site. That was the best rated position of the most common interaction pattern, characterized by favorable values of the scoring functions (IFDScore, docking score) and the presence of important (according to the literature) binding interactions (
Table 6).
As depicted in
Figure 6, HC-030031 was arranged in a similar manner at each of the designated binding sites. In every complex except B, the methylxanthine core was located in the space between the pre-S1 helix and S4–S5 linker, forming hydrogen bonds and/or π-π stackings with Trp-711. Additional stabilizing interactions were hydrogen bonds with Glu-854 and His-719, π-π stackings with Phe-1024 for the majority of complexes, and cation-π interactions with Arg-975 in the G complex. In all complexes, the methylxanthine core was perpendicular to the Trp-711 residue, excluding the G complex where it was located parallel to its plane. The substituent in position 7 of the ligand was arranged in two variants: it pointed “up” towards the transmembrane domain, which is represented by the D, E, and G complexes, and “down” towards the pre-S1 helix, which was illustrated by the remaining complexes. Interestingly, regardless of the variant, the carbonyl group of the amide bond formed a hydrogen bond with the main chain of Asn-855. In complex B only, the ligand’s substituent in position 7 was parallel to the helix formed by residues Leu-1016–Phe-1024, and a hydrogen bond was formed with Trp-711.
The final complexes, collected in
Figure 6, were subjected to MD simulations (15 ns) to assess the stability of the binding interactions. The C, D, and G complexes turned out to be the most stable over the entire simulation (
Figure 7). The others showed large fluctuations in RMSD and no permanent conformations were established. Considering the stability of the binding interactions formed by the ligand with the protein, comparably persistent bonds occurred in the three complexes mentioned above. In the case of complex G, MD simulation results showed that after system relaxation, there was a change in the arrangement of the substituent in position 7 of HC-030031. Nevertheless, it H-bonded to the side chain of Asn-855 (which reflects the mutagenetic study results [
17]), while maintaining the other characteristic interactions with Trp-711, Phe-853, etc. Moreover, this structure turned out to be the most stable one in MD (RMSD value below 4.0 Å); therefore, this representation of the HC-030031-TRPA1 structure was used for further research regarding complex G (
Figure 7). The π-π stacking interaction of the xanthine pyrimidinedione ring with Trp-711 persisted for 100% of the simulation time, as did the hydrogen bonds with Asn-855, Glu-854, and Trp-711, which were observed for 99%, 94%, and 96% of the simulation time, respectively. The least established contact was the π-π stacking interaction with Phe-853 (17%). In complex D, hydrogen bonds with His-719, Glu-854, and Asn-855 were maintained for 96%, 95%, and 85% of the simulation time, respectively. The π-π stacking interactions with Trp-711 were observed in 84% of the simulation time. In complex C, the most stable interactions were hydrogen bonds with Asn-855, Trp-711, and Leu-1023 (99%, 85%, and 82% of the simulation time, respectively) as well as π-π stacking with Trp-711 and Phe-853 (86% and 85% of the simulation time). It was also characterized by high stability throughout the whole MD simulation in terms of RMSD (the protein 2.9 Å and the ligand 3.9 Å), similar to complex D (maximum RMSD of 3.2 Å for the protein and 2.5 Å for the ligand). During the simulations of the remaining complexes, ligand RMSD fluctuations up to 10.5 Å occurred. The above-described docking and MD results showed that complexes C, D, and G represent the most suitable binding modes of HC-030031. However, during the simulations of complexes C and G only, the substituent in position 7 of the ligand formed a permanent hydrogen bond with Asn-855 (99% of simulation time). Therefore, these two complexes were selected for further investigation—the protein structures served as models for docking and MD studies (referred to as models C and G).
The comparison of the models developed as part of the present study with the cryo-EM structure of the TRPA1 ion channel bound to the hypoxanthine derivative compound 21 recently published by Terrett et al. (PDB ID: 7JUP, released 31 March 2021, after the described molecular modeling studies) revealed that in the selected binding site of complex G, the binding mode of the methylxanthine moiety was analogous to that of the hypoxanthine moiety from the experimental model (
Figure 8B) [
21]. In contrast, in the case of complex C, methylxanthine was located perpendicular to the hypoxanthine and above its site (
Figure 8A). Both complexes differed substantially from the 7JUP representation in terms of the location of the large substituent—the amide moiety of HC-030031 was oriented upright, while the heterocyclic moiety of compound 21 was placed to the right (
Figure 8A,B).
In order to test the selected representations of the binding mode of methylxanthine antagonists in TRPA1, retrospective virtual screening studies were performed. The results (
Figure 9C,D) showed that both models C and G recognized all 22 active ligands. Nonetheless, model G performed better than C in distinguishing the actives from the decoys, as indicated by the enrichment factor EF1% (33 vs. 4.6, respectively). In the 1% ligand database set (11.22 ligands), in the case of model G, nine active ligands were classified, whereas model C found only one active compound. The BEDROC parameter indicated the explicit usefulness of model G in virtual screening, as most of the active ligands were ranked before inactive decoys—BEDROC
α=20 0.785 (vs. 0.185 for model C). As shown in the enrichment plot, model G also had the largest area under the accumulation curve (AUAC = 0.96), which confirms that it can be considered to be the best representation of the antagonist binding site. The predicted binding mode of HC-030031 in model G, showing key interactions with Asn-855 and Trp711, is depicted in
Figure 9A (for details, see
Supplementary Materials: HC-030031-TRPA1_complex_G_relaxed.pdb file).
To compare the binding mode of HC-030031 itself in the newly available 7JUP TRPA1 conformational model with those found in the models developed as part of the present study, its structure was docked to a preprocessed 7JUP using Glide SP (
Figure 9B). Moreover, the latter conformational model was tested in retrospective virtual screening under the same conditions as the previous models. The results showed that 7JUP, used as a model in VS, ranked all the active compounds, but only one of them was found in the first 1% of the compound library. This was reflected in the VS parameters, which were only slightly better than for model C, but distinctly lower than for model G (
Figure 9C,D).
To verify the stability of the predicted interactions and confirm the selection of the most probable representation of the binding mode of methylxanthine antagonist in TRPA1, additional 100 ns MD simulations were performed. The RMSD graphs show that complex G was characterized by the highest conformational stability of the ligand and fair stability of the ion channel (
Figure 10). HC-030031 and the target protein in complex G established their conformations and were not subject to large RMSD fluctuations. The durability of the interactions was also favorable—the hydrogen bond and π-π stacking interactions with Trp-711 were observed for 98% and 99% of the simulation time. The hydrogen bond between the amide substituent in position 7 of the ligand and Asn-855 was also stable for over 80% of the simulation time, with the only difference being that it was mediated half the time by a water molecule. In the case of complex C, a hydrogen bond with Asn-855, as well as interactions with Trp-711, were established for shorter periods (55% and 31% of the simulation time), which confirmed the lowest ligand stability in the comparison. The results of the MD simulation of the 7JUP-HC-030031 complex took mid-range values. The RMSD fluctuations were slightly higher than for complex G, while the π-π stacking interactions with Trp-711 were less persistent (81% of the simulation time).