Conceptual DFT, QTAIM, and Molecular Docking Approaches to Characterize the T-Type Calcium Channel Blocker Anandamide

The anandamide is a relevant ligand due to its capacity of interacting with several proteins, including the T-type calcium channels, which play an important role in neuropathic pain and depression disorders. Hence, a detailed characterization of the chemical properties and conformational stability of anandamide may provide valuable information to understand its behavior in a biological context. Herein, conceptual DFT and QTAIM analyses were performed to theoretically characterize the chemical reactivity properties and the structural stability of conformations of anandamide, using the BP86/cc-pVTZ level of theory. Global reactivity description, based on conceptual DFT, indicates that the hardness increases and the electrophilicity index decreases for both, the hairpin and U-shape conformers relative to the extended conformers. Also, an increase in the chemical potential value and a decrease in the electronegativity and the electrophilicity index is observed in the ethanolamide open ring conformers in comparison with the corresponding closed ring structures. In addition, regarding the characterization of local reactivity descriptors, the maximum values of the Fukui and Parr functions indicate that the most probable location for a nucleophilic attack is either the hydroxyl oxygen located in the ethanolamide closed ring conformers or the carbonyl oxygen present in the open ring conformers. The most probable location for an electrophilic attack is in the alkyl double bond region in all anandamide conformers. According to the QTAIM results, the intramolecular hydrogen bond formation stabilizing the structure of anandamide has interaction energy values for the closed ring conformations of 12.33–12.46 kcal mol⁻¹, indicating a strong interaction. Lastly, molecular docking calculations determined that a region in the pore, denominate as pore-blocking, is a probable site for the interaction of anandamide with the human Ca_v3.2 isoform of the T-type calcium channel family. The pore-blocking site contains hydrophobic residues where the non-polar part in the final alkyl region of anandamide established mainly alkyl-alkyl interactions, while the polar part (the ethanolamide group) interacts with the polar residue S900. The information based on conceptual DFT presented may aid in the design of drugs with similar chemical characteristics as those identified in anandamide so as to bind anandamide-interacting proteins, including the T-type calcium channels.

GRAPHICAL ABSTRACT

GRAPHICAL ABSTRACT

Introduction

Anandamide is a fatty acid neurotransmitter that is synthesized enzymatically in the brain from arachidonic acid (AA) and ethanolamide substrates (Devane and Axelrod, 1994); however, other synthetic pathways involving the N-arachidonoyl phosphatidylethanolamine (PE) precursor and the phosphodiesterase enzyme, have been also proposed (Sugiura et al., 2002). Although anandamide is an endogenous endocannabinoid ligand known to bind mainly to the cannabinoid receptors, it is also capable of directly modulating a variety of ion channels, such as potassium channels, sodium channels, TRPV1 (Transient Receptor Potential Vanilloid type 1), and the T-type calcium channels (Van Der Stelt and Di Marzo, 2005; Oz, 2006). Along these lines, it has been proved that blocking both, the T-type calcium channel and the cannabinoid receptors is an important therapeutic strategy for treating neuropathic and inflammatory pain (You et al., 2011; Gadotti et al., 2013; Bladen et al., 2015). Moreover, the T-type calcium channels and the CB1/CB2 cannabinoid receptors are involved in disorders associated with neuropathic pain, mental pain, suicidal behavior, and depression (Pacher et al., 2006; Todorovic and Jevtovic-Todorovic, 2013; Tibbs et al., 2016; Colino et al., 2018; Snutch and Zamponi, 2018; Yang et al., 2018). Among the three isoforms that constitute the T-type calcium channels (Ca_v3.1, Ca_v3.2, and Ca_v3.3), the Ca_v3.2 contributes significantly to the nociceptive pathway which is responsible for the physiological and pathological mechanism of pain transmission. It was demonstrated by electrophysiological experiments that anandamide directly inhibits the T-type calcium channels during neural activities with a preference for the Ca_v3.2 isoform displaying IC₅₀ values for the Ca_v3.1, Ca_v3.2, and Ca_v3.3 channels of 4.15 µM, 330 nM, and 1.10 µM, respectively (Chemin et al., 2001). Using electrophysiology recordings, it was found that the hydroxyl group and the alkyl chain from anandamide play important roles in the inhibition mechanism of the T-type calcium channel by anandamide. In detail, it was found that the chain length and the presence, position, and all-cis isomerism of the alkyl chain double bonds, are all important factors for the degree of anandamide inhibition. Interestingly, the inhibition can occur even though the alkyl chain contains either 18 or 22 carbons instead of 20 carbons. Also, it was observed that a decrease in the degree of unsaturation in anandamide reduces its blocking effect, with a fully saturated alkyl chain displaying negligible inhibitory effect. The inhibition increases when double bonds are near the carboxyl group and have no effect when a trans-configuration and triple bonds are considered (Chemin et al., 2007).

Experimental X-ray structures found the alternated form of anandamide (cis-trans-cis-trans) and all-trans anandamide in interaction with its intracellular transporter, the fatty acid binding protein FABP5 in mouse (mFABP5) and human (hFABP5), respectively (Sanson et al., 2014). Also, both isomer molecules the synthesized all-trans anandamide and the natural cis-anandamide are equally good substrates for fatty acid amide hydrolase FAAH enzyme (Ferreri et al., 2008). Nevertheless, it has been observed that endogenous trans and cis-trans of AA isomerization originate from diet and by the action of the nitrogen dioxide radicals (NO₂) (Zghibeh et al., 2004). This could indicate that the alternated (cis-trans-cis-trans) and all-trans anandamide forms may be present in certain conditions but the most relevant form during receptor interaction is the all-cis anandamide.

Regarding the interaction of anandamide with the cannabinoid receptors, a mutagenesis study demonstrated the importance of the amino acid F3.25 (following B&W nomenclature) for binding anandamide in the binding site of the CB1 receptor while a computational docking study showed interactions between anandamide, in a hairpin conformation, with the same receptor (McAllister et al., 2003). In a crystallographic study where the structure of the CB1 receptor was solved, the authors used the X-ray structure to dock various agonist molecules, including anandamide and found that the molecule prefers a hairpin conformation in the binding site of the receptor (Hua et al., 2016). Although there is significant information regarding the interaction of anandamide and various proteins, there is no information regarding the interaction of this important compound with the T-type calcium channels. Herein we used computational methods to characterize the reactivity of anandamide in the ligand-receptor context, evaluate its structural stability, and study the interaction of anandamide with the structure of the Ca_v3.2 channel. Conceptual DFT is used for the chemical reactivity interpretation by global and local reactivity descriptors, which are used to identify the possible interacting regions of anandamide at the Ca_v3.2 channel. QTAIM analyses were performed to theoretically characterize the structural stability of the preferred conformations of anandamide (Rangel-Galván et al., 2022a). These calculations were made using the BP86/cc-pVTZ level of theory. Finally, molecular docking calculations are used to characterize the main amino acids interacting in the Ca_v3.2/anandamide complex.

Computational Methods

Conceptual DFT Analysis

The conceptual DFT approach (Domingo et al., 2016; Frau and Glossman-Mitnik, 2017; Frau and Glossman-Mitnik, 2018) was used to evaluate the global reactivity descriptors, such as chemical potential (μ), electronegativity (χ), hardness (η), softness (s), and electrophilicity index (ω), from the Frontier molecular orbitals energies, E_HOMO and E_LUMO, using the following equations: μ = (E_HOMO + E_LUMO) ⁄ 2; χ = −(E_HOMO + E_LUMO) ⁄ 2; η = E_LUMO−E_HOMO; s = 1 ⁄ η, ω = μ² ⁄ 2η. In addition, the local reactivity descriptors, such as the Fukui functions $\mathit{f}\left(\mathit{r}\right)\mathit{ }$ for electrophilic ${\mathit{f}}^{-}\left(\mathit{r}\right)$ and nucleophilic ${\mathit{f}}^{+}\left(\mathit{r}\right)$ attacks, and the dual descriptor ${\mathit{f}}^{\left(2\right)}\left(\mathit{r}\right)$ was calculated using the following equations: ${\mathit{f}}_{\mathit{k}}^{-}={\mathit{q}}_{\mathit{k}}\left(\mathit{N}\right)-{\mathit{q}}_{\mathit{k}}\left(\mathit{N}-1\right)$; ${\mathit{f}}_{\mathit{k}}^{+}={\mathit{q}}_{\mathit{k}}\left(\mathit{N}+1\right)-{\mathit{q}}_{\mathit{k}}\left(\mathit{N}\right)$; ${\mathit{f}}_{\mathit{k}}^{\left(2\right)}={\mathit{f}}_{\mathit{k}}^{+}-{\mathit{f}}_{\mathit{k}}^{-}$, where ${\mathit{q}}_{\mathit{k}}$ are the electronic population of the kth atom of the molecule with $\mathit{N}-1$, $\mathit{N}$, and $\mathit{N}+1$ electrons (Yang and Mortier, 1986; Morell et al., 2005). Hirshfeld population analysis was carried out by using the Multiwfn program (Lu and Chen, 2012). The Parr functions $\mathit{P}\left(\mathit{r}\right)$ for electrophilic ${\mathit{P}}^{-}\left(\mathit{r}\right)$ and nucleophilic ${\mathit{P}}^{+}\left(\mathit{r}\right)$ attacks were performed using the equations: ${\mathit{P}}_{\mathit{k}}^{-}={\mathit{\rho }}_{\mathit{k}}^{\mathit{rc}}$; ${\mathit{P}}_{\mathit{k}}^{+}={\mathit{\rho }}_{\mathit{k}}^{\mathit{ra}}$, where ${\mathit{\rho }}_{\mathit{k}}^{\mathit{rc}}\mathit{ }$ and ${\mathit{\rho }}_{\mathit{k}}^{\mathit{ra}}$ is the atom spin density (ASD) of the kth atom at the cation and the anion of the molecule, respectively (Chamorro et al., 2013; Domingo et al., 2013). The data of the optimized conformers of anandamide were obtained from the BP86 functional (Becke, 1988) and cc-pVTZ basis set (Dunning, 1989) in chloroform solvent, as previously reported (Rangel-Galván et al., 2022a). DFT calculations have been shown to be a reliable tool to analyze reactivity descriptors within the framework of conceptual DFT (De Proft et al., 2003; Vivas-Reyes et al., 2003; Vivas-Reyes et al., 2008). Calculations were carried out using the Gaussian16 program (Frisch et al., 2016).

QTAIM Analysis

Topological parameters, based on the QTAIM approach (Matta and Boyd, 2007; Hernández-Paredes et al., 2017; Bayat and Fattahi, 2019), were calculated, such as electronic density, $\rho \left(\mathit{r}\right)$, the Laplacian, ${\nabla }^2\rho \left(r\right)$, the Lagrangian kinetic energy, G, the Hamiltonian kinetic energy, H, the potential energy density, V, the interaction energy, E_{H … Y}, the interatomic distance D_inter, and the delocalization indexes, DI, in order to characterize the hydrogen bonds and other intramolecular interactions in the anandamide conformers. The intramolecular hydrogen bond in the ethanolamide (EA) group was evaluated in the anandamide conformers with the closed ring. These topological properties were calculated using the equations: $H\left(\mathit{r}\right)=G\left(\mathit{r}\right)-V\left(\mathit{r}\right)$, and $E_{H\cdots Y}=\frac{1}{2}V\left(\mathit{r}\right)$. The QTAIM calculations were performed using the AIMAll (Version 17.11.14) program (Tood, 2017).

Molecular Docking Analysis

Molecular docking calculations were carried out for the Ca_v3.2 calcium channel and the conformers of anandamide. The rigid blind docking was performed using the AutoDockVina (version 1.1.2) program (Trott and Olson, 2010). AutoDockVina uses a genetic algorithm as a searching method and the binding free energy parameter (−ΔG) as the scoring function. The binding free energy is calculated through the equation: $c={\displaystyle \sum _{i<j}{f}_{t_i{t}_j}\left(r_{ij}\right)}$, where each atom $i$ is assigned an atom type $t_i$, and $f_{t_i{t}_j}$ are a symmetric set of interaction functions occurring at the interatomic distance ${\mathit{r}}_{ij}$. The sum can be seen as the addition of the intermolecular and intramolecular contributions of all the pairs of atoms interacting with each other. The optimization algorithm finds the global minimum $c$ in terms of the free energy values obtained for each conformation (Trott and Olson, 2010). The grid dimension space was 90 Å × 110 Å × 110 Å, containing the entire receptor structure. The default exhaustiveness value of 8 and 80 were used with 10 resulting poses. The structures of anandamide conformers, used as ligands, were obtained from the optimized structures at the BP86/cc-pVTZ level of theory in chloroform solvent (Rangel-Galván et al., 2022a). The Ca_v3.2 structure, used as the receptor, was taken from the homology model previously reported (Rangel-Galván et al., 2022b). The ligand Gasteiger charges were calculated and hydrogen atoms were added with Autodock tools (Sanner, 1999). The interactions were visualized with PyMOL v2.0 (Schrödinger, 2017).

Results and Discussion

Conceptual DFT Analysis

Six minimum energy structures for anandamide were selected for the conceptual DFT analysis: extended with closed EA ring (E_closed), extended with open EA ring (E_open), U-shape with closed EA ring (U_closed), U-shape with open EA ring (U_open), hairpin with closed EA ring (H_closed), and hairpin with open EA ring (H_open). Global reactivity descriptors for the six conformers of anandamide E_closed, E_open, U_closed, U_open, H_closed, and H_open, were evaluated: chemical potential (μ), electronegativity (χ), hardness (η), softness (s), and electrophilicity index (ω), according to conceptual DFT approach (Domingo et al., 2016; Frau and Glossman-Mitnik, 2017). Table 1 summarized the HOMO and LUMO energies and the global reactivity descriptors for the conformers of anandamide.

TABLE 1

TABLE 1. HOMO and LUMO energies and global reactivity descriptors (eV) of the conformers of anandamide obtained at the BP86/cc-pVTZ level of theory in chloroform.

For the E_closed/E_open, U_closed/U_open, and H_closed/H_open shapes of anandamide, in Table 1 it is observed that there are no significant changes when anandamide changes the curvature of this structure. Some slight differences are found in hardness, softness, and the electrophilicity index. The hardness increases a 4.6/3.5 and 7.4/6.3% for U_closed/U_open and H_closed/H_open shapes, respectively, relative to the E_closed/E_open conformers. The softness suffers a decrement keeping the same proportion. Finally, a decrease of the electrophilicity index of 3.3/3.6 and 10.6/12.0% is observed for U_closed/U_open and H_closed/H_open shapes, respectively, with respect to the E_closed/E_open conformers. The effect of the ethanolamide (EA) open ring conformers in comparison with the corresponding closed shapes is seen in slight differences, an increase in chemical potential, and a decrease in electronegativity and electrophilicity index. For example, for the electrophilicity index, the decrease represents a 2, 2.3, and 3.5% for E_open, U_open, and H_open with respect to their corresponding closed shapes.

In addition to anandamide as a dual ligand for the T-type calcium channels and the cannabinoid receptors, the NMP compounds have been shown to be capable of binding both proteins (You et al., 2011; Gadotti et al., 2013; Bladen et al., 2015), which is relevant in the treatment of pain disorders.

Taking into account the conceptual DFT analysis previously executed for the NMP compounds, the global reactivity descriptor that changes the most when anandamide and NMP compounds are compared (NMP-7 and H_closed shape for representation), is the hardness of 51%, from 4.76 eV in the H_closed conformation of anandamide and 3.14 eV in the NMP-7 ligand (Rangel-Galván et al., 2022b). Anandamide has a higher resistance to changing electronic distribution compared to the NMP compounds (Rangel-Galván et al., 2022b). It is worth mentioning that significant changes in the hardness index have been observed when a ligand-receptor interaction process is modeled (Aliste, 2000). The electrophilicity index is another uneven descriptor; a higher value corresponds to the NMP compounds with respect to anandamide conformers with a difference of 37.6%. The electrophilicity index results are well correlated to the receptor affinity properties and biological activity (Parthasarathi et al., 2004).

Respect the local reactivity descriptors, Table 2 shows the condensed Fukui functions, $f^{+}\left(r\right)$ and $f^{-}\left(r\right)$, the dual descriptor, $f^{\left(2\right)}\left(r\right)$, and the Parr functions, $P^{-}\left(r\right)$ and $P^{+}\left(r\right)$, the conformers of anandamide with a closed ring of ethanolamide (EA). For E_closed, U_closed, and H_closed shapes of anandamide, values of Fukui functions were found in the ranges f ⁺ = 0.007–0.071 and f ^– = 0.007–0.053 for E_closed, f ⁺ = 0.012–0.080 and f ⁻ = 0.008–0.057 for U_closed, and f ⁺ = 0.005–0.081 and f ^– = 0.009–0.052 for H_closed. In addition, for open ring the values were found in the ranges f ⁺ = 0.020–0.079 and f ^– = 0.022–0.059 for E_open, f ⁺ = 0.023–0.087 and f ^– = 0.017–0.064 for U_open, and f ⁺ = 0.022–0.089 and f ^– = 0.028–0.060 for H_open, as shown in Supplementary Table S1. The most relevant values are in the atom O22 of the hydroxyl group in the closed ring conformers, O21 of the carbonyl group in the open ring conformers in the ethanolamide (EA) group for nucleophilic attacks, and C12 in the middle alkyl region (Alkyl-M) for electrophilic attack for all the conformers. For the Parr functions, it is found a correspondence in the location of the highest values with the values found in the Fukui indices. Figure 1 presents the isosurfaces for Fukui functions with the major value for f ⁺ and f ^– for all shapes of anandamide. In Figure 1, it is observed that f ⁺ distributions are located on the hydroxyl oxygen O22 in closed conformers, and in the carbonyl oxygen O21 in open conformers, as shown in Supplementary Figure S1 in the SM section, also in the vinylic carbons on the Alkyl-M region for all conformers. While the f ^– distribution is located on the carbonyl carbon C2 in the closed conformers, it is also found on the vinylic carbons in the Alkyl-M region for all conformers. This finding is in accord with the experimental data which show that the double bonds region for anandamide is an essential factor for the T-type calcium channel blocking mechanism (Chemin et al., 2014).

TABLE 2

TABLE 2. Fukui functions, $f^{+}\left(r\right)$ and $f^{-}\left(r\right)$, dual descriptor, $f^{\left(2\right)}\left(r\right)$, and Parr functions, $P^{-}\left(r\right)$ and $P^{+}\left(r\right)$ the conformers of anandamide: E_closed, U_closed, and H_closed, obtained at the BP86/cc-pVTZ level of theory in chloroform.

FIGURE 1

FIGURE 1. Fukui functions, f ⁺ and f ^–, of the conformers of anandamide: E_closed, U_closed, and H_closed, obtained at the BP86/cc-pVTZ level of theory in chloroform.

QTAIM Analysis

The QTAIM analysis was focused to characterize the intramolecular hydrogen bond of the closed ring formed in the ethanolamide group in the conformers E_closed, U_closed, and H_closed of anandamide. This interaction of the hydrogen bond generates a ring structure of seven atoms in the EA group. The stability of the conformers with a closed ring of anandamide by the hydrogen bond formation effect is analyzed by means of interaction energy for each closed ring anandamide conformer from electronic density obtained from the BP86/cc-pVTZ level of theoretical calculations in chloroform. Table 3 and Supplementary Table S2 show the values of electronic density, $\rho \left(\mathit{r}\right)$, the Laplacian, ${\nabla }^2\rho \left(r\right)$, the Lagrangian kinetic energy, G, the Hamiltonian kinetic energy, H, the potential energy density, V, the interaction energy, E_{H … Y}, the interatomic distance, D_inter, and the delocalization indexes, DI, of the main bond critical points (BCP) of the anandamide conformers. Atom labels correspond to Figure 2.

TABLE 3

TABLE 3. Topological parameters (a.u.), E_{H … Y} (kcal mol⁻¹), and interatomic distances (D_inter, Å) of the conformers of anandamide: E_closed, U_closed, and H_closed, obtained at the BP86/cc-pVTZ level of theory in chloroform.

FIGURE 2

FIGURE 2. Molecular graphs of the conformers of anandamide: E_closed, U_closed, and H_closed, obtained at the BP86/cc-pVTZ level of theory in chloroform.

From the results, it is observed the non-covalent interaction corresponds to the hydrogen bond in the EA group in O21⋯H58-O22 with ρ(r) = 0.043 and ∇²ρ(r) = ∼ 0.096 a.u. for conformers E_closed, U_closed, and H_closed. These values are in agreement with those reported in the literature of 0.002–0.035 and 0.024–0.139 a.u., respectively (Popelier, 2000). On the other hand, the sign of H(r) parameter indicates, in the three closed conformers, that the accumulation of charge density on the hydrogen bond is a stabilizing bond (H(r) < 0) (Cremer and Kraka, 1984). In addition, the DI parameters have values in the range of 0.1106–0.1110 a.u. for the intramolecular hydrogen bond O21⋯H58−O22 in the three closed conformers, indicating that is an interaction <1.0 corresponding to a non-covalent interaction, while the DI for BCP between H58−O22 have values in the range of 0.5371–0.5402 a.u., indicating that this bond has a minor strength than a single bond (value close to 1.0 a.u.), due to the H58 is also contributing to the hydrogen bond formation with the atom O21. The interaction energy of the intramolecular hydrogen bond O21⋯H58−O22 corresponds to 12.33–12.46 kcal mol⁻¹ and it forms an RCP with ρ(r) = 0.0115–0.0116 a.u. for the three conformers, being slightly major the E_{H … Y} value in the conformer U_closed. It indicates that this hydrogen bond has a stabilizing effect larger than in the other conformers with the closed ring. The value of ρ(r) in the RCP of ethanolamide indicates that the ring formation stabilizes in the same order as the structures in the three conformers of the closed ring.

The non-covalent interactions H⋯H in the Alkyl-M region with a value of ρ(r) in the range of 0.011–0.020 a.u. generate the formation of four ring structures with ρ(r) in the RCP of 0.010–0.015 a.u. for the three closed conformers. The interaction energies between H⋯H are very small indicating weak interactions with values of DI of 0.03 a.u. approximately. Only in the conformer H_closed, is observed the non-covalent interaction C₆−H₃₇C₁₀ with values of ρ(r) in the BCP and RCP of 0.0135 and 0.0092 a.u., respectively, and a DI value of 0.045 a.u. In addition, the conformer H_closed forms an additional ring structure for the interactions between C₁₇H₄₅−O₂₄ and C₂₀H₅₃−O₂₄ with the value of ρ(r) = 0.0013 a.u. in the RCP. On the other hand, the DI values between atoms C5 = C6, C8 = C9, C11 = C12, and C14 = C15 in the Alkyl-M region are 1.77 a.u., corroborating the cis double bonds formation in the three closed conformers. Figure 2 and Supplementary Figure S2 show the molecular graphs of the anandamide conformers. The purple dots represent the bond critical points (BCP) and the green points represent the ring critical points (RCP).

Molecular Docking Analysis

Given the importance of mixed T-type/cannabinoid blockage in the pain diseases context, in this molecular docking analysis, and as a comparison, we consider the five proposed sites of the complex formed by the Ca_v3.2 channel and other dual blockers, the NMP compounds. The sites named S6DI, S6DII, S6DIII, S5DIV, and pore-blocking (Figure 3), are located in the transmembrane region, where aromatic and hydrophobic amino acids are prevalent (Rangel-Galván et al., 2022b). In this work, were analyzed the conformers E_closed, U_closed, and H_closed in interaction with the Ca_v3.2 calcium channel. In addition, the conformers E_open, U_open, and H_open were included to confirm our previous observations that the closed ring structures are more stable than their open counterparts (Rangel-Galván et al., 2022a) and probably more like to participate in the interaction with biological macromolecules, specifically with the Ca_v3.2 calcium channel. Figure 3 shows the anandamide conformers as ligands and the Ca_v3.2 channel as the receptor, showing the main interacting segments.

FIGURE 3

FIGURE 3. Molecular structures. (A) Anandamide conformers E_closed, U_closed, and H_closed, and (B) Ca_v3.2 channel showing the main interacting segments.

In the S5DIV site (Figure 4), the U_closed and U_open conformers place their polar head groups into the S4DIII segment, where the polar interactions are formed with the hydrogen N23−H⋯O of U_closed and the carbonyl oxygen of L806 (backbone), and the hydrogen O22−H⋯O of U_open and the carbonyl oxygen of R807 (both residues are located at 2.6 Å from the ligands). Also, is observed an unfavorable acceptor-acceptor interaction with O21 of U_closed and U_open conformers and the side chain carbonyl oxygen of D767 residue (Figure 4). The U_closed conformer forms an additional hydrogen bond, formed by hydrogen O22−H⋯O and the side chain carbonyl oxygen of acidic residue D767, however, this interaction does not contribute to the affinity of the ligand for the calcium channel with interaction energy of −9.2 kcal mol⁻¹ for both conformers. The non-polar portion of both conformers (U_closed and U_open) is placed in the S5DIV segment where the hydrophobic residues are located forming alkyl–alkyl interactions with the L815, L1151, L1154, and V1249 residues. In the X-ray structures of the TRPV5, TRPV2, and TRPV1 channels, which are close members of the T-type calcium channels, in complex with econazole (Hughes et al., 2018), resiniferatoxin (Zubcevic et al., 2018), and capsazepine (Gao et al., 2016), respectively, we observed ligand binding sites located in equivalent positions to that defined as the segment S5DIV in the Ca_v3.2.

FIGURE 4

FIGURE 4. Molecular interactions of U_open (cyan) and U_closed (yellow) anandamide conformers with Ca_v3.2. (A) 3D perspective of S5DIV site, U_closed and U_open conformers forming two hydrogen bonds with residues L806 and R807, U_closed additionally forms a hydrogen bond with D767 (B) 2D perspective interaction for each anandamide conformer in the S5DIV site.

As for the pore-blocking site, we identified a common hydrophobic region formed by the residues V1251, V1254, M948, F949, V945, L946, I331, and M330, where the non-polar tail (Alkyl-F) of the anandamide conformer U_open and the pentyl group of the NMP-181 compound established alkyl-alkyl interactions. In these molecular poses, the carbonyl oxygen of S900 and T568 residues in Ca_v3.2, form a hydrogen bond with the polar head (C2’−H⋯O) of the conformer U_open of anandamide and the amine group (C7−H⋯O) of the NMP-181, respectively. The conformer U_closed, in the pore-blocking site, is located very similar to the conformer U_open (Figure 5) but the EA group is oriented towards the segment P of the DI forming a polar interaction (C−H⋯O22) with O22 of anandamide and the carbon of G1206. In this context, it can be observed an equivalence of the interaction modes of the functional groups of anandamide and the NMP ligands (Rangel-Galván et al., 2022b) with the channel Ca_v3.2. Electrophysiological experimental registers show a blocking preference of the inactivation phase of the Ca_v3.2 channel by both anandamide (Chemin et al., 2001) and the NMP compounds (You et al., 2011; Gadotti et al., 2013; Bladen et al., 2015) was the MFV residues, located in the site S6-DIII, contribute to the inactivation phase of the Ca_v3 channels (Marksteiner et al., 2001). In the case of the Ca_v3.2, the corresponding residues are M948, F949, and V950 which are located in the pore-blocking site. Also, in a recent study, the Ca_v3.1/Z944 complex was solved by cryo-EM (Zhao et al., 2019), where common hydrophobic interacting residues stabilize the ligand binding pose, including the residue T921 (which is equivalent to T586 of the Ca_v3.2 located in the pore-blocking site). This ligand binding site was proposed also for the genistein/Ca_v3.3 complex using a combination of in vitro and in silico technics for the study (Rangel-Galván et al., 2021). Another recent study mapping the binding site of the Ca_v3.1 channel with a small cyclic peptide PnCs1 using docking and molecular dynamics showed a pocket located mainly in the pore region and including some fenestration (Depuydt et al., 2021). Based on the aforementioned experimental evidence, it is possible that the most probable pose for this type of ligand is located in the pore-blocking site.

FIGURE 5

FIGURE 5. Molecular interactions of U_closed, U_open (green), and NMP-181 (orange) with Ca_v3.2. (A) 3D perspective of U_open and NMP-181 at the pore-blocking site in the Ca_v3.2 channel; (B) 2D perspective interaction for U_closed and U_open anandamide conformer in the pore-blocking site.

In the pose S6DII, the E_closed and E_open anandamide conformers (Figure 6A) form a π-σ interaction with residue F161 while the Alkyl-F region forms an alkyl interaction with residues M330 and L333. The H_closed and H_open conformers form a π−σ interaction with the residues F316 and F1197 in the S6DI site (Figure 6B) and with the F943 in the S6DIII site (Figure 6C).

FIGURE 6

FIGURE 6. Important residues interaction of the Ca_v3.2 channel and the anandamide conformers: (A) S6DII site and the E_closed and E_open conformer, (B) S6DI site and the H_closed and H_open conformers, (C) S6DIII site and the H_closed and H_open anandamide conformers with π interactions mainly with the F943 residue.

The energy values are summarized in Table 4. According to this analysis, the higher interaction energy in the Ca_v3.2/anandamide complex corresponds to the E_closed conformer in the S6DI site. Nevertheless, due to anandamide flexibility is more probable for anandamide to prefer folded conformers. Indeed, the X-ray data of anandamide interacting with its protein transporter (FABP5) show a hairpin shape conformer (Sanson et al., 2014) and according to MD simulations, anandamide prefers curved shapes in the pocket binding FAAH interaction (Palermo et al., 2013).

TABLE 4

TABLE 4. Interaction energies ΔG (kcal mol⁻¹) of the conformers of anandamide with the channel Ca_v3.2.

It is observed that there is a negligible difference in the values for the interaction energy related to the formation of a seven-atom ring in the EA group (open versus closed conformations). Only in the S6DI site, a preference for the closed ring conformers is obtained with a difference not greater than 0.7 kcal mol⁻¹. In addition, our results indicate that better interaction energy for the U-shape conformers (U_closed/U_open) with respect to the hairpin shape conformers (H_closed/H_open) is observed. Along these lines, the drug Z944 is placed in a U-shape form in the binding pocket of the T-type calcium channel (Zhao et al., 2019).

Conclusion

Using conceptual DFT and QTAIM approaches with the BP86/cc-pVTZ level of theory, the structural stability and chemical reactivity properties of six preferred conformers of the endocannabinoid anandamide were analyzed. Our results indicate slight differences for U and H shapes relative to extended forms, in the global reactivity descriptors, concluding an increase in hardness, and a decrease in softness and the electrophilicity index. From these parameters, a decrease in the electrophilicity index was observed in the case of the open ring conformers with respect to their corresponding closed ring counterparts. Also, the hardness and the electrophilicity indexes change when the values for anandamide are compared with those of the NMP compounds. According to the local reactivity descriptors (Fukui and Parr functions), the most probable locations for electrophilic and nucleophilic attacks are located in the ethanolamide group and in the Alkyl-M region for all the anandamide conformers considered in this study. Molecular docking analysis showed important π-σ interactions of anandamide with the side chain of phenylalanine residues. From our results, we observed that, despite their marked structural differences, the NMP compounds (NMP-4, NMP-7, and NMP-181) and anandamide may share a similar physiological activity profile regarding ligand/receptor interactions. The carbazole group is an important region of electronic density accumulation for the NMP compounds in the same way that the Alkyl-M region is for anandamide. According to the literature and the results obtained, the pore-blocking site of the Ca_v3.2 calcium channel could be a probable binding site for the anandamide molecule.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors.

Author Contributions

Conceptualization and design, MC and FM; methodology, MR-G, MC, FM, and JP-A; software, MC, JP-A, NC, and FM; validation, MR-G, and NC; writing the original draft preparation, MR-G, MC, and FM; writing, reviewing and editing, JP-A and NC. All authors have read and agreed to the published version of the manuscript.

Funding

Vicerrectoría de Investigación y Estudios de Posgrado (VIEP-BUAP, Mexico) (VIEP2022 project), and the PRODEP Academic Group BUAP-CA-263 (SEP, Mexico).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

MR-G thanks CONACYT-México for financial support [Ph.D. fellowship No. 286497]. Authors thank the Laboratorio Nacional de Supercómputo del Sureste de México (LNS-BUAP) of the CONACYT network of national laboratories, for the computer resources and support provided and the PRODEP Academic Group BUAP-CA-263 (SEP, Mexico).

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2022.920661/full#supplementary-material

References

Aliste, M. P. (2000). Theoretical Study of Dopamine. Application of the HSAB Principle to the Study of Drug-Receptor Interactions. J. Mol. Struct. THEOCHEM 507, 1–10. doi:10.1016/S0166-1280(99)00253-5

CrossRef Full Text | Google Scholar

Bayat, A., and Fattahi, A. (2019). Influence of Remote Intramolecular Hydrogen Bonding on the Acidity of Hydroxy‐1,4‐Benzoquinonederivatives: A DFT Study. J. Phys. Org. Chem. 32, e3919–10. doi:10.1002/poc.3919

CrossRef Full Text | Google Scholar

Becke, A. D. (1988). Density-functional Exchange-Energy Approximation with Correct Asymptotic Behavior. Phys. Rev. A 38, 3098–3100. doi:10.1103/PhysRevA.38.3098

PubMed Abstract | CrossRef Full Text | Google Scholar

Bladen, C., McDaniel, S. W., Gadotti, V. M., Petrov, R. R., Berger, N. D., Diaz, P., et al. (2015). Characterization of Novel Cannabinoid Based T-type Calcium Channel Blockers with Analgesic Effects. ACS Chem. Neurosci. 6, 277–287. doi:10.1021/cn500206a

PubMed Abstract | CrossRef Full Text | Google Scholar

Chamorro, E., Pérez, P., and Domingo, L. R. (2013). On the Nature of Parr Functions to Predict the Most Reactive Sites along Organic Polar Reactions. Chem. Phys. Lett. 582, 141–143. doi:10.1016/j.cplett.2013.07.020

CrossRef Full Text | Google Scholar

Chemin, J., Monteil, A., Perez-Reyes, E., Nargeot, J., and Lory, P. (2001). Direct Inhibition of T-type Calcium Channels by the Endogenous Cannabinoid Anandamide. EMBO J. 20, 7033–7040. doi:10.1093/emboj/20.24.7033

PubMed Abstract | CrossRef Full Text | Google Scholar

Chemin, J., Nargeot, J., and Lory, P. (2007). Chemical Determinants Involved in Anandamide-Induced Inhibition of T-type Calcium Channels. J. Biol. Chem. 282, 2314–2323. doi:10.1074/jbc.M610033200

PubMed Abstract | CrossRef Full Text | Google Scholar

Chemin, J., Cazade, M., and Lory, P. (2014). Modulation of T-type Calcium Channels by Bioactive Lipids. Pflugers Arch. - Eur. J. Physiol. 466, 689–700. doi:10.1007/s00424-014-1467-5

PubMed Abstract | CrossRef Full Text | Google Scholar

Colino, L., Herranz-Herrer, J., Gil-Benito, E., Ponte-Lopez, T., del Sol-Calderon, P., Rodrigo-Yanguas, M., et al. (2018). Cannabinoid Receptors, Mental Pain and Suicidal Behavior: a Systematic Review. Curr. Psychiatry Rep. 20, 1–9. doi:10.1007/s11920-018-0880-4

PubMed Abstract | CrossRef Full Text | Google Scholar

Cremer, D., and Kraka, E. (1984). Chemical Bonds without Bonding Electron Density ? Does the Difference Electron-Density Analysis Suffice for a Description of the Chemical Bond? Angew. Chem. Int. Ed. Engl. 23, 627–628. doi:10.1002/anie.198406271

CrossRef Full Text | Google Scholar

De Proft, F., Vivas‐Reyes, R., Biesemans, M., Willem, R., Martin, J. M. L., and Geerlings, P. (2003). Density Functional Study of the Complexation Reaction of Sn(CH 3 ) 3 X (X = F, Cl, Br and I) with Halide Anions. Eur. J. Inorg. Chem. 2003, 3803–3810. doi:10.1002/ejic.200300044

CrossRef Full Text | Google Scholar

Depuydt, A.-S., Rihon, J., Cheneval, O., Vanmeert, M., Schroeder, C. I., Craik, D. J., et al. (2021). Cyclic Peptides as T-type Calcium Channel Blockers: Characterization and Molecular Mapping of the Binding Site. ACS Pharmacol. Transl. Sci. 4, 1379–1389. doi:10.1021/acsptsci.1c00079

PubMed Abstract | CrossRef Full Text | Google Scholar

Devane, W. A., and Axelrod, J. (1994). Enzymatic Synthesis of Anandamide, an Endogenous Ligand for the Cannabinoid Receptor, by Brain Membranes. Proc. Natl. Acad. Sci. U.S.A. 91, 6698–6701. doi:10.1073/pnas.91.14.6698

PubMed Abstract | CrossRef Full Text | Google Scholar

Domingo, L. R., Pérez, P., and Sáez, J. A. (2013). Understanding the Local Reactivity in Polar Organic Reactions through Electrophilic and Nucleophilic Parr Functions. RSC Adv. 3, 1486–1494. doi:10.1039/c2ra22886f

CrossRef Full Text | Google Scholar

Domingo, L., Ríos-Gutiérrez, M., and Pérez, P. (2016). Applications of the Conceptual Density Functional Theory Indices to Organic Chemistry Reactivity. Molecules 748, 1–22. doi:10.3390/molecules21060748

PubMed Abstract | CrossRef Full Text | Google Scholar

Dunning, T. H. (1989). Gaussian Basis Sets for Use in Correlated Molecular Calculations. I. The Atoms Boron through Neon and Hydrogen. J. Chem. Phys. 90, 1007–1023. doi:10.1063/1.456153

CrossRef Full Text | Google Scholar

Ferreri, C., Anagnostopoulos, D., Lykakis, I. N., Chatgilialoglu, C., and Siafaka-Kapadai, A. (2008). Synthesis of All-Trans Anandamide: A Substrate for Fatty Acid Amide Hydrolase with Dual Effects on Rabbit Platelet Activation. Bioorg. Med. Chem. 16, 8359–8365. doi:10.1016/j.bmc.2008.08.054

PubMed Abstract | CrossRef Full Text | Google Scholar

Frau, J., and Glossman-Mitnik, D. (2017). Conceptual DFT Descriptors of Amino Acids with Potential Corrosion Inhibition Properties Calculated with the Latest Minnesota Density Functionals. Front. Chem. 5, 1–8. doi:10.3389/fchem.2017.00016

PubMed Abstract | CrossRef Full Text | Google Scholar

Frau, J., and Glossman-Mitnik, D. (2018). Conceptual DFT Study of the Local Chemical Reactivity of the Colored BISARG Melanoidin and its Protonated Derivative. Front. Chem. 6, 1–9. doi:10.3389/fchem.2018.00136

PubMed Abstract | CrossRef Full Text | Google Scholar

Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., CheesemanScalmani, J. R. G., et al. (2016). Gaussian 16, Revision B.O1. Wallingford: Gaussian, Inc.

Google Scholar

Gadotti, V. M., You, H., Petrov, R. R., Berger, N. D., Diaz, P., and Zamponi, G. W. (2013). Analgesic Effect of a Mixed T-type Channel inhibitor/CB2 Receptor Agonist. Mol. Pain 9, 1744–8069. doi:10.1186/1744-8069-9-32

PubMed Abstract | CrossRef Full Text | Google Scholar

Gao, Y., Cao, E., Julius, D., and Cheng, Y. (2016). TRPV1 Structures in Nanodiscs Reveal Mechanisms of Ligand and Lipid Action. Nature 534, 347–351. doi:10.1038/nature17964

PubMed Abstract | CrossRef Full Text | Google Scholar

Hernández-Paredes, J., Carrillo-Torres, R. C., Hernández-Negrete, O., Sotelo-Mundo, R. R., Glossman-Mitnik, D., Esparza-Ponce, H. E., et al. (2017). Experimental and Theoretical Study on the Molecular Structure, Covalent and Non-covalent Interactions of 2,4-dinitrodiphenylamine: X-Ray Diffraction and QTAIM Approach. J. Mol. Struct. 1141, 53–63. doi:10.1016/j.molstruc.2017.03.087

CrossRef Full Text | Google Scholar

Hua, T., Vemuri, K., Pu, M., Qu, L., Han, G. W., Wu, Y., et al. (2016). Crystal Structure of the Human Cannabinoid Receptor CB1. Cell. 167, 750–762. doi:10.1016/j.cell.2016.10.004

PubMed Abstract | CrossRef Full Text | Google Scholar

Hughes, T. E. T., Pumroy, R. A., Yazici, A. T., Kasimova, M. A., Fluck, E. C., Huynh, K. W., et al. (2018). Structural Insights on TRPV5 Gating by Endogenous Modulators. Nat. Commun. 9, 1–11. doi:10.1038/s41467-018-06753-6

PubMed Abstract | CrossRef Full Text | Google Scholar

Lu, T., and Chen, F. (2012). Multiwfn: A Multifunctional Wavefunction Analyzer. J. Comput. Chem. 33, 580–592. doi:10.1002/jcc.22885

PubMed Abstract | CrossRef Full Text | Google Scholar

Marksteiner, R., Schurr, P., Berjukow, S., Margreiter, E., Perez‐Reyes, E., and Hering, S. (2001). Inactivation Determinants in Segment IIIS6 of Ca V 3.1. J. Physiol. 537, 27–34. doi:10.1111/j.1469-7793.2001.0027k.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Matta, C. F., and Boyd, R. J. (2007). An Introduction to the Quantum Theory of Atoms in Molecules: From Solid State to DNA and Drug Design. 1st ed. Bonn, Germany, Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA. doi:10.1002/9783527610709.ch1

CrossRef Full Text | Google Scholar

McAllister, S. D., Rizvi, G., Anavi-Goffer, S., Hurst, D. P., Barnett-Norris, J., Lynch, D. L., et al. (2003). An Aromatic Microdomain at the Cannabinoid CB1 Receptor Constitutes an Agonist/Inverse Agonist Binding Region. J. Med. Chem. 46, 5139–5152. doi:10.1021/jm0302647

PubMed Abstract | CrossRef Full Text | Google Scholar

Morell, C., Grand, A., and Toro-Labbé, A. (2005). New Dual Descriptor for Chemical Reactivity. J. Phys. Chem. A 109, 205–212. doi:10.1021/jp046577a

PubMed Abstract | CrossRef Full Text | Google Scholar

Oz, M. (2006). Receptor-independent Actions of Cannabinoids on Cell Membranes: Focus on Endocannabinoids. Pharmacol. Ther. 111, 114–144. doi:10.1016/j.pharmthera.2005.09.009

PubMed Abstract | CrossRef Full Text | Google Scholar

Pacher, P., Bátkai, S., and Kunos, G. (2006). The Endocannabinoid System as an Emerging Target of Pharmacotherapy. Pharmacol. Rev. 58, 389–462. doi:10.1124/pr.58.3.2

PubMed Abstract | CrossRef Full Text | Google Scholar

Palermo, G., Campomanes, P., Neri, M., Piomelli, D., Cavalli, A., Rothlisberger, U., et al. (2013). Wagging the Tail: Essential Role of Substrate Flexibility in FAAH Catalysis. J. Chem. Theory Comput. 9, 1202–1213. doi:10.1021/ct300611q

PubMed Abstract | CrossRef Full Text | Google Scholar

Parthasarathi, R., Subramanian, V., Roy, D. R., and Chattaraj, P. K. (2004). Electrophilicity Index as a Possible Descriptor of Biological Activity. Bioorg. Med. Chem. 12, 5533–5543. doi:10.1016/j.bmc.2004.08.013

PubMed Abstract | CrossRef Full Text | Google Scholar

Popelier, P. L. (2000). Atoms in Molecules: An Introduction. 1st ed. Harlow, Great Britain: Prentice Hall College Div.

Google Scholar

Rangel-Galván, M., Rangel, A., Romero-Méndez, C., Dávila, E. M., Castro, M. E., Caballero, N. A., et al. (2021). Inhibitory Mechanism of the Isoflavone Derivative Genistein in the Human Cav3.3 Channel. ACS Chem. Neurosci. 12, 651–659. doi:10.1021/acschemneuro.0c00684

PubMed Abstract | CrossRef Full Text | Google Scholar

Rangel-Galván, M., Castro, M. E., Caballero, N. A., Perez-Aguilar, J. M., and Melendez, F. J. (2022a). NMR and IR Spectroscopic Analysis of the Preferred Conformations of Neurotransmitter Anandamide. [Non-Published Work].

Google Scholar

Rangel-Galván, M., Castro, M. E., Perez-Aguilar, J. M., Caballero, N. A., Rangel-Huerta, A., and Melendez, F. J. (2022b). Theoretical Study of the Structural Stability, Chemical Reactivity, and Protein Interaction for NMP Compounds as Modulators of the Endocannabinoid System. Molecules 27, 414–416. doi:10.3390/molecules27020414

CrossRef Full Text | Google Scholar

Sanner, M. F. (1999). Python: A Programming Language for Software Integration and Development. J. Mol. Graph. Model. 17, 57–61. Available at: https://www.academia.edu/download/25505223/10.1.1.35.6459.pdf.

PubMed Abstract | Google Scholar

Sanson, B., Wang, T., Sun, J., Wang, L., Kaczocha, M., Ojima, I., et al. (2014). Crystallographic Study of FABP5 as an Intracellular Endocannabinoid Transporter. Acta Cryst. D. Biol. Crystallogr. 70, 290–298. doi:10.1107/S1399004713026795

PubMed Abstract | CrossRef Full Text | Google Scholar

Schrödinger, L. (2017). The PyMOL Molecular Graphics System; Version 2.0. Palo Alto, CA, USA: DeLano Sci. LCC.

Google Scholar

Snutch, T. P., and Zamponi, G. W. (2018). Recent Advances in the Development of T-type Calcium Channel Blockers for Pain Intervention. Br. J. Pharmacol. 175, 2375–2383. doi:10.1111/bph.13906

PubMed Abstract | CrossRef Full Text | Google Scholar

Sugiura, T., Kobayashi, Y., Oka, S., and Waku, K. (2002). Biosynthesis and Degradation of Anandamide and 2-arachidonoylglycerol and Their Possible Physiological Significance. Prostagl. Leukot. Essent. Fat. Acids (PLEFA) 66, 173–192. doi:10.1054/plef.2001.0356

PubMed Abstract | CrossRef Full Text | Google Scholar

Tibbs, G. R., Posson, D. J., and Goldstein, P. A. (2016). Voltage-Gated Ion Channels in the PNS: Novel Therapies for Neuropathic Pain? Trends Pharmacol. Sci. 37, 522–542. doi:10.1016/j.tips.2016.05.002

PubMed Abstract | CrossRef Full Text | Google Scholar

Todorovic, S. M., and Jevtovic-Todorovic, V. (2013). Neuropathic Pain: Role for Presynaptic T-type Channels in Nociceptive Signaling. Pflugers Arch. - Eur. J. Physiol. 465, 921–927. doi:10.1007/s00424-012-1211-y

PubMed Abstract | CrossRef Full Text | Google Scholar

Tood, A., K. (2017). AIMAll (Version 17.11.14). TK Gristmill Software, Overland Park KS, USA (aim.tkgristmill.com). Available at: aim.tkgristmill.com (Accessed April 10, 2022).

Google Scholar

Trott, O., and Olson, A. J. (2010). AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. J. Comput. Chem. 31, 455–461. doi:10.1002/jcc.21334.AutoDock

PubMed Abstract | CrossRef Full Text | Google Scholar

Van Der Stelt, M., and Di Marzo, V. (2005). Anandamide as an Intracellular Messenger Regulating Ion Channel Activity. Prostagl. Other Lipid Mediat. 77, 111–122. doi:10.1016/j.prostaglandins.2004.09.007

CrossRef Full Text | Google Scholar

Vivas-Reyes, R., De Proft, F., Biesemans, M., Willem, R., and Geerlings, P. (2003). A DFT Study of Tin‐ and Crown‐Ether‐Based Host Molecules Capable of Binding Anions and Cations Simultaneously. Eur. J. Inorg. Chem. 2003, 1315–1324. doi:10.1002/ejic.200390171

CrossRef Full Text | Google Scholar

Vivas-Reyes, R., Mercado, L. D., Anaya-Gil, J., Marrugo, A. G., and Martinez, E. (2008). Theoretical Study to Evaluate Polyfuran Electrical Conductivity and Methylamine, Methoxy Substituent Effects. J. Mol. Struct. THEOCHEM 861, 137–141. doi:10.1016/j.theochem.2008.04.019

CrossRef Full Text | Google Scholar

Yang, W., and Mortier, W. J. (1986). The Use of Global and Local Molecular Parameters for the Analysis of the Gas-Phase Basicity of Amines. J. Am. Chem. Soc. 108, 5708–5711. doi:10.1021/ja00279a008

PubMed Abstract | CrossRef Full Text | Google Scholar

Yang, Y., Cui, Y., Sang, K., Dong, Y., Ni, Z., Ma, S., et al. (2018). Ketamine Blocks Bursting in the Lateral Habenula to Rapidly Relieve Depression. Nature 554, 317–322. doi:10.1038/nature25509

PubMed Abstract | CrossRef Full Text | Google Scholar

You, H., Gadotti, V. M., Petrov, R. R., Zamponi, G. W., and Diaz, P. (2011). Functional Characterization and Analgesic Effects of Mixed Cannabinoid receptor/T-type Channel Ligands. Mol. Pain 7, 1744–8069. doi:10.1186/1744-8069-7-89

PubMed Abstract | CrossRef Full Text | Google Scholar

Zghibeh, C. M., Raj Gopal, V., Poff, C. D., Falck, J. R., and Balazy, M. (2004). Determination of Trans-arachidonic Acid Isomers in Human Blood Plasma. Anal. Biochem. 332, 137–144. doi:10.1016/j.ab.2004.04.030

PubMed Abstract | CrossRef Full Text | Google Scholar

Zhao, Y., Huang, G., Wu, Q., Wu, K., Li, R., Lei, J., et al. (2019). Cryo-EM Structures of Apo and Antagonist-Bound Human Cav3.1. Nature 576, 492–497. doi:10.1038/s41586-019-1801-3

PubMed Abstract | CrossRef Full Text | Google Scholar

Zubcevic, L., Le, S., Yang, H., and Lee, S.-Y. (2018). Conformational Plasticity in the Selectivity Filter of the TRPV2 Ion Channel. Nat. Struct. Mol. Biol. 25, 405–415. doi:10.1038/s41594-018-0059-z

PubMed Abstract | CrossRef Full Text | Google Scholar