Cannabidiol interactions in the voltage-gated calcium channel by molecular docking: role its neuronal inhibitory mechanism Interações do canabidiol no canal de cálcio dependente de voltagem por docking molecular: papel no seu mecanismo inibitório neuronal

Objective: to analyzer the interactions of cannabidiol in the Ca V 3.2 through molecular docking. Methodology: this is a research with in silico approach, which CBD and gabapentin (GBP) were employed as test substances, and Ca V 3.2 channel the target protein. Molecular docking experiments were realized by Dockthor. The drugs simulations were classified in order of highest affinity in the channel. The binding energy scores were linked using Student t-test by GraphPad Prism software, the values were significantly different when p < 0.05. Results: the spatial positions into CBD or GBP and Ca V 3.2 were 1.000,000 conformers. Our data showed that the binding energies of Ca V 3.2 channel and CBD or GBP were - 6.493 ± 0.07 and - 6.842 ± 0.19 kcal/mol, respectively. Those values did not show statistically difference (p = 0.08), suggesting that both drugs bind similarly the Ca V 3.2, however both chemicals connected the distinct sites. Conclusions: CBD binds to Ca V 3.2, which corroborates its blockade channel. Those data support the analgesic effect of CBD through the neuronal inhibitory pathway.


INTRODUCTION
Pain is defined as an uncomfortable emotional and sensitive experience, associated or similar to potential or real tissue lesion. (1)(2) The patient is affected by pain consequences, because health activities, social life and workday are limited. (3) Despite high prevalence of disorder related to pain, it effective handling is a challenge. (4) A putative analgesic is the cannabidiol (CBD), a phytocannabinoid presents at Cannabis sativa. CBD acts without psychoactive and is not cognitive depressive, which promotes drug safe. (5,6) CBD have been applied in many diseases that involves membrane excitability, therefore its targets including channels, such as voltage-gated sodium (Na V ) channels, voltage-gated potassium (K v ) channels, voltage-gated calcium (Ca V ) channels, and transient receptor potential (TRP) channels. (7)(8)(9)(10)(11) The low voltage-gated calcium channel family is important in the peripheral processing of nociceptive signals since they play a crucial role in controlling neuronal excitability. (12) Three subtypes of Ca V channels have been identified, namely Ca V 3.1, Ca V 3.2, and Ca V 3.3. Gene regional distribution analysis demonstrated that the Ca V 3.2 subtype is predominantly found at sites essential for pain transmissions. (13,14) Blockers of Cav3.2 are putative candidates to treatment of chronic and acute pain. (15)(16)(17)(18)(19) CBD was able to abolish fully conductance via Ca V 3.1, 3.2 and 3.3 T-type channels using patch clamp electrophysiology, although mechanism molecular details of CBD on Ca V has not been elucidate yet. (9) Therefore, molecular docking insights understand the function-structure relation in a pharmacological target and its ligand-protein binding. (20) Cannabidiol interactions in the voltage-gated calcium channel by molecular docking Objetivo: analisar as interações do canabidiol (CBD) no Ca V 3.2 através de docking molecular. Metodologia: trata-se de uma pesquisa do tipo in silico, que o CBD e a gabapentina (GBP) foram utilizadas como substâncias teste e o canal Ca V 3.2 como proteína alvo. Os experimentos de docking molecular foram realizados no Dockthor. As simulações dos fármacos foram classificadas em ordem de maior afinidade no canal. As energias de ligação foram comparadas usando o teste "t" no programa GraphPad Prism, os valores foram significantemente diferentes quando p < 0,05. Resultados: as posições entre CBD e GBP foram 1.000,00 conformações. Os dados mostraram que as energias de ligação no Ca V 3.2 e CBD ou GBP foram -6,493 ± 0,07 kcal/mol e -6,842 ± 0,19 kcal/mol, respectivamente. Esses valores não apresentaram diferença estatística significante (p = 0,08), mostrando que ambos têm afinidade similar no canal, apesar de posicionamentos distintos. GBP is a synthetic analogue of the neurotransmitter gamma-aminobutyric acid with anticonvulsant activity, a blockade neuronal Ca V and has also become popular alternatives to opioids for pain and are widely recommended as first-line agents for the treatment of neuropathic pain. (21,22) GBP presents molecular formula C 9 H 17 NO 2 and molecular weigth 171.24 g/mol, the CBD has C 21 H 30 O 2 and 314.5 g/mol. (23) This study aimed to analyzer the CBD interactions in the Ca V 3.2 channel by molecular docking insights, and to compare the GBP.

RESULTS AND DISCUSSION
The spatial positions into CBD or GBP and Ca V 3.2 were 1,000,000 conformers. The top three were selected, and the binding energy of each drug and the channel was calculated and shown in Table 1. It is generally determined that the molecular docking is more stable if presents lower biding energy. (25) Our data showed that the binding energies of Ca V 3.2 channel and CBD or GBP were -6.493 ± 0.07 and -6.842 ± 0.19 kcal/mol, respectively (Table 1). Those values did not show statistically difference, suggesting that both drugs bind similarly the Ca V 3.2. In fact, the energy scores are < − 5 kcal/mol, it determines the spontaneous biding into ligand and receptor. (26) Arruda GEJ, Silva Júnior GJ, Mendes NN, Mendes GN, Fernandes LCL, Costa AEA, et al. Table 1. Docking binding energy of CBD and GNP in the CaV3.2.

Ligand
Affinity (kcal/mol) CBD -6.493 ± 0.07 GBP -6.842 ± 0.19 Being: p = 0.08 (CBD vs. GBP). * CBD: cannabidiol; GBP: gabapentin The best CBD`s and GBP's position were presented in Fig. 1, which may be correspond to blockade channel (Fig. 1B). Visualizing the 3D structures, both chemicals connected the distinct sites in Ca V 3.2, however they exhibited same binding energy (Table 1). In similar manner, CBD and carbamazepine showed same energy on Na V 1.7. (27) Others phytocannabinoids, as cannabigerolic acid and cannabidivarin, have been shown inhibit Ca V 3.2 using whole-cell patch clamp recordings. (28) Cannabidiol interactions in the voltage-gated calcium channel by molecular docking The helices of CaV3.2 and CBD binding sites are presented in Figure 2A. Specifically, the CDB makes H-bond with ASP690, ASP421 and LYS423 residue (Fig. 2B), whose the distance were 2.38, 1.42 and 1.88 Å, respectively. However, GBP did not present H-bond with residue (data not shown), suggesting it has less stability than CBD in CaV3.2. The CBD binding site and others channels have been different, such as NaV1.7 involves the THR180 and a bacterial NaV channel the M175 residue. (10,27)   GBP acts by blockade neuronal CaV and is a recommended first-line agent for treating neuropathic pain, despite its efficacy rate is reportedly low, and the risk of adverse events is high. (29,30) On the other hands, our data shown that CBD mechanism in CaV3.2 may be an analgesic alternative. The binding are capable of blocking the entry of calcium into the neuronal terminal, which prevents neurotransmitter exocytosis, and thus, communication for the conduction of the painful stimulus.

CONCLUSIONS
The cannabidiol interacts CaV3.2 channel residues, corroborating its blockade, while gabapentin interacts with others residues. Those findings reinforce neuronal inhibition promoted by cannabidiol that may be an alternative drug to treat neuropathic pain. It can be used as a reference for future research.

CONFLICT OF INTERESTS
Not applicable