Abstract
Objective
The present study aimed to investigate five triazole compounds as P2X7R inhibitors and evaluate their ability to reduce acute inflammation in vivo.
Material
The synthetic compounds were labeled 5e, 8h, 9i, 11, and 12.
Treatment
We administered 500 ng/kg triazole analogs in vivo, (1–10 µM) in vitro, and 1000 mg/kg for toxicological assays.
Methods
For this, we used in vitro experiments, such as platelet aggregation, in vivo experiments of paw edema and peritonitis in mice, and in silico experiments.
Results
The tested substances 5e, 8h, 9i, 11, and 12 produced a significant reduction in paw edema. Molecules 5e, 8h, 9i, 11, and 12 inhibited carrageenan-induced peritonitis. Substances 5e, 8h, 9i, 11, and 12 showed an anticoagulant effect, and 5e at a concentration of 10 µM acted as a procoagulant. All derivatives, except for 11, had pharmacokinetic, physicochemical, and toxicological properties suitable for substances that are candidates for new drugs. In addition, the ADMET risk assessment shows that derivatives 8h, 11, 5e, and 9i have high pharmacological potential. Finally, docking tests indicated that the derivatives have binding energies comparable to the reference antagonist with a competitive inhibition profile.
Conclusions
Together, the results indicate that the molecules tested as antagonist drugs of P2X7R had anti-inflammatory action against the acute inflammatory response.
Supplementary Information
The online version contains supplementary material available at 10.1007/s00011-022-01664-1.
Keywords: P2X7 receptor, Antagonists, Paw edema, Synthetic substances, Triazoles
Introduction
Inflammation is a physiological response that plays an important role in the maintenance of tissue homeostasis [1]. The inflammatory response is triggered by the detection of infection or tissue damage through the recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), respectively [2]. In sterile inflammation, endogenous molecules, such as ATP, uric acid, and some cytoplasmic and nuclear proteins released by damaged cells, are DAMPs [3]. These signaling molecules recognize a set of specific receptors triggering the inflammatory process by the activation of blood vessels, release of soluble mediators, and recruitment of leukocytes to the site of inflammation [4]. After the ceasing or elimination of the cause of inflammation, the reestablishment of tissue homeostasis initiates the resolution phase. However, whether the resolution of inflammation does not occur properly and whether the cause of inflammation is not eradicated by the acute inflammatory response, a chronic inflammatory process that can lead to tissue damage and dysfunction, has been established [5].
Extracellular ATP is now a well-established DAMP signal that exerts its inflammatory effects through activation of plasma membrane receptors expressed by immune cells, known as purinergic receptors [6]. In general, the action of ectonucleotidases keeps extracellular ATP at low concentrations [7]. However, several lines of evidence demonstrate the accumulation of extracellular ATP at sites of inflammation and infection [8]. Under these conditions, ATP can reach high concentrations sufficient to stimulate purinergic receptors, and trigger a series of proinflammatory responses [9]. Thus, these subtypes of receptors have been considered relevant pharmacological targets for the development of novel anti-inflammatory drugs to avoid or mitigate the side effects of traditional COX inhibition-based therapy. Among them, the P2X7 receptor (P2X7R) is the subtype most directly involved in inflammatory responses, regulating the release of proinflammatory cytokines, such as IL-1β [10].
P2X7R is an ATP-gated ion channel that is preferentially permeable to mono- and divalent cations [11]. This receptor has pharmacological characteristics that differentiate it from other purinergic receptors in the P2X family. Its activation requires ATP concentrations of approximately 100 µM (EC50 ≥ 100 µM) [12–14], which are higher than those observed to activate other family subtypes. In addition, P2X7R treatment with high agonist concentrations or sustained stimulation evokes the formation of a high-conductance nonselective pore, which allows the passage of molecules of up to 900 Da across the cell plasma membrane. These cell modifications result in disruptions in cell ionic homeostasis [15–18]. This receptor also has another important and exclusive characteristic, which is the ability to initiate the release of intracellular ATP on a large scale that is associated with the intrinsic ability to form pores or due to the association with hemichannels of pannexins, promoting a positive-feedback loop in purinergic signaling and an increase in inflammation [13, 19].
P2X7R activation in several cells induces the production and secretion of different inflammatory mediators, such as TNF-α, MPC-1 (monocyte chemoattractant protein-1), and IL-6, as well as the cleavage of metalloproteinases, CD23, selectin-1, CD27, and matrix [20–24] (induced by lipopolysaccharide (LPS) and elevates mRNA levels for iNOS (inducible nitric oxide synthase). In mast cells, it induces an increase in the expression of TNF-α, IL-4, IL-6, and IL-13 [25–28]. Furthermore, P2X7R participates in the formation of the NLRP3 inflammasome, which is responsible for the maturation and release of IL-1β and IL-18 [21]. Since P2X7R is involved in several inflammatory dysfunctions and there is an urgent need for the development of new anti-inflammatory drugs with novel mechanisms of action, this receptor has become an attractive molecular target to circumvent the toxicity issues associated with traditional anti-inflammatory therapy.
A method for classifying the P2X7R antagonist is according to the binding site of the molecules into orthosteric ligands and allosteric ligands. The first category is composed of molecules that bind to the ATP-binding site, while the second category of ligands are defined by molecules that bind to the receptor in a different region, decreasing its endogenous ligand effectiveness [29]. To date, no selective agonists for this receptor have been described with effective therapeutic action [29]. Therefore, it is urgently necessary to search for new and selective human P2X7R antagonists. Previously, our research group assessed the antagonistic activity of a series of 1,2,3-triazole compounds and identified that some derivatives were able to potentially block the formation of the P2X7R pore in dye uptake assays performed in mammalian cells (J774. G8 cells and peritoneal macrophages). These molecules were also able to inhibit the release of IL-1β mediated by P2X7R. In addition, molecule 6c was able to decrease ATP-induced dye uptake, and molecule 9e partially inhibited intracellular dye uptake, giving them the potential to antagonize P2X7R. In addition, our previous molecular docking studies indicated the ATP-binding pocket as a potential binding site for the 1,2,3-triazole compounds [30]. We named the synthetic compounds with inhibitory activity on mP2X7R and hP2X7R antagonists 5e, 8h, 9i, 11, and 12. The choice criterion was the previous results demonstrating antagonistic in vitro action against P2X7R at nanomolar concentrations [30].
Methodology
In vivo studies
Male mice (Swiss Webster), weighing approximately 30.0 g, treated with a PURINA-LABINA balanced ration, water "ad libitum" and a light–dark cycle of 12 h were used in the paw edema assays. These tests are carried out in accordance with CEUA-FIOCRUZ with license number 043/18.
Single-dose toxicity
Triazole toxicity was evaluated by the in vivo test, according to [31], with modifications. All triazoles (1000 mg/kg) or saline solution was injected intraperitoneally (i.p.) into the abdominal region of the mice. Then, behavior and mortality were observed for 24 h.
Paw edema assay
For paw edema induction, the mice received a subplantar injection in one of the hind paws with ATP (10 mM/paw). A solution of 0.9% NaCl was applied to the contralateral paw. One hour before the application of the phlogistic agent, the following treatments were performed: G1 and G2—negative control groups (NaCl 0.9% intraperitoneal); G3—positive control group (Diclofenac 10 mg/kg/intraperitoneal); and G4, G5, G6—test groups (tested compounds at 100, 500, and 1000 ng/kg/intraperitoneal), respectively. After 60 min of preincubation with antagonists and 60 min after the application of ATP, the paw volume was evaluated with the aid of a plethysmometer device (UGO-BASILE). All substances administered to the hind paw were administered at a volume of 20 μL/paw, and all substances administered into the peritoneum were administered at a volume of 100 μL/intraperitoneal.
Peritonitis
For the peritonitis assay, the animals were treated intraperitoneally with 100 µL of the molecules tested at concentrations of 10 mg/kg (carrageenan), 10 mg/kg (dexamethasone), and 0.1 mg/kg (5e, 8h, 9i, 11, and 12). A one X solution of PBS (phosphate-buffered saline) at pH 7.4 (applied in a volume of 100 µL) and carrageenan were used as negative and positive controls. The groups that were pretreated before receiving the inflammation inducer received dexamethasone, 5e, 8h, 9i, 11, and 12 with pretreatment and 1 h later received carrageenan. Three hours after the first administration, intraperitoneal fluid was collected, and the differential and total leukocyte counts were determined. The total leukocyte count was performed using a Neubauer chamber. For the differential leukocyte count, we fixed the slide containing the microtube samples in methanol for 5 min and then stained it with Giemsa solution for 15–20 min. Differential counting was performed by counting 100 cells per slide, including mast cells, eosinophils, macrophages, lymphocytes, and neutrophils.
Platelet aggregation tests
Platelet aggregation was monitored turbidimetrically in a platelet aggregometer (Model 490 2D—Chrono-log Corporation, Pennsylvania, USA) according to [32], using platelet-rich plasma (PRP) obtained from healthy volunteer donors. Blood samples from healthy volunteer donors were collected through venipuncture using 3.8% w/v sodium citrate (citrate/blood 1:9) as an anticoagulant. The donors declared that they did not have any disorder related to hemostasis and that they did not use any medication that could affect the results. Blood was centrifuged at 1800 rpm for 12 min at room temperature to obtain platelet-rich plasma (PRP) in the supernatant. Platelet-poor plasma (PPP) was obtained by centrifuging the remaining blood at 2,500 rpm for 12 min. Assays were performed using 300 µL of PRP kept at 37 °C for 1 min in siliconized glass cuvettes under constant agitation. Then, platelet aggregation was initiated by the addition of the physiological agonist ADP (15 µM). The 100% platelet aggregation was obtained with the supramaximal platelet response to the addition of agonist after 6 min of reaction, and 0% platelet aggregation was determined by transmittance caused by PRP alone prior to the addition of agonist. Plasma with saline was used to adjust the apparatus (basal), and then, the treatments were realized. To evaluate the effect of the derivatives on platelet aggregation, they were preincubated with 300 µL of PRP for 5 min at 37 °C under constant agitation. Then, the agonist was added, and platelet aggregation was monitored. As a control, PRP was incubated with 0.15 M NaCl, and the compound was incubated in the absence of ADP in both cases. The same procedure was performed as described above. As a result, it was observed that compounds in the absence of ADP were not capable of inducing platelet aggregation (data not shown). Following a pattern of graphic demonstration present in the literature, a platelet aggregation of 100% was obtained as a response of PRP to the addition of the agonist, with nongraphic representation of the percentage of inhibition of platelet aggregation obtained by compounds [32].
Coagulation assays
All coagulation tests were performed in a Multichannel Coagulometer (Model KC4A micro—Amelung—Lemgo, Germany). The plasma was obtained from healthy volunteer donors who declared to not have any hemostasis or bleeding disorders and to not use any medication that could affect the results obtained in coagulation assays. Blood was collected by venipuncture using 3.8% w/v sodium citrate, as an anticoagulant (1:9, anticoagulant: blood), and centrifuged at 3000 rpm for 10 min at room temperature for subsequent removal of the plasma. Then, we pooled and stored the plasma of at least three different donors in plastic tubes at −20 °C until use.
Prothrombin time test
The prothrombin time (TP) test assesses the extrinsic pathway and common pathways of the coagulation cascade. In this assay, the Soluplastin kit (Wiener Lab, Rosario, Argentina) was used, following the manufacturer's instructions. Plasma (100 µL) was maintained for 2 min at 37 °C, and the reaction was started by the addition of thromboplastin with calcium (100 µL). Then, 50 µL of the derivatives was incubated with plasma for 5 min at 37 °C, and 100 µL of thromboplastin with calcium was added to trigger coagulation of plasma. We monitored the clotting time (in seconds) in the coagulometer and compared it with data obtained in a control tube containing saline.
Activated partial thromboplastin time (aPTT) test
The activated partial thromboplastin time (APTT) test assesses the initiation or propagation pathway of the coagulation cascade. In this assay, the APTT kit (Wiener Lab) was used according to the manufacturer’s instructions. The pool of plasma (100 µL) was preincubated with 100 µL of activated cephalin for 10 min at 37 °C in the absence or presence of the derivatives. The reaction was started by the addition of 100 µL of CaCl2 (8.3 mM, final concentration, previously heated to 37 °C), and coagulation was monitored in seconds in the coagulometer. Similarly, saline was added to the reaction medium, as a control.
Plasma recalcification time test
The recalcification time test evaluates calcium-dependent coagulation cascade factors through the addition of CaCl2 to plasma. The pool of plasma pool (100 µL) was incubated with saline or with the derivatives at 37 °C for 10 min, 50 µL of CaCl2 (12.5 mM, final concentration) was added to the reaction medium, and the clotting time was monitored in the coagulometer.
Toxicity hemocompatibility
The toxicity of the triazoles was evaluated by the hemocompatibility test, according to [33], with modifications. All the compounds (100 μg/mL) or saline (negative control) were incubated with a 13% (v/v) red blood cell suspension for 3 h at 37 °C. Then, the samples were centrifuged for 3 min at 1800 rpm, and lysis of the cells was detected by measuring hemoglobin at an absorbance of 578 nm using a microplate reader (SpectraMax, Model M4, Molecular Devices, California, USA). One hundred percent hemolysis (positive control) was achieved by adding Triton X-100 (1%, v/v) or water to the red blood cell suspension.
Statistical analysis
Analysis of calcium, paw edema, and peritonite assay data
Statistical comparisons are represented as the mean ± SD (standard deviation), as shown in the text. The statistical significance of the differences between means was tested by one-way ANOVA followed by Tukey's test. A bicaudal p < 0.05 was considered significant. The results were plotted using GraphPad Prism version 5.0.
Analysis of platelet aggregation and coagulation data
The results are expressed as the mean ± SEM (standard error of the mean) of the indicated number of experiments performed. The results obtained were analyzed by Student's t test using the GraphPad Prism 6 program. Values of p < 0.05 were considered significant.
In silico studies
Pharmacokinetic and toxicological profile of triazoles
ADMET Predictor® (Simulation Plus) predicted the pharmacokinetic and toxicological profiles [34]. The structures of the triazole-derived molecules that were evaluated are shown in Fig. 1.
Fig. 1.
1,2,3-Triazole compounds to be evaluated in this work for antagonistic activity against P2X7R
Analysis of the binding potential of triazoles in the P2Y12 receptor
To complement the platelet aggregation and coagulation assays, we performed molecular docking to explore the P2Y12 purinergic receptor as a potential additional target for the triazole derivatives. Since the P2Y12 receptor is a purinergic receptor that is well known to be involved in the coagulation process [35, 36] and represents an important pharmacological target for the development of antithrombotic drugs [37], blind molecular docking was performed against the entire structure of the P2Y12 receptor to identify whether this receptor could be a target for triazole derivatives. For that, we selected the crystal structure deposited under the PDB code of 4PXZ, since it is the only structure available to date of P2Y12 in complex with an antagonist, AZD1283. To validate and compare the results, redocking was performed with the ligand AZD1283. The methods used to perform the molecular docking are described in the work by [38].
Results
Paw edema assays
As mentioned before, P2X7R is directly associated with the inflammatory response, since its activation on several cells induces the production and secretion of different inflammatory mediators, such as TNF-α, nitric oxide, and several proinflammatory cytokines, such as IL-1β, IL-6, and IL-18. The experimental model selected to study the potential in vivo anti-inflammatory effect of the derivatives was ATP-induced paw edema. The molecules selected for this study were 5e, 8h, 9i, 11, and 12, which exhibited potent antagonist activity toward P2X7R in previous in vitro studies [38]. We tested all compounds at different doses: 100, 500, and 1000 ng/kg. As shown in Fig. 1, treatment with 10 mM ATP induced a 30% increase in paw edema compared to the saline group, corroborating its action as a physiological agent in this experimental model (Fig. 2A). In addition, all tested compounds at different doses exhibited significant inhibitory effects on paw edema formation compared to the reference drug diclofenac (Fig. 2B–F). Additionally, treatment with selective P2X2 and P2X4 receptor antagonists did not inhibit the ATP effect (Supplemental Figure 1) [39, 40].
Fig. 2.
Inhibition of ATP-induced paw edema formation. A Paw edema was induced by the injection of 10 mM ATP (positive control) and saline solution was used as a negative control. We administered the tested compounds (100, 500, and 1000 ng/kg) or diclofenac (10 mg/kg) 1 h before edema formation. B Compound 8h. C Compound 12. D Compound 5e. E Compound 9i. F Compound 11. The volume of the paws was read using a plethysmometer device (UGO-BASILE) 60 min after the administration of the phlogistic inducer. Graph showing the decrease in the percentage of paw edema in the groups treated with only saline, 10 mM ATP, 0.8% diclofenac and the tested compounds (100, 500, and 1000 ng/kg) (**p < 0.01 and ***p < 0.001 compared to ATP treatment). Three paw edema experiments were performed on different days
Carrageenin-induced peritonitis model
Previously, we evaluated the anti-inflammatory effect of triazoles on a mouse paw edema model [31]. Thus, we used a carrageenan-induced peritonitis model to investigate the inhibitory action of triazole derivatives in the recruitment of inflammatory cells to the peritoneal cavity. Treatment with carrageenan-induced peritonitis and saline was used as a negative control. Before carrageenan stimulation, the animals received the molecules (0.1 mg/kg) and dexamethasone (10 mg/kg) via intraperitoneal injection. Three hours later, we collected the intraperitoneal fluid, and determined the total and differential leukocyte counts. The experimental group that received carrageenan as a phlogistic agent exhibited an intense inflammatory response characterized by an increase in white blood cells compared to the saline group (Fig. 3). This effect was reversed by pretreatment with dexamethasone. Nonetheless, the groups that received the triazole derivatives in association with carrageenan presented a significant decrease in the number of total leukocytes, particularly 5e and 9i, suggesting in vivo anti-inflammatory action (Fig. 3). Treatment with selective P2X2 and P2X4 receptor antagonists did not inhibit this effect (Supplemental Figure 2).
Fig. 3.
Inhibition of peritonitis by treatment with 5e, 8h, 9i, 11, and 12 molecules. Total leukocytes. The graph shows the decrease in total leukocytes in the groups treated with dexamethasone, 5e and 5e + carrageenan, 8h and 8h + carrageenan, 9i and 9i + carrageenan, 11 and 11 + carrageenan, and 12 and 12 + carrageenan. **p < 0.05 and ***p < 0.001 compared to carrageenan treatment. Three experiments were performed for each molecule on different days to build this graph
We evaluated the effect of treatments on each cell population and observed that pretreatment with 5e, 8h, 9i, 11, and 12, all at a concentration of 10 mg/kg, inhibited the effect of carrageenan, maintaining the number of mononuclear cells and decreasing the number of segmented cells (Table 1), including in the group that received dexamethasone (Table 1; Fig. 3K, L).
Table 1.
Effect of triazoles on the differential counting of cells after carrageenan-induced peritonitis in mice
| Treatments | % Mononuclear cells | % Neutrophils |
|---|---|---|
| Saline | 81 ± 4ª | 16 ± 10ª |
| 10 mg/kg Carrageenan | 28 ± 8 | 67 ± 10 |
| 10 mg/kg dexamethasone | 90 ± 6ª | 9 ± 7ª |
| Carrageenan + dexamathasone | 72 ± 9ª | 27 ± 9ª,g |
| 10 mg/kg 5e | 89 ± 5ª,b | 11 ± 7ª,b |
| 10 mg/kg 8h | 96 ± 5ª,c | 5 ± 4ª,c |
| 10 mg/kg 9i | 93 ± 3ª,d | 6 ± 3ª,d |
| 10 mg/kg 11 | 84 ± 2ª,e | 16 ± 4ª,e |
| 10 mg/kg 12 | 86 ± 5ª,f | 7 ± 3ª,f |
| Carrageenan + 5e | 56 ± 8ª | 40 ± 12ª |
| Carrageenan + 8h | 76 ± 7ª | 17 ± 11ª |
| Carrageenan + 9i | 65 ± 10ª | 40 ± 10ª |
| Carrageenan + 11 | 55 ± 10ª | 37 ± 4ª |
| Carrageenan + 12 | 71 ± 10ª | 40 ± 5ª |
Triazoles’ effect on differential counting cells after carrageenan-induced peritonitis in mice
ap > 0.005 compared with Carrageenan treatment
bp > 0.05 compared with Carrageenan + 5e
cp > 0.05 compared with Carrageenan + 8h
dp > 0.05 compared with Carrageenan + 9i
ep > 0.05 compared with Carrageenan + 11
fp > 0.05 compared with Carrageenan + 12
gp > 0.05 compared with Carrageenan + dexamethasone
Coagulation and platelet aggregation
Nonsteroidal anti-inflammatory drugs (NSAIDs) can hinder platelet aggregation via selective or nonselective inhibition of cyclooxygenase COX-1 and COX-2 isoenzymes and, therefore, the synthesis of prostaglandins, prostacyclin, and thromboxane [42]. In this sense, these drugs can increase the risk of bleeding, especially gastrointestinal bleeding. In addition, there are reports of an increased risk of bleeding in patients who use anticoagulants in association, such as patients with atrial fibrillation [41, 42].
The effect of derivatives on coagulation was investigated using three in vitro tests, prothrombin time (PT), activated partial thromboplastin time (APTT), and recalcification test (RC), which are regularly used in the clinic to assess procoagulant or anticoagulant disorders; the effect of the derivatives at two concentrations on the PT test. Derivatives 5e (15.8 ± 1 s, p = 0.075), 8h (16.4 ± 2 s, p = 0.056), 9i (15.8 ± 1 s, p = 0.057), 11 (15.4 ± 1 s, p = 0.055), and 12 (15.5 ± 1 s, p = 0.056) added at concentrations of 1 µM or 5e (15.7 ± 2 s, p = 0.064), 8h (15.4 ± 0.7 s, p = 0.064), 9i (15.2 ± 0.06 s, p = 0.058), 11 (15.2 ± 0.5 s, p = 0.065), and 12 (15.4 ± 0.8 s, p = 0.056) at a concentration of 10 µM did not cause alterations in the prothrombin time compared to the control group (15 ± 2 s). Treatment with the selective P2X2 receptor antagonist, NF770, at concentrations of 1 µM (15.5 ± 1 s, p = 0.068) and 10 µM (15.7 ± 1 s, p = 0.06) did not inhibit this effect. In a similar manner, treatment with the P2X4 receptor antagonist, 5-BDBD, at concentrations of 1 µM (15.4 ± 0.7 s, p = 0.054) and 10 µM (15.7 ± 0.5 s, p = 0.057) did not inhibit.
All derivatives at 1 and 10 µM for 8h, 11, and 12 significantly prolonged the clotting time in the aPTT test. Treatment with 1 µM 5e (44.5 ± 0.6 s, p = 0.043), 8h (47.8 ± 0.7 s, p = 0.044), 9i (44.5 ± 0.7 s, p = 0.046), 11 (51.5 ± 0.8 s, p = 0.045), and 12 (51.4 ± 0.7 s, p = 0.046) augmented the anticoagulant activity compared with control (41.3 ± 0.7 s). Treatment with 10 µM for 8h (48.3 ± 0.7 s), 11 (48.5 ± 1 s), and 12 (52 ± 0.9 s) increased the clotting time, in contrast to 5e (48.6 ± 9 s, p = 0.07) and 9i (50.65 ± 10 s, p = 0.09). Treatment with the selective P2X2 receptor antagonist, NF770 at concentrations of 1 µM (40.9 ± 1 s, p = 0.062) and 10 µM (42.2 ± 1 s, p = 0.057) did not inhibit this effect. In a similar manner, treatment with the P2X4 receptor antagonist, 5-BDBD, at concentrations of 1 µM (41.9 ± 0.6 s, p = 0.06) and 10 µM (42.5 ± 0.8 s, p = 0.054) did not inhibit.
The effect of the derivatives on the plasma recalcification time was also evaluated. The incubation of plasma with the derivatives 1 µM 5e (284.4 ± 4 s, p = 0.038), 1 µM 8h (284.7 ± 20 s, 0.044), 1 µM 9i (244.3 ± 11 s, p = 0.042), 1 µM 11 (277.7 ± 8 s, p = 0.037) or 10 µM (336.6 ± 36 s, p = 0.022), 1 µM 12 (260.2 ± 10 s, p = 0.047) or 10 µM (281.2 ± 12 s, p = 0.031) prolonged the clotting time when compared to the control (196.9 ± 9 s). On the other hand, derivative 5e, at concentration of 10 µM, decreased the clotting time (189.1 ± 3 s, p = 0.048) when compared to the control, indicating that it might act as a procoagulant agent at this concentration. Treatment with the selective P2X2 receptor antagonist, NF770, at concentrations of 1 µM (200.4 ± 12 s, p = 0.055) and 10 µM (198 ± 8 s, p = 0.054) did not inhibit this effect. In a similar manner, treatment with the P2X4 receptor antagonist, 5-BDBD, at concentrations of 1 µM (204.6 ± 9 s, p = 0.062) and 10 µM (195 ± 7 s, p = 0.059) did not inhibit.
The triazoles increased the closing time in a mechanism independent of the P2X2 and P2X4 receptors. Therefore, we decided to evaluate the effect of the triazole derivatives on platelet aggregation induced by ADP that binds to the P2Y12 purinergic receptor on platelets. This receptor is also involved in the coagulation process [43]. As show in Fig. 4, the derivatives (1 µM) inhibited ADP-induced platelet aggregation, as did clopidogrel (1 µM), which is a selective antagonist of the P2Y12 receptor.
Fig. 4.
Effect of the derivatives or clopidrogel at a concentration of 1 µM on platelet aggregation of platelet-rich plasma (PRP) induced by ADP. The results are expressed as the mean ± SEM (n = 9)
Analysis of the binding potential of triazole derivatives molecules in the P2Y12 receptor target
Based on the results that suggested an anticoagulant action of the compounds and the absence of an effect of other P2X receptor subtypes such as P2X2 and P2X4 receptors, we performed molecular docking to analyze the binding potential of triazole molecules to the P2Y12 receptor. Redocking assays were performed, and the results were able to reproduce most of the interactions of AZD1283 with its binding site, particularly the interactions between the piperidinyl and benzylsulfonyl moieties of the ligand with helices VI and VII (Fig. 5). The redocking reproduced the hydrogen bonding of the carboxyl group with the Arg256 residue and the hydrophobic interaction with the Phe251, Tyr105, Tyr259, and Lys276 residues (Fig. 5).
Fig. 5.
Representative redocking result for the AZD1283 ligand against the P2Y12 receptor. The P2Y12 receptor is shown in cartoon and colored cyan. The AZD1283 conformation derived from the crystal structure (PDB: 4NTJ) is depicted in green, and the AZD1283 conformation derived from redocking is depicted in pink (Color figure online)
The docking results of the triazole derivatives indicated that the most populated cluster is in the same AZD1283 binding pocket. In addition, the triazole derivatives and AZD1283 presented similar values of binding energy (Supplemental Table 1), indicating a potential affinity of the derivatives for the P2Y12 receptor and a competitive profile for the triazole derivatives.
It is important to mention that molecular docking estimates the binding energy via a scoring function. However, despite being a method efficient in the identification of ligands and nonligands, the scoring functions of docking are not able to discriminate between the interactions of ligands with less than 1 kcal/mol [44]. As the binding energy evaluated for all compounds in this work was less than 1 kcal/mol, we can only infer that they might present similar binding affinity.
Interestingly, the favorable conformation of all triazoles presented a similar binding mode, corroborating the affinity toward this binding site.
In all triazole derivatives, the benzyl group was oriented in the same direction as the benzylsulfonyl group of the ligand AZD1283 toward helices VI and VII, thus performing hydrophobic interactions with residues Phe251, Tyr105, Tyr259, and Lys276. It was also possible to note a hydrogen-bond interaction with residue Arg256, and such a hydrogen bond might have an important contribution to the antagonist bioactive conformation (Fig. 6).
Fig. 6.
Representation of the most favorable conformation for each triazole derivative. Green represents the binding mode of the ligand AZD1283 derived from the crystal structure (PDB: 4NTJ). Each triazole derivative is represented in pink (Color figure online)
These data are relevant, because the in vivo administration of triazoles could affect both receptors. However, the inhibitory effect of triazoles on the P2X7 receptor occurs in at nanomolar concentrations and dosages [45], and on the P2Y12 receptor at micromolar concentrations.
In vitro and in vivo toxicity of triazole analogs
Toxicity in red blood cells
The toxicity of triazoles was assayed through an in vitro hemocompatibility test using RBCs. The treatment of cells with Triton X-100 or water lysed 100% RBCs (positive groups), while treatment with saline (negative control) did not result in lysis. All triazoles (100 μg/mL) lysed approximately 3% of red blood cells (data not shown), and according to [33], hemolysis below 10% means that the compound or molecule is devoid of toxicity; thus, triazoles can be considered nonhemolytic or nontoxic molecules. However, it is worth noting that the concentration of triazoles in this toxicity test (100 μg/mL) was approximately 10–100 times higher than any tested concentration in the assays of coagulation or platelet aggregation.
Single-dose toxicity
We performed an assessment of mortality resulting from the probability of survival for the 1000 mg/kg dose [31]. This demonstrated that there was no lethal or behavioral toxic effect after inoculation of the triazole compounds during the 24 h of observation.
In silico study
Evaluation of the physical–chemical and pharmacokinetic properties
As we intend to use triazoles as anti-inflammatory drugs, we compare them in silico with commercial anti-inflammatory drugs to have a clearer forecast of the feasibility of progressing or not advancing these studies. The evaluation of the physicochemical properties indicates that most triazole derivatives have similar profiles to other commercially available anti-inflammatory drugs (Supplemental Table 2). The exception was 11, which violated one Lipinski’s Five Rule (7.158 LogPb) [34].
The pharmacokinetic parameters of the triazole analogs are shown in Supplemental Table 3. As the reference drugs (diclofenac, ibuprofen, and naproxen), all derivatives had a high probability of crossing the blood–brain barrier, and none acted as a substrate of P-glycoprotein. Regarding effective jejunal permeability, only derivatives 5e and 11 presented values lower than the reference drugs. All derivatives presented higher values of volume of distribution in humans (Vd) than commercial drugs, with emphasis on 11, which presented a Vd approximately 4 times higher, possibly due to two chlorines in the aromatic ring. Finally, the ADMET risk prediction indicated that derivatives 8h, 9i, 5e, and 9i have a high pharmacological profile, particularly since they presented lower values than commercial anti-inflammatory drugs.
Toxicological profile
Supplemental table 4 presents the values of the toxicological parameters analyzed for the triazoles. Through the evaluation of the TOX_Risk parameter, all the tested triazole analogs exhibited low toxicological risk (Table 4). Although none of the derivatives had the potential to block the cardiac potassium channel (hERG), some of the derivatives presented an elevated probability of causing alterations in liver enzymes, suggesting potential hepatotoxicity, although further studies are necessary to confirm this possibility. Derivatives 5e and 9i showed high mutagenic potential, which could explain the increase in the TOX_Risk parameter when compared to the other derivatives. However, its values are still lower than those of commercial anti-inflammatory drugs.
Discussion
P2X7R has been widely studied as a target for anti-inflammatory disorders. The experimental models used in this work helped us to understand the mechanism of action of triazole-derived molecules toward P2X7R, as previously reported [45, 46], by showing their inhibitory potential against P2X7R-mediated inflammatory processes in vivo.
In general, the inflammatory process is characterized by tissue responses, such as pain, heat, redness, edema, and loss of function, the so-called cardinal signs. Activation of P2X7R induces the production and secretion of different inflammatory mediators that promote tissue responses, such as edema. To study the effect of the triazole compounds on the P2X7R-mediated inflammatory response, we evaluated the experimental model of paw edema using ATP as the phlogistic agent.
The present study indicated satisfactory results from the in vivo model of paw edema. Triazole-derived molecules tested as P2X7R antagonists 8h, 12, 5e, 9i, and 11 showed significant anti-inflammatory action in the acute inflammation protocol. In this assay, we used a paw edema model strictly triggered by purinergic activation, using 10 mM ATP as an inflammation inducer [45]. All tested concentrations caused a significant reduction in paw edema, comparable to 0.8% diclofenac, a currently marketed standard anti-inflammatory drug.
The anti-inflammatory action of triazole derivatives in the in vivo model of paw edema has been previously demonstrated in studies in vitro and in vivo [38], from which the molecules to be tested were selected. The protocol performed by the authors induced paw edema by carrageenan and ATP, measuring the edema 30 min after their applications in the mouse paw. The 9d triazole inhibited the paw edema, with an ID50 value of 79.84 ng/kg, in the edema induction by ATP at 1 mM. Using carrageenan as an edema inducer, 9d also inhibited the formation of paw edema with an ID50 value of 94.35 ng/kg. Additionally, the authors also carried out an oral treatment in which they induced edema formation by ATP and carrageenan and observed the inhibition of paw edema with greater potency than intraperitoneal treatment, obtaining ID50 values of 59 and 80.49 ng/kg, respectively.
In another work by Faria et al., the same model of ATP-induced paw edema was used to study the antagonistic potential of boronic acid derivatives. The substances NO-01 and NO-12 had a better effect than BBG and a similar effect to A740003, a selective P2X7R inhibitor, in reducing paw edema. The results presented in this work show that triazole derivatives could reduce paw edema at a lower dose than substances NO-01 and NO-12. Our results agree with other studies in the literature that indicate an anti-inflammatory potential for this class of compounds. In studies by Almasirad et al., another family of 1,2,3-triazole derivatives was also tested. Six triazole derivatives were tested at a concentration of 50 μmol/kg using the carrageenan-induced paw edema model, and the anti-inflammatory action of three of these compounds was observed.
A hallmark of inflammation is the migration of immune cells to the injured site. As purinergic signaling is involved in this cellular response, we used a carrageenan-induced peritonitis model to analyze the action of triazole molecules in inhibiting or reducing cell migration into the peritoneal cavity. The use of carrageenan as an inflammatory inducer was shown in studies by de Souza et al. [47], which demonstrated the capacity of carrageenan to generate an inflammatory response in rat and mouse paws. Moreover, carrageenan is frequently used in experimental models for the induction of peritonitis [48, 49]. The molecules 8h, 12, 5e, 9i, and 11 (0.1 mg/kg) reduced the recruitment of cells into the peritoneum when compared to control treatment, indicating that they can decrease the inflammatory process in vivo [47]. In the present study, carrageenan in the peritonitis model induced histological alterations comparable to those observed in previous studies [50, 51], in which an inflammatory response with an elevated leukocyte infiltrate in the peritoneum was reported. As expected, the administration of saline did not cause peritonitis, whereas in the dexamethasone-administered group, there was inhibition of the effect of carrageenan. Interestingly, the molecules 8h, 12, 5e, 9i, and 11 presented an inhibition profile comparable to that of the dexamethasone group. In addition, when administered alone, the triazole compounds did not induce alterations in peritoneal leukocytes.
The triazole lysed approximately 3% of red blood cells; a result considered a very low profile of toxicity. According to [33], hemolysis below 10% means that the compound or molecule is devoid of toxicity, and thus, triazoles can be considered nonhemolytic or nontoxic molecules. Moreover, toxicity assessment was performed in vivo, observing the probability of mortality at a dose of 1000 mg/kg [31]. The derivatives were neither lethal nor induced behavioral toxic effects in mice during the 24 h of observation.
A fundamental step in drug research is the evaluation of the ADMET properties of new chemical entities. These in silico analyses allow us to predict the viability of promising hit compounds to advance throughout the different stages of drug development, and together with the biological results, they are essential to determine the efficacy and safety of a new drug candidate [52]. The analysis of ADME properties showed a high pharmacological potential for most of the triazole compounds evaluated. Only 11 presented a violation of Lipinski's rule of five by exhibiting an inadequate log P (LogP) value, indicating that this compound could have a low oral absorption [53]. Interestingly, all derivatives showed a high probability of crossing the blood–brain membrane and a low probability of interacting with P-glycoprotein (P-gp), two desirable characteristics for new drug candidates [54]. Furthermore, all derivatives, except for 5e and 11, had a total volume of distribution comparable to that of the reference drugs, such as diclofenac and ibuprofen. Finally, the prediction of toxicity by the ADMET and TOX_Risk parameters together indicates that the derivatives have a low potential to cause toxic effects, such as elevation of liver enzymes or blockade of the cardiac potassium channel (hERG).
The anti-inflammatory drugs currently marketed have the COX-2 enzyme as a therapeutic target. They were developed to avoid the side effects of previous anti-inflammatory drugs, which had COX-1 as a pharmacological target. However, several further studies showed that drugs that block COX-2 also lead to serious side effects, such as cardiovascular and thrombotic effects [55], since this enzyme is responsible for the generation of prostacyclins, substances that promote an antithrombotic effect, vasodilation, and reduce platelet aggregation and adhesion [56, 57].
In this work, we tested molecules aiming to identify new substances with anti-inflammatory action that target P2X7R, therefore acting in a pathway independent of COX-2 blockade. Therefore, we expected to minimize the mentioned side effects, such as thromboembolic side effects.
Therefore, it is of great importance to test these molecules in platelet aggregation and coagulation assays, since there is evidence of P2X7R involvement in thrombus formation [57]. In this sense, the molecules tested as P2X7R antagonists, 8h, 12, 5e, 9i, and 11, showed an anticoagulant effect through some current clinical tests, such as aPTT and RC. Only substance 5e used at a concentration of 10 µM acted as a procoagulant and may be a promising substance when compared to other anti-inflammatory drugs that increase the risk of bleeding.
P2Y12 receptor is a purinergic receptor subtype directly involved in the coagulation process. Therefore, we tested whether the triazole analogs could also inhibit platelet aggregation. Our results indicated that these triazoles exhibited a similar inhibition profile to Clopidrogel, which is an inhibitor of the P2Y12 receptor, suggesting the possibility that these triazoles could also act as P2Y12 receptor antagonists. It is noteworthy that the docking assays performed for the selected triazoles indicated binding energy values comparable to AZD1283, a high-affinity antagonist of the P2Y12 receptor, corroborating our in vitro results and indicating a competitive profile in relation to the reference drug.
Conclusion
In the present study, the molecules 5e, 8h, 9i, 11, and 12 exhibited an anti-inflammatory action in ATP-induced acute inflammation and ameliorated paw edema. Molecules 5e, 8h, 9i, 11, and 12 had similar action to the anti-inflammatory drug diclofenac. Molecules 5e and 8h also showed an anti-inflammatory action on peritonitis induced by carrageenan, comparable to the reference drug dexamethasone. Regarding platelet aggregation results, triazole derivatives tested as P2X7R antagonists had good prospects in coagulation assays. The substances 5e, 8h, 9i, 11, and 12 had an anticoagulant effect, and the molecule 5e (10 µM) acted as a procoagulant, being a good alternative to replace anti-inflammatory drugs that present risks of bleeding. All derivatives, except for 11, had adequate pharmacokinetic, physicochemical, and toxicological properties. In addition, the ADMET risk assessment indicates that derivatives 8h, 12, 5e and 9i have high pharmacological potential. Docking assays for the P2Y12 receptor indicated that the derivatives exhibit binding energies comparable to the reference antagonist with a competitive inhibition profile. However, the affinity for this binding, when compared with the concentration and dose to inhibit the P2X7R-mediated function, is more than 100 times smaller. Finally, 8h, 12, 5e, and 9i reversed the inflammatory response through P2X7R inhibition and caused anticoagulant action on P2Y12R. This series may be relevant to combat COVID-19 infection involvement for acting on inflammation and blood aggregation.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary file1 (TIF 74 KB) Supplemental Figure 1. Inhibition of ATP-induced paw edema formation. A. Paw edema was induced by the injection of 10 mM ATP (positive control) and saline solution was used as a negative control. We administered the tested 5-BDBD and NF770 (1 mg/kg) or diclofenac (10 mg/kg) one hour before edema formation. The volume of the paws was read using a plethysmometer device (UGO-BASILE) 60 minutes after the administration of the phlogistic inducer. Graph showing the decrease in the percentage of paw edema in the groups treated with only saline, 10 mM ATP, 0.8% diclofenac and the tested compounds (1 mg/kg) (*** p<0.001 compared to ATP treatment). Three paw edema experiments were performed on different days.
Supplementary file2 (TIF 153 KB) Supplemental Figure 2. Inhibition of peritonitis by treatment with 1 mg/kg 5-BDBD and 1 mg/kg NF770 molecules. Differential counting. One hundred cells per slide were counted, and macrophages, lymphocytes, monocytes (mononuclear cells) and neutrophils were found. Three experiments were performed for each molecule on different days to build this graph. *p<0.05, **p<0.05 and ***p<0.001 compared to saline treatment. #p<0.05, ##p<0.05 and ###p<0.001 compared to saline treatment. Three experiments were performed for each molecule on different days to build this graph.
Acknowledgements
We thank the IOC, LABTOXo, and LAPSA for their support.
Author contributions
N.G.P., A.D.S., B.Q.M., and J.S.P. performed the biological assays. N.L.R. and C.R.R. performed the in silico assays. D.T.G.G., V.F.F., and F.C.S. synthetized the molecules. D.T.G.G., F.C.S., and V.F.F. prepared the figures, coordinated the synthesis assays, wrote, and revised the paper. J.S.P., A.D.S., A.C.S., A.L.F., and R.X.F. revised the biological assays and wrote and revised the paper.
Funding
The fellowships granted by CNPq (301873/2019–4, 316568/2021–0, and 306011/2020–4), CAPES (Financial Code 001), and FAPERJ (E-26/203.246/2017, E-26/211.025/2019, E-26/200.982/2021, E-26/203.191/2017, E-26/202.800/2017, E-26/010.101106/2018, E-26/200.870/2021, E-26/201.369/2021, and SEI-260003/001178/2020) are gratefully acknowledged.
Data availability statement
The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files.
Declarations
Conflict of interest
The authors declare that they have no conflicts of interest.
Ethical approval
All procedures performed in studies involving animals followed the institution's ethical standards or practice at which the studies were conducted.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplementary file1 (TIF 74 KB) Supplemental Figure 1. Inhibition of ATP-induced paw edema formation. A. Paw edema was induced by the injection of 10 mM ATP (positive control) and saline solution was used as a negative control. We administered the tested 5-BDBD and NF770 (1 mg/kg) or diclofenac (10 mg/kg) one hour before edema formation. The volume of the paws was read using a plethysmometer device (UGO-BASILE) 60 minutes after the administration of the phlogistic inducer. Graph showing the decrease in the percentage of paw edema in the groups treated with only saline, 10 mM ATP, 0.8% diclofenac and the tested compounds (1 mg/kg) (*** p<0.001 compared to ATP treatment). Three paw edema experiments were performed on different days.
Supplementary file2 (TIF 153 KB) Supplemental Figure 2. Inhibition of peritonitis by treatment with 1 mg/kg 5-BDBD and 1 mg/kg NF770 molecules. Differential counting. One hundred cells per slide were counted, and macrophages, lymphocytes, monocytes (mononuclear cells) and neutrophils were found. Three experiments were performed for each molecule on different days to build this graph. *p<0.05, **p<0.05 and ***p<0.001 compared to saline treatment. #p<0.05, ##p<0.05 and ###p<0.001 compared to saline treatment. Three experiments were performed for each molecule on different days to build this graph.
Data Availability Statement
The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files.