New Insights in Purinergic Therapy: Novel Antagonists for UTP- Activated P2Y Receptors from Natural Products

P2Y2 and P2Y4 receptors are physiologically activated by UTP and are widely expressed in many cell types in humans. They promote an increase in intracellular calcium via PLCβ/ IP3 and act on ion flux and water secretion. P2Y2 plays an important role in inflammation and proliferation of tumor cells, which could be attenuated with the use of antagonists. However, little is known about the physiological functions related to P2Y4 due to the lack of selective ligands for these receptors, which can be solved through the search of novel compounds with antagonistic activity. In the present study, we have applied a methodology of calcium measurement to identify new antagonist candidates for these receptors. Firstly, we established optimal conditions for calcium assay using J774.G8, a murine macrophage cell line, which expresses functional P2Y2 and P2Y4 receptors. J774.G8 cells were loaded with 2 μM of Fluo-4 to test its sensitivity in responding calcium stimuli. ATP and ionomycin, known as inductors of intracellular calcium rise, were used to stimulate cells. The EC50 obtained were 11 μM and 103 nM, respectively. Subsequently, investigation of P2Y2 and P2Y4 expression was performed. These cells responded with EC50 of 1.021 μM to the UTP stimulation. Screening assays were performed and a total of 100 extracts from Brazilian natural products were tested. JA2, RA3, and RB3 extracts stood out for their ability to inhibit UTP-induced responses without causing cytotoxicity and presented IC50 of 32.32 μg/mL, 14.99 μg/mL, and 12.98 μg/mL, respectively. Collectively, our results point to the discovery of potential antagonists candidates from natural products for UTPactivated receptors.


Introduction
Purinergic receptors are plasma membrane receptors activated by extracellular purines and pyrimidines.These receptors are subdivided into two families, according to their main physiological ligand: P1 receptors, which are activated by adenosine, and P2 receptors, that are activated by nucleotides.P2 receptors are classified into two classes: ionotropic P2X receptors (P2XR) and metabotropic P2Y receptors (P2YR) [1].
P2Y2 and P2Y4 receptors are physiologically activated by uridine triphosphate (UTP) and promote an increase in intracellular calcium via PLC/IP3 [2].They are expressed in many cell types such as epithelial cells, monocytes, macrophages, neutrophils, cardiomyocytes and organs, including brain, heart, kidneys, liver, spleen, and muscle [3].They have effects on chloride secretion in epithelial cells from airway and eyes, and they also induce water secretion in epithelial cells from the bowel.These functions are important to promote surface lubrification and mucus hydration, which can improve the treatment of diseases such as cystic fibrosis, dry eye disease and chronic constipation [2,4].Recently, pharmaceutical companies have been applying efforts to search new P2Y2 agonists able to treat diseases associated with this receptor.As a result of these studies, Diquafosol ® was implemented to treat dry eye disease, however, it is only approved in Japan and South Korea [5,6].
P2Y4R participates in Na + , K + , and Cl -regulation processes, but its functions are not completely characterized because it is expressed in the same tissues and cell types that P2Y2R, where they are activated by the same physiological ligand, UTP [16][17][18][19].
Therefore, the lack of selective ligands impairs the discovery of new functions associated with these receptors.However, this problem can be solved with the search for novel compounds with antagonistic activity on P2Y2 and P2Y4 receptors.
Natural products are an important source for discovery of new molecules with antagonistic activity.They offer the vantage of millenary use in the treatment of several diseases, such as inflammation, parasitic, pain, and recently, cancer, constituting the basis of traditional oriental medicines [20][21][22].Among the thirteen drugs approved for global marketing between 2005 and 2007, five were classified as natural products, while the others were semi-synthetic or derived, which reinforces its importance for clinical use [23].In addition, it is already described in the literature that some biological species have compounds with antagonistic activity on P2 receptors, such as emodin from Rheum officinale Bail and amentoflavone from Rheedia longifolia Planch & Triana on P2X7R [24,25].However, due to a large number of samples for testing, implementation of high-throughput screening methodologies is necessary to meet this demand.
In this scenario, techniques of intracellular calcium measurement emerge as a tool for screening of new antagonists, since P2Y2 and P2Y4 receptors are coupled to calcium signaling pathway [26].These methodologies have demonstrated importance in high-throughput screenings due to the advantage of evaluation of many compounds in a short period of time [27,28].Kaulich et al. tested a series of forty flavonoids using calcium assays to discover novel lead antagonists for P2Y2R.They found that some flavonoids could inhibit P2Y2R and suggest the use of their structures to develop new compounds with antagonistic activity [29].Recently, Ito et al. applied a protocol of intracellular calcium measurement in a high-throughput screening campaign to search novel antagonists for P2Y6 receptor and found one compound, TIM-38, with potential activity [30].
Thus, the aim of this study was to improve a protocol based on intracellular calcium measurement that allows the discovery of natural product compounds with antagonist activity on P2Y receptors activated by UTP.Firstly, we establish optimal conditions of protocol to execute the experiments using a cell line that express P2Y2 and P2Y4 receptors.After that, we performed a screening of a group of 100 extracts from natural products aiming at validating our protocol and discover possible hits.We found that nine extracts partly inhibited calcium mobilization in J774.G8 cell line, but only one significantly blocked this activity in peritoneal macrophages and did not present cytotoxicity.This natural extract may constitute a potential lead in the development of new antagonists for UTP-activated receptors.

-Establishment of a protocol for detection of intracellular calcium mobilization
Our primary aim was to discover new antagonists for P2Y receptors activated by UTP, so a protocol for detection of intracellular calcium mobilization was first established.We made a search in the literature to know concentrations of calcium indicator, Fluo-4, which were used in calcium assays and that could be applied in our protocol.We found in the literature a range of Fluo-4 used in calcium assays between 2 and 4 μM [34][35][36][37].In order to use low doses of dye, the first step was to optimize the concentration of Fluo-4 to our conditions of the assay, making it more economical to high throughput screening.In this context, we investigated concentrations ranging from 1 to 8 μM.We used ATP [100 μM] to stimulate the cells due to its activity as a main physiological P2 receptor agonist, which is widely expressed in J774.G8 cells.As observed in Figure 1A, there is no significant difference in the quantification of calcium response between concentrations between 1 to 4 μM.Concentrations of 6 and 8 μM have significative difference only compared to 1 μM.Furthermore, our results are in a concentration range of Fluo-4 used in literature (2-4 μM) [34][35][36][37].A similar result was obtained with stimulation of cells with ionomycin, a calcium ionophore that promotes a massive transport of this ion from the extracellular to intracellular medium.All concentrations evaluated have the similar profile of quantification of calcium response (Figure 1B).Thus, the selected concentration of Fluo-4 to use in our experiments was of 2 μM because calcium responses in this concentration did not differ from higher concentrations as well as being in literature range.As we could observe in Figure 1C, cells loaded with Fluo-4 at a concentration of 2 μM show a homogenous dye scattering in their cytosol and after a stimulus with ionomycin, they become more fluorescence.
Next, two assays were performed to verify Fluo-4 sensibility in established concentration (2 μM).Cells were stimulated with increasing concentrations of ATP and ionomycin and Fluo-4 fluorescence increased in a concentration-dependent manner, as shown in Figures 1D and 1E.EC50 found for ATP and ionomycin were 11 μM and 103 nM, respectively.Therefore, in this primary step, we found that Fluo-4 concentration at 2 μM was ideal for our assay since cells presented homogenous dye loading and sensibility to respond to calcium stimulus in increasing concentrations.

-Assessment of quality of calcium responses induced by P2 receptors
In order to characterize the quality of calcium responses induced by P2 receptors through ATP stimulation acquired by FlexStation III, we analyzed original records.
Original records showed that DPBS addition alone did not cause any signal variation, as expected (Figure 2A).ATP stimulus, on the other hand, increased intracellular calcium levels, which can be noted by fluorescence peak demonstrated in Figure 2B.This effect was reversed with the addition of EGTA, a calcium chelating agent, confirming that the fluorescence is associated with calcium, as shown in Figure 2C.Meanwhile, when cells were stimulated with ionomycin, they also increase their fluorescence (Figure 2D).However, it was not possible to observe a decline in intracellular levels during the selected time to perform this experiment, i.e., 90 seconds.More time probably will be required to observe this decline, which could decrease the number of wells read in a short period of time.Therefore, with these experiments, we validated our protocol of calcium measurement in FlexStation III.All these receptors, except the P2Y12R, are associated with signaling pathways of intracellular calcium increase via opening of a non-selective cation ion channel (P2X) or PLCβ/IP3 system activation (P2Y).Then, we investigated the response of P2R subtypes expressed in J774.G8 cells using selective agonists.As shown in Figure 3C, this cell line expresses P2X and P2Y receptors, such as P2Y2 and P2Y4 activated by UTP, P2Y1, and P2Y6, which were activated by ADP and UDP, respectively.Cells also express P2X7 that promotes calcium response since stimulation with BzATP (P2X7 selective agonist).ATP and αβmeATP, non-selective agonists were used to observing general calcium responses of P2 and P2X receptors, respectively.These results confirm expression of P2 receptors observed in RT-PCR, including UTP-activated P2Y receptors, i.e., P2Y2 and P2Y4, which are our target subtypes.Interestingly, we observed that all agonists, with exception of ADP, present a lower response without extracellular calcium, which possible points participation of store-operated calcium channels.In case of ATP and αβmeATP, this response could be ionotropic channels.After characterization of UTP calcium responses and inhibition profile of P2Y2 and P2Y4 antagonists, we calculated the z'-factor of our assay.Z'-factor is a statistical parameter used to measure the robustness of assays.We applied the mathematical equation of z'-factor and we obtained a value of 0.645.Therefore, z'-factor of our assay was above 0.5 and it is within the optimal range (z' ≥ 0.5).

-Characterization of UTP-induced calcium responses and screening of natural products
Finally, we performed the screening of 100 extracts from natural products, which data are summarized in Table 1.Nine extracts partially inhibited calcium mobilization induced by UTP: JA2, JA5, JB2, JB7, P2B6, P5C1, P5F3, RA3 and RB3.
The same experiment was repeated using peritoneal macrophages from mice, a primary cell that expresses P2R.Using these cells, we observed that only JA2 extract was able to significantly inhibit UTP-calcium responses (Figure 5A).JA2 presented mean in peritoneal macrophages less than in J774.G8 (21.07 ± 18.24 compared to 52.97 ± 29.48), which could reflect differences between cell lines and primary cells.RA3 and RB3 extracts have not been tested in peritoneal macrophages due to sample scarcity.
Then, cells were treated with increasing concentrations of JA2

Discussion
P2Y2 and P2Y4 are purinergic receptors physiologically activated by UTP, which promote an increase of intracellular calcium via PLCβ/ IP3 when activated [2].
They are expressed in many cell types such epithelial cells and leukocytes, and even in organs such as brain, heart, kidneys, liver, spleen, and muscle [3], where they play important roles.Likewise, these P2Y receptors are associated with certain diseases such as cystic dry eye disease, Alzheimer, and cancer [2,4,7].Thus, pharmaceutical companies have been developing drugs that act on these P2Y receptors, especially P2Y2, in order to treat these related diseases [5,6,38].However, the lack of selective ligands that act on P2Y2 and P2Y4 receptors compromises the discovery of new functions associated with these receptors, which can be solved by the search of new antagonist compounds [19].Therefore, the aim of this study was to improve a methodology for the detection of intracellular calcium mobilization to discover new molecules with antagonistic activity on UTP-activated P2Y receptors.For this reason, we established a protocol to evaluate intracellular calcium mobilization.
First, the concentration of calcium indicator Fluo-4 was optimized.Fluo-4 is widely used in high-throughput screening assays of metabotropic receptors that induce an increase of intracellular calcium [39].We chose a 2 μM concentration because of the apparent homogenous cellular loading in cells and due to the fact that there were no significant differences between it and other concentrations.In addition, it is important to use the lowest concentration that results in a good signal, thereby reducing reagent consumption and expenses during screening [33].Gee and coworkers (2000) also demonstrated that the use of the lowest concentrations of calcium indicators can reduce the buffer effect on calcium, as well as minimize the levels of toxic products such as formaldehyde and acetic acid, which are produced through the hydrolysis of acetoxymethyl esters [40].Furthermore, by using this concentration we demonstrated that cells were able to react to different stimuli that increase intracellular calcium levels for Hek 293 and CHO-K1 cells, respectively [39].This could be a sum of response from various P2R that could be activated by ATP such as P2Y2, P2Y11, P2X1, P2X2 and P2X7 subtypes.The latter could have displaced the EC50 since its activation occurs in concentrations higher than 100 μM.Meanwhile, EC50 found for ionomycin (103 nM) was lower than that obtained by Valentin et al. (2011), i.e., 1.5, μM when they performed calcium assay on microplate reader with human endothelial cells of the umbilical vein (Huvec) [41].
We also observed some expected characteristics of calcium signals, such as the uniformity of the basal signal, the peak of the signal after ATP stimulus, indicating an increase of [Ca 2+ ]i and the return of this signal to basal level after response time, which in calcium events usually occurs in seconds.In addition, a decrease of calcium responses in the presence of EGTA, a calcium chelating agent, was also observed.A similar response profile was obtained through calcium imaging (data not shown).
Ionomycin stimulation also induced an increase in intracellular calcium levels, but during the period of measurement of the experiment (90 seconds), it was not possible to observe the return to basal levels.However, longer time of measurement of ionomycin response could be required to observe this return.But increasing the time of wells measurement could cause a diminution in a number of samples read in an established period of time.Therefore, we opted to maintain the set time for 90 seconds.Moreover, we calculated z'-factor, a measure of robustness of assay, and we found a value of 0.645, which indicates that our protocol was adequate for use.Ito and coworkers (2017) performed a high-throughput screening assay to identify novel antagonists for P2Y6R using P2Y6-1321N1 cells and obtained a z'-factor of 0.80 [30].Valentin et al. (2011) used primary cells (Huvec) to perfume high-throughput screening based on calcium measurement and obtained a z'-factor of 0.60 [41].These findings suggest that use of transfected cells could improve the performance of the assay.However, the use of transfected cells can add a cost to screening, and therefore it is suggested that it is used in a second moment in order to verify the response of hits.Taken together, these evaluated characteristics suggest that our protocol was appropriated for use.
Furthermore, all these receptors are associated with signaling pathways of intracellular calcium increases via the opening of a non-selective cation ion channel (P2X) or PLCβ/IP3 (P2Y), with exception of P2Y12.Therefore, we characterized these subtypes using the following agonists: ATP, a non-selective P2 receptor agonist, UTP, a P2Y2 and P2Y4 receptors agonist, ADP for P2Y1 receptor, UDP for P2Y6 receptor, BzATP, a selective agonist of P2X7 and αβmeATP for P2X receptors.These data showed functional expression of these receptors.The calcium responses decreased in absence of extracellular calcium, which suggests the participation of store-operated calcium channels and ionotropic receptors in amplification of intracellular calcium mobilization [46].
Then, we obtained the profile of P2Y2 and P2Y4 agonist UTP and antagonists (Suramin, RB2, and PPADS).J774.G8 cells were stimulated with increasing concentrations of UTP and we found an EC50 of 1.021 μM.It is similar to that cited by King and coworkers (1998): 1.1 μM for P2Y2 and 0.20 μM for P2Y4 receptors [47].In relation to the antagonists, we observed that Suramin and RB2 partially block the intracellular UTP-induced calcium mobilization.King et al. (1998) cited that PPADS (100 μM) and RB2 (100 μM) inhibited approximately 30% of P2Y4R, while Suramin is ineffective [47,48].Von Kügelgen (2006) cited that P2Y2 is blocked by Suramin and RB2, but not by PPADS [48].We also hypothesize that participation of store-operated calcium channels could have influenced some level of their inhibition.
It is worth mentioning that our work was one of the pioneers in the search for novel antagonists using natural products, which present a diversity in a number of species and chemical structures, both acquired over millions of years of evolution.Thus, we tested one hundred extracts from a natural library at a cut-off concentration, established at 50 μg/mL.At first, nine extracts demonstrated a partial inhibition of UTPinduced calcium responses in J774.G8 cells.After repeat this experiment with a primary cell, i.e., peritoneal macrophages, that express P2Y2 and P2Y4 receptors [44], we found that only JA2 extract inhibit this activity.Other two extracts were not tested in these cells due to sample scarcity.However, aiming at observing the occurrence of this inhibition at higher doses, we treated J774.G8 cells with increasing concentrations of extracts.Calcium responses were inhibited in a concentration-dependent manner, suggesting its potential ability to block UTP-activated P2Y receptors.We asked if this inhibition could be due to cell toxicity.To answer this question, we performed a cell viability assay applying MTT technique and observed that they did not promote cytotoxicity.
Collectively, our results point to the discovery of potential antagonists from natural products for P2Y2 and P2Y4 receptors, which may significantly contribute to purinergic pharmacology.

-Extracts
Extracts used in this work were collected from Brazilian biomes (Amazon, Cerrado, Caatinga, Atlantic Forest, Pantanal, and Pampas) and were obtained through extraction with methanol.They were deposited at Bioprospecting Platform from the Laboratory of Chemistry of Natural Products.JA2, RA3, and RB3 extracts vouchers numbers are EX294, EX9355, and EX9356, respectively.For the experiments, they were resuspended in a solution of 1 mL Dulbecco's PBS containing 0.5% DMSO.The extracts were tested using a single-blind method, i.e., the professional responsible to perform screening experiments did not know the name of biological species that originated the samples [31].Because of this, they were named with a code.supplemented with 10% fetal bovine serum, at 37°C in a 5% CO2 atmosphere.The medium was changed every three days and the cells were adjusted to a concentration of 2x10 6 cells per 150 cm 2 cell culture flask (Corning, New York, USA).Cell viability was analyzed by the trypan blue assay and the calcium assays were conducted only when the range of viability was above 90%.

-Calcium assays
J774.G8 cells were plated in a concentration of 2x10  (2009) [28].The wells which signal did not return to approximately 30% of the baseline after stimulation were discarded as control of quality.

-Observation of cells loading in fluorescence microscope
J774.G8 cells were load with Fluo-4 as previously described.Then, cells were observed in fluorescence microscope Nikon Eclipse TE 2000-S (Nikon, Tokyo, Japan), using 20X objectives.Fluo-4 was excited using a mercury lamp and a set of filters: exciter: 480/30 and barrier: 535/40.Ionomycin was added after 40 s of observation and pictures were captured with microscopy digital camera Infinity 3 (Lumenera, Ontario, Canada).

-RNA extraction, RT-PCR and electrophoresis
The mRNA was isolated from the J774.G8 cell line from a concentration of 5x10

-Animals
The experiments with animals were approved by Ethics Committee on the use of Animals from Oswaldo Cruz Institute under identification code L-037/2017, approved on November 24 2017 and follow ethical principles in animal experimentation of Brazilian College of Animal Experimentation.Healthy male C57BL/6 mice with a weight between 25 and 30g were obtained from Oswaldo Cruz Central Bioterium.The animals were housed under conditions of 12h/12h light and dark cycle with free access to food and water.

-Peritoneal macrophage isolation
Animals were euthanized by CO2 asphyxiation.Then, 5 mL of RPMI medium was injected into the peritoneal cavity.Peritoneal cells were collected and macrophages were isolated by centrifugation at 1500 RPM for 10 minutes.Cell viability was analyzed by the trypan blue assay.Peritoneal macrophages were plated in a concentration of 4x10 5 cells/well in a 96-well black-wall and clear-bottom plate and maintained in culture with RPMI supplemented with 10% fetal bovine serum, at 37°C in a 5% CO2 atmosphere for 24h.Calcium assays were conducted using the same conditions previously described.

-Cell viability measurement
J774.G8 cells were plated in a concentration of 2x10 5 cells/well in a 96-well plate (Corning, New York, USA), and maintained in culture with DMEM supplemented with 10% fetal bovine serum, at 37°C in a 5% CO2 atmosphere for 24h.Next, cells were treated with extracts [50 μg/mL] and antagonists PPADS [300 μM] and RB-2 [20 μM] for 1, 6 or 24h.Cells treated with Triton-x (0.1%) were considered as a negative control of viability, while our positive control was obtained with a cell that did not receive any treatment.Subsequently, the medium was replaced by 180 μl of DMEM without phenol red and each well received a volume of 20 μl of MTT solution [100 μg/well] and the plate was incubated for 3h at 37°C in a 5% CO2 atmosphere.After this time, the plate was centrifuged at 1500 RPM for 1 minute.Then, supernatants were collected and formazan crystals were dissolved in 100 μl of DMSO as described [32].
The absorbance of the wells was measured in FlexStation III equipment, using λ: 570 nm.

-Data analysis
Each sample was measured in triplicate and all experiments were performed on at least three independent days.All data are presented as means ± S.D.M., while the curves are presented as means ± S.E.M.In order to test if samples follow a Gaussian distribution, the D' Agostino and Pearson normality test was used.If data follow a Gaussian distribution, an appropriate parametric test was applied, if not, an appropriate non-parametric test was applied.Used tests were specified in figure legends.P values of 0.05 or less were considered significant.Graphs and statistical analyses were performed by GraphPad Prism version 7 (GraphPad Software, San Diego, California, USA).We also assessed the quality of our calcium assay by calculating the z'-factor.Z'-factor is a valuable tool to evaluate the robustness and suitability of HTS assays.Z'-factor values above 0.5 consider assay as excellent and equal a 1, an ideal assay.However, values below 0.5, consider assay as a double assay that needs to be reformulated [33].This parameter was calculated using the following equation: Z'= 1 -(3SD of positive control + 3SD of negative control) (mean of positive controlmean of negative control) where cells stimulated with UTP at [10 μM] were considered as a positive control (concentration used in screening experiments), while cells stimulated with DPBS only were considered as negative control.

Figure 2 : 2 . 3 -
Figure 2: Original records of calcium signals obtained on a FlexStation III.Original records of calcium signals obtained on a FlexStation III equipment after addition of 50 μl of DPBS (A), ATP [100 µM] (B and C) or Ionomycin (D) at 25 seconds and EGTA [20 mM] (54 seconds) (C) (indicated by the arrows) in J774.G8 cells stained with 2 µM of Fluo-4.Representative images from three independent experiments performed in triplicate.
CO2 atmosphere.Extracellular Fluo-4 was then removed by three consecutive washes with 200 μl of DPBS and then, cells received a final volume of 150 μl of DPBS.Cells were treated with antagonists for 30 minutes.The plate was then placed into the FlexStation III equipment (Molecular Devices, California, USA), which performed the addition of the agonists in each well, diluted in 50 μl of DPBS.The mobilization of intracellular calcium was measured in real time prior, during and after the addition of the agonists at λ (excitation): 485 nm and λ (emission): 525 nm.Total measurement runtime was 90 seconds and agonists were added at the time of 20 seconds.The readings were conducted every 1.52 seconds and a total of 60 readings were obtained for each well.The ΔFU, i.e., the difference between fluorescence peak associated with the maximum concentration of intracellular calcium ([Ca 2+ ]i) and basal fluorescence of calcium was determined as mentioned in the study by Hansen and Bräuner-Osborne

preprints.org) | NOT PEER-REVIEWED | Posted: 30 January 2018 doi:10.20944/preprints201801.0285.v1
Peer-reviewed version available at Journal of Medicinal Food 2018; doi:10.1089/jmf.2018.0087 , RA3, and RB3 extracts and we found IC50 equal to 32.32 μg/mL, 14.99 μg/mL, and 12.98 μg/mL, respectively (Figures5B, C, and D).Aiming at verifying if the inhibition of calcium responses would come from possible extracts cytotoxicity, tests of viability applying MTT technique were performed.As can be seen in Figure5E, only RB3 extract caused a slight decrease in cell viability, but it did not be significative.Taking together, we characterized JA2, RA3, and RB3 extracts as potential antagonists candidates for UTPstimulated receptors.The next step is to characterize the active molecules from candidate extracts.Preprints (www.