E. coli (APEC), is an economically important bacterial disease of poultry. APEC are a subgroup of extra intestinal pathogenic E. coli (ExPEC) and poultry are considered potential sources of foodborne ExPEC to humans. Currently, APEC infections in poultry are controlled by antibiotics and/or vaccination; however, their effect is limited due to emergence of antibiotic resistant strains and infections with heterologous serotypes. Therefore, novel approaches are needed. Here, using the bioluminescent quorum sensing (QS) autoinducer 2 (AI-2) indicator Vibrio harveyi BB170, we screened the cell free culture supernatant of APEC O78 prepared from cultures grown in the presence of 4,182 small molecules . A total of 69 SMs inhibited > 75% of APEC O78 AI-2 activity in the indicator bacteria. Ten SMs that showed highest AI-2 inhibition were selected for further studies. Most of these SMs inhibited the AI-2 activity of other APEC serotypes and significantly reduced APEC O78 biofilm formation and motility. Most compounds showed minimal toxicity on human intestinal cells (Caco-2), chicken macrophage (HD-11), and chicken and sheep red blood cells, and reduced APEC survival in HD-11 and THP-1 macrophages. The SMs induced no or minimal toxicity and conferred protection against APEC in wax moth larval model. SMs affected the expression of APEC O78 QS, virulence, biofilm and motility associated genes providing insight on their potential mode(s) of action. Further testing in chickens will facilitate development of these SMs as novel therapeutics to control APEC in poultry and thereby also reduce zoonotic transmission.Colibacillosis caused by avian pathogenic  E. coli (APEC), is a significant bacterial disease of poultry worldwide [Escherichia coli (ExPEC). APEC- like bacteria can be transmitted to humans through consumption of contaminated poultry and fresh produce contaminated with poultry litter [Colibacillosis, caused by avian pathogenic orldwide ,2. APEC y litter . Additioy litter . Even thy litter ,6.Avian colibacillosis is characterized by yolk sac infection, swollen-head syndrome, septicemia, and inflammation of different organs such as pericarditis, perihepatitis, airsacculitis, salpingitis, arthritis, and peritonitis ,7. APEC E. coli) [Currently, APEC infections in poultry are controlled by a commercially available modified-live vaccine (Poulvac E. coli) . HoweverE. coli) . In addiE. coli) . Thus, tluxS which mediates the synthesis of AI-2 has also been shown to regulate motility, biofilm formation, virulence and pathogenesis of many bacterial pathogens, including APEC [luxS and/or AI-2 activity using novel intervention such as QS small molecule inhibitors (QSI) can be a potential strategy for novel antibacterial development. As the QSI do not interfere with the metabolic processes of a bacterial cell such as protein synthesis, DNA metabolism, cell wall formation which are the targets for the development of drug resistance [APEC possesses several virulence factors that have been determined to be involved in different stages of pathogenesis, including P fimbriae, type IV pili, curli, invasins, iron acquisition systems, serum complement resistance factors, anti-phagocytic factors, hemolysin E, outer membrane proteins A, and vacuolating autotransporter toxin ,9,10. Pasistance , they dosistance .Vibrio harveyi, a marine Gram-negative bioluminescent bacterium, regulates its luminescence through QS [V. harveyi regulates bioluminescence via two-component signaling systems, AI-1 and AI-2, each is composed of a sensor–autoinducer pair [V. harveyi AI-2 indicator is capable of detecting the AI-2 of many bacteria that produce similar AI-2 molecules and stimulate light production following the addition of cell-free culture supernatants from these bacteria [E. coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Vibrio cholerae have been reported to produce signaling molecules that stimulate bioluminescence production in V. harveyi [rough QS . V. harvcer pair ,19. V. hbacteria . For exa harveyi .V. harveyi BB170 AI-2 indicator bacteria (AI-1+ and AI-2-) [in vitro to evaluate their toxicity in human intestinal cells, chicken macrophages, chicken and sheep red blood cells (RBCs) and determine their efficacy against APEC infection in chicken and human macrophages and in wax moth  larva model. Further, the expression of several QS, virulence, biofilm and motility associated genes were assessed to provide insight on their potential mode(s) of action on APEC O78. We identified 10 small molecules (SMs) that modulated APEC infection in vitro and in vivo. These SMs can serve as leads for future application in APEC control in poultry and thereby also reduce potential zoonotic transmission of APEC to humans.Here, we used d AI-2-)  to identV. harveyi AI-2 indicator. Inhibition of bioluminescence by SM treated APEC O78 culture supernatants was compared to that of the DMSO treated control. A total of 69 compounds inhibited ≥ 75% of the bioluminescence activity. These 69 compounds were further tested for AI-2 mediated bioluminescence inhibition in four independent experiments and 10 compounds (referred hereafter as AI-2 inhibitors) that showed highest AI-2 inhibition (75–98%) were selected for further studies  was screened for APEC O78 growth inhibition. The results showed that 4,122 molecules did not impact the growth of APEC O78 (no elevated OD) . Superna studies . HoweverFurther, the selected AI-2 inhibitors displayed differential effect on the inhibition of AI-2 activity of multiple APEC serotypes. Most compounds resulted in ≥ 75% inhibition of AI-2 activity of APEC O2 with the exception of C3 and C9, while only C1 and C6 resulted in ≥ 75% inhibition of AI-2 activity of APEC O1. Only C6 exhibited ≥ 75% inhibition of AI-2 activity of O8, O15, O18, and O35. Whereas C8 exhibited ≥ 75% inhibition of AI-2 activity of O109, while C6 and C10 exhibited ≥ 75% inhibition of AI-2 activity of O115 .10.1080/Biofilms play a crucial role in APEC virulence and enhance the bacterial resistance to antimicrobials and immune clearance, leading to failure of antimicrobial therapy ,22. TherMotility and chemotaxis allow bacteria to migrate towards or away from favorable environments in response to stimulus and thus contribute to bacterial fitness and virulence . The effThe toxicity of the selected AI-2 inhibitors was assessed on Caco-2 and HD-11 cells using lactate dehydrogenase (LDH) assay to quantitatively measure the LDH released into the media from damaged cells after exposure to AI-2 inhibitors as an indicator for cellular cytotoxicity and cytolysis. Our results showed that, when treated with 100 µM of SMs, C8 and C4 exhibited 2% and 12% cytotoxicity, respectively; while C6 and C10 exhibited 27% cytotoxicity and C1-C3, C7, and C9 displayed 36% to 42% cytotoxicity on Caco-2 cells. Further, in HD-11 cells, C5 and C8 exhibited less than 10% cytotoxicity, while C2, C4, C6, and C7 exhibited cytotoxicity between 12% and 18%, and C1, C3, C9 and C10 exhibited toxicity between 22% and 27% .10.1080/Additionally, toxicity of the selected AI-2 inhibitors to sheep and chicken RBCs was also tested using 100 µM of each compound. Notably, all compounds exhibited no hemolytic activity against sheep or chicken RBCs with the exception of C3, which exhibited 20.6 % and 6.4% hemolytic activity in sheep and chicken RBCs, respectively .The invasion of APEC into phagocytic cells facilitates its intracellular survival and systemic spread to different organs which is critical for APEC pathogenesis . TherefoAPEC-O1) that carries pap operon (putative virulence genes of APEC), invasion determinant protein encoding gene tia and iron-regulated outer membrane protein encoding gene ireA which are likely to contribute to increased virulence of APEC O1; thus, the lower efficacy of SMs [Similarly, in THP-1 cells, C1- C3, and C5- C9 exhibited 100% clearance of APEC O78 , while C10 significantly reduced its intracellular survival . C1- C3, C9 and C10 exhibited 100% clearance of APEC O2 , and C4- C8 significantly reduced its intracellular survival . Similarly, C1, C3, C9 and C10 significantly reduced the intracellular survival of APEC O1  . Interesy of SMs .in vivo efficacy of the AI-2 inhibitors was evaluated using the wax moth larva model, which has been previously used to evaluate drug efficacy and bacterial pathogenesis [The ogenesis . TreatmeTreatment of the wax moth larvae with the AI-2 inhibitors also reduced APEC O78 colony forming units (CFU) in both dead and live larvae in comparison to DMSO control. At 72 h post treatment, the bacterial load in dead larvae in all treated groups, except those treated with C1 and C7 that showed no larval death, were significantly reduced  in comparison to DMSO treated control. However, in surviving larvae, C2 and C8 resulted in 100% clearance of APEC , while C4-C6 and C10 significantly reduced the bacterial load  in comparison to dead larvae. Further, at 48 h post treatment, C2, C5, C6 and C8 treated groups showed no larval death while the rest of the compounds resulted in significant reduction of the APEC O78 load  in comparison to DMSO treated controls . Howeverin vivo studies in the natural host.Notably, all AI-2 inhibitors showed no or low toxicity in larvae. The survival of larvae treated with C1 and C6 was 100%, whereas the survival of larvae was more than 75% for the rest of the AI-2 inhibitors . These rQuorum sensing inhibitors have been proposed to affect the expression of QS regulated and virulence- associated genes . TherefoluxS), which mediates AI-2 synthesis. Notably, C4 up-regulated the expression of 5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase gene (pfs), which also mediates AI-2 synthesis , carbohydrate transport sn-glycerol-3-phosphate import ATP-binding protein , cytoplasmic ferredoxin-NADP reductase , and thiazole biosynthesis protein   .10.1080/fimC; C1- C6, and C8- C10), iron uptake chelate protein D , ferric yersiniabactin uptake A , serum survival protein , and vacuolating autotransporter toxin . However, C2- C4 and C9 up-regulated the expression of iss , transcriptional regulator protein , outer membrane protein G precursor , and tyrosine-phosphatase protein . Both hha and rcsB are located in the cytosol and were shown to be involved in biofilm formation, while ompG and wzb contributes to exopolysacchride formation. Likewise, genes associated with chemotaxis and motility, such as cytoplasmic flagellar synthesis chaperone protein , chemotaxis protein , flagellar motor protein B  and flagellar biosynthesis protein  were also down-regulated by AI-2 inhibitors (nlpC).AI-2 inhibitors also affected the expression of biofilm formation, motility and exopolysaccharide formation associated genes. The AI-2 inhibitors down-regulated the expression of genes encoding the hemolysin-expression modulating protein , and carbon storage regulator A , and UDP-N-acetylmuramoylalanine- D-glutamate ligase . However, C2-C4, and C7- C10 up-regulated the expression of major outer membrane lipoprotein precursor gene (lpp)  .Principal component and multivariate analysis were performed to determine the gene expression profiles of APEC treated with the AI-2 inhibitors. The expression profiling divided the AI-2 inhibitors into 2 groups . The first group composed of C5, C7, C8 and C10 (0.59 ≤ r ≤ 0.84), while the second group composed of C3, C4, C6 and C9 (0.49 ≤ r ≤ 0.66). The C9 and C8 showed high correlation with other AI-2 inhibitors  and they connected these two groups together . FurtherV. harveyi BB170 AI-2 indicator bacteria, 10 novel QS AI-2 inhibitors that did not inhibit APEC growth but interfered with QS- regulated processes including virulence factors, biofilm formation, motility, exopolysaccharide synthesis, stress survival, cell division and pathogenesis in APEC [The increase in antibiotic resistant APEC , the lim in APEC . Use of luxS. Further, most of the SMs also down-regulated the expression of QS- regulated and virulence-associated genes such as pfs, iucD, fyuA, vat, ompA, iss, and fimC  rcsBdivision ,37; 3) bynthesis ,38; and ynthesis . These fynthesis . Our resed genes . Additioed genes . Therefois genes ,43.P. aeruginosa through the reduction of QS-regulated virulence factors such as protease, pyoverdin and chitinase and their associated proteins [agr QS system in S. aureus [V. cholerae virulence factors such as cholera toxin and toxin coregulated pilus [fimC, which plays an important role in APEC adherence to the cells through the regulation of adhesion of type 1 fimbriae [fyuA and lucD) which are involved in biofilm formation [iss which plays a role in resistance against serum complements [vat which regulates the motility, agglutination, and biofilm formation of APEC [G. mellonella survival and reduced APEC loads in surviving larvae  ironormation , 3) iss plements , and 4)  of APEC . This ing larvae ).luxS expression, while C4 did not, suggesting a potential association between the chemical structure and the mode of action of the compounds in APEC. Four compounds  possess high structural similarities between each other . C5 and C10 are both composed of a phenylpropyl piperazinyl phenyl groups and showed similarity in their effect on expression profile . These in vitro and in vivo. C1, C2 and C10 contain piperidine functional group (score = 0.61) but C1 displayed no effect on biofilm formation , C6 contains nitrobenzene sulfonamide functional group. However, C6 and C7 have some structural similarity , which might explain their similar effect both ormation  and lowe and C10 . This mi and C10 . Previouectively ,53.in vitro and in vivo and the inhibitory activity seems to be related to piperazine based functional group. Discovery of novel antimicrobials that do not affect bacterial growth is less likely to impose a selective pressure for resistance development by the bacteria and represents an ideal antibiotic independent approach. Further in vivo studies in chickens and specific target identification of these potential leads will facilitate development of these AI-2 inhibitors as novel therapeutics to augment APEC control in poultry and prevent its potential zoonotic transmission. Because of their small size and convenient synthesis scheme, we expect the cost of large scale synthesis of these inhibitors to be equivalent or less than the cost associated with synthesis of current antibiotics used in the industry to treat APEC infections. In addition, since human pathogenic ExPECs genetically resemble APEC, our future studies will also test the effect of these inhibitors against human pathogenic ExPECs particularly those that are multidrug resistant.The potential AI-2 inhibitors identified in this study differentially affected the pathobiology of APEC and the expression of QS regulated virulence, biofilm, and motility genes. C5, C6, C7 displayed highest efficacy both A library of 4,182 compounds (yactives) were selected via pre-screening of 81,320 compounds and were obtained from Chembridge, Inc.  . These cR) was isolated by plating the APEC O78 on LB agar containing 50 µg/mL of rifampicin [E. coli DH5α (Invitrogen) was grown overnight in LB broth at 37 °C with shaking at 200 rpm. V. harveyi BB170  and V. harveyi BB120  were kindly provided by Dr. Bonnie L. Bassler (Princeton University) and were grown in AB medium at 30 °C aerobically with shaking as described previously [APEC serotypes O1 (GenBank accession no. NC_008563), O2 (GenBank accession no. NZ_CP006834), and O78 (GenBank accession no. CP004009) were kindly provided by Dr. Tim Johnson . Other APEC serotypes such as O8, O15, O18, O35, O109, and O115 were kindly provided by Drs. Lisa K. Nolan and Catherine M. Logue . Luria-Bertani broth  was used for routine propagation of APEC serotypes. APEC serotypes were routinely grown in LB broth overnight at 37 °C with shaking at 200 rpm. Rifampicin resistant APEC O78  of 0.05 (7 × 107 CFU/mL) in fresh LB broth. One hundred micro-liters of the culture was transferred to a 96-well plate and 1µL  of the compound was added to each well using a pin tool [TM Tecan plate reader  and the growth was kinetically monitored every 30 min by measuring the OD at 600 nm. Compounds that resulted in no growth inhibition (no elevated OD) were chosen for screening in AI-2 bioluminescence inhibition assay.Overnight culture of APEC O78 prepared in LB broth was adjusted to an optical density (ODpin tool . ChloramV. harveyi BB170 (AI-1+ and AI-2-) was grown overnight in AB medium at 30 °C aerobically with shaking [V. harveyi BB170 culture was distributed into each well of a 96-well plate and mixed with 20 µL of the cell free culture supernatant, the plate was then incubated at 30 °C for 2.5 h in the dark and the bioluminescence was measured using in vivo imaging system . The incubation time was determined based on the preliminary studies which showed optimal induction of bioluminescence at 2.5 h (data not shown). Cell-free culture supernatants collected from overnight cultures of V. harveyi BB120 (AI-1+andAI-2+) and E. coli DH5α (a strain lacks AI-2) were used as controls [The AI-2 bioluminescence assay was performed as described previously ,19. Briecontrols . Four inThe selected compounds were also tested for inhibition of AI-2 production in other APEC serotypes such as O1, O2, O8, O15, O18, O35, O109, and O115 that are commonly implicated in colibacillosis using the same procedure described above.Effect of the selected AI-2 inhibitors on biofilm formation was assessed using the crystal violet (CV) assay . Briefly600 of 0.05 and 100 µL was transferred to each well of a 96 well plate and grown in the presence of 100 µM of each compound at 37 °C for 10 h. The culture was then adjusted to an OD600 of 0.05 and used for the motility assay. The motility assay was performed in a 48-well plate using semisolid agar media (0.4% LB agar) containing 0.01% tetrazolium chloride and 100 µM of each compound. One microliter of the OD adjusted culture was stabbed onto the middle of the agar and the plate was incubated at 37 °C for 6 h. The motility was assessed by measuring the diameter of the halo zone in comparison to DMSO treated control. Two independent experiments were conducted with duplicate wells in each experiment.The effect of selected AI-2 inhibitors on APEC motility was performed as described previously [5 cells/well) were grown in a 96-well plate in minimal essential medium (MEM) supplemented with 20% fetal bovine serum , 1% non-essential amino acid  and 1mM sodium pyruvate, at 37 °C in a humidified 5% CO2 incubator for 48 h until a complete monolayer was formed. For LDH assay, cells were washed twice with media containing no FBS/no antibiotics and incubated with 150 µL of fresh media containing 100 μM of each compound at 37 °C for 24 h in a humidified 5% CO2 incubator. Fifty microliters of supernatant was analyzed using LDH Cytotoxicity Assay Kit (Thermo Scientific). Two independent experiments were conducted in triplicate wells in each experiment.The selected 10 AI-2 inhibitors were evaluated for their toxicity to Caco-2 (ATCC® HTB-37™) using LDH assay . Briefly5 cells/well) were grown in Iscove’s modified Dulbecco’s medium  supplemented with 2 mM glutamine and 10% FBS at 37 °C in a humidified 5% CO2 for 48 h until a monolayer was completely formed. LDH assay was conducted as described above. Two independent experiments were conducted in triplicate wells in each experiment.Toxicity was also determined on HD-11 cells (CVCL-4685). The HD-11 cells (1.4 × 10The hemolytic activity of the selected AI-2 inhibitors was determined as described before . Briefly7 CFU (MOI = 100) of mid-log phase APEC strains  at 37 °C for 1 h. Cells were washed with PBS and treated with gentamicin (150 μg/mL) for 1 h to kill the extracellular bacteria [The effect of the selected AI-2 inhibitors on the survival of APEC in macrophage cells was tested as described before . Brieflybacteria . The cel5 cells/well) were grown in a 96-well plate in a 5% CO2 incubator at 37 °C for 48 h, infected with APEC (MOI = 100), treated with each compound and the intracellular bacteria was determined as above.Similarly, the intracellular survival assay was also performed in THP-1 cells (ATCC® TIB-202™) as described above. The THP-1 cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% FBS and 2 mM glutamine. In order to differentiate the THP-1 monocyte to macrophage, 100 nM phorbol myristate acetate  was added to the media. The cells (1.4 × 10G. mellonella caterpillars (larvae) in the final instar stage (fifth instar) were obtained from Vanderhorst, Inc. , stored in wood shavings in a petri dish in the dark and used within 7 days of receiving. Larvae with 15–25 mm length, 250–350 mg weight, having a creamy color with minimal speckling and no grey markings were used in this study. The infection was performed as described previously [4) as described previously and each larva (n = 10) was inoculated with 8.5 µL  of the AI-2 inhibitors into the hemocoel via the last left proleg using PB600-1 repeating dispenser (Hamilton) attached to insulin syringe . Larvae were placed inside sterile petri dishes and incubated for 2 h in the dark at 37 °C. Then, larvae were infected with 8.5 µL of (4.25 × 104 CFU) of RifR APEC O78 in 10 mM MgSO4 on the right hind proleg. RifR APEC O78 was generated as described above by plating APEC on LB agar plate containing 50 µg/ mL rifampicin for specific monitoring of APEC population inside the larvae. The AI-2 inhibitors exhibited similar AI-2 inhibition against RifR APEC O78 as that of parent wild-type APEC O78 (data not shown). Infection dose of RifR APEC O78 to larvae was identified based on preliminary study . Larvae were incubated at 37°C in the dark and the survival was monitored daily for 72 h. Infected larvae treated with buffer mix and 75 mg/kg of chloramphenicol were used as controls.eviously ,60. For For the quantification of APEC inside the larvae, dead and live larvae were surface sterilized with 70% ethanol, homogenized in PBS. The suspension was tenfold serially diluted and plated on MacConkey agar plates supplemented with 50 µg/mL of rifampicin. The plates were then incubated overnight at 37 °C and APEC load was enumerated. Two independent experiments were conducted using larvae (n = 10) obtained in different batches.G. mellonella larvae  were inoculated with 8.5 µL of SMs  into the hemocoel via the last left proleg. Post inoculation, larvae were placed inside sterile petri dishes and incubated in the dark at 37 °C for 72 h and larval survival was monitored every 24 h. Two independent experiments were conductedTo confirm that the death of the larvae was not due to the compounds, intrinsic toxicity the AI-2 inhibitors were assessed in a separate experiment. 2 First Strand Kit (Qiagen). The qRT-PCR was performed using SensiMixTM SYBR® Hi-ROX qPCR Master Mix (Bioline) according to manufacturer instructions in a realplex2 mastercycler (Eppendorf) with 55 oC annealing temperature. Gene-specific primers were designed using PrimerQuest Tool and obtained from integrated DNA technologies (IDT). The primers used with target genes’ description are listed in The effect of the selected AI-2 inhibitors on the expression of QS-regulated and virulence factors genes of APEC was determined using qRT-PCR. APEC O78 was grown in the presence of 100 µM of each compound in a 96-well plate at 37 °C for 10 h. APEC O78 culture treated with 1µL of 100% DMSO was used as control. Total RNA was extracted from duplicate wells for each compound (200 µL) using a miRNeasy Mini Kit (Qiagen). RNA quality and quantity was determined by nanodrop 2000 C spectrophotometer (Thermo scientific). Traces of DNA were removed using Genomic DNA removal mix (Qiagen). Approximately, 5 μg of purified RNA was used to synthesize cDNA using the Qiagen RTData from biofilm formation, motility, qRT-PCR, toxicity, hemolytic activity, intracellular survival, and wax moth infection assays were expressed as the mean ± standard deviation. ANOVA followed by Tukey test was used to analyze these data and a P-value < 0.05 was used to determine statistically significant differences between means. A fold change of ± 1.5 ≥ or ≤ 1.5 and a P-value ≤ 0.05 were used to determine statistically significant differences in gene expression. The differences between the gene expression profiles were analyzed using a principal component analysis (PCA) on JMP PRO 13 software (SAS Institute). Statistical analyses of the wax moth infection studies were performed using GraphPad Prism 5 software  and plotted using the Kaplan-Meier graph.


These studies lay the foundations for the structure-based design of new generations of TRPC1/4/5 modulators.TRPC1/4/5 channels are non-specific cation channels implicated in a wide variety of diseases, and TRPC1/4/5 inhibitors have recently entered clinical trials. However, fundamental and translational studies require a better understanding of TRPC1/4/5 channel regulation by endogenous and exogenous factors. Although several potent and selective TRPC1/4/5 modulators have been reported, the paucity of mechanistic insights into their modes-of-action remains a barrier to the development of new chemical probes and drug candidates. Xanthine-based modulators include the most potent and selective TRPC1/4/5 inhibitors described to date, as well as TRPC5 activators. Our previous studies suggest that xanthines interact with a, so far, elusive pocket of TRPC1/4/5 channels that is essential to channel gating. Here we report the structure of a small-molecule-bound TRPC1/4/5 channel—human TRPC5 in complex with the xanthine Pico145—to 3.0 Å. We found that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a bound phospholipid. Our findings explain the mode-of-action of xanthine-based TRPC1/4/5 modulators, and suggest a structural basis for TRPC1/4/5 modulation by endogenous factors such as (phospho)lipids and Zn Wright et al. report the first structure of a human TRPC1/4/5 channel in complex with a xanthine-based TRPC5 inhibitor Pico145. They find that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a phospholipid. This study provides insights into the mechanism-of-action of xanthine-based TRPC1/4/5 modulators. Based on sequence similarity, the mammalian TRPC proteins can be further divided into sub-groups: TRPC1/4/5, TRPC3/6/7 and TRPC27, the latter of which is encoded by a pseudogene in humans8. TRPC4 and TRPC5 are the most closely related TRPC proteins9 (70% sequence identity) and can form functional, homomeric TRPC4:C4 and TRPC5:C5 channels10. TRPC1  is not thought to form functional homomeric channels but is widely expressed and an important contributor to heteromeric TRPC1/4/5 ion channels15.Transient Receptor Potential Canonical (TRPC) proteins form homo- or heterotetrameric, non-selective cation channels permeable by NaTrpc4/5 genes16 and global expression of a dominant-negative mutant TRPC512 do not cause catastrophic phenotypes in rodents, TRPC1/4/5 channels have been implicated in a wide range of physiological and pathological mechanisms17. These findings have driven the development of potent and selective TRPC1/4/5 modulators as chemical probes and drug candidates18, and clinical trials have been started by Hydra Biosciences/Boehringer Ingelheim (the TRPC4/5 channel inhibitor BI 135889 for treatment of anxiety/CNS disorders) and Goldfinch Bio .Although disruption of the 6 and may include mediation by endogenous and dietary lipids22 and metal ions such as Zn2+23. In addition, structurally diverse pharmacological modulators have been reported18, including inhibitors suitable for studies of TRPC1/4/5 in cells, tissues and animal models such as the xanthines Pico145 26 and HC-07026, the pyridazinone derivative GFB-843827, and the benzimidazole ML20428. However, structural insight into the mode-of-action of small-molecule TRPC1/4/5 modulators is lacking, and no small-molecule-binding sites have been identified.Physiological activation and modulation of TRPC1/4/5 channel activity is complex50 values in the picomolar to the low nanomolar range and displays the highest potency against heteromeric channels24. Pico145 is orally bioavailable and has been used successfully for studies of TRPC1/4/5 channels in vivo30. Detailed characterisation of Pico145 and the related xanthine AM237—a partial TRPC5 agonist that inhibits other TRPC1/4/5 channels31—revealed that: (1) Pico145 and AM237 act rapidly and reversibly in outside-out excised patch recordings, highlighting a membrane-delimited effect31; (2) Pico145 is a competitive antagonist of (−)-englerin A (EA)24 and AM23731, and also inhibits channel activation by sphingosine-1-phosphate (S1P), carbachol and Gd3+25, although picomolar concentrations of Pico145 can potentiate Gd3+-induced TRPC4 currents as well24; (3) Pico145 concentration-dependently inhibits photoaffinity labelling of TRPC5 by the xanthine-based photoaffinity probes Pico145-DAAlk and Pico145-DAAlk2, with IC50 values in the same range as inhibition of TRPC5-mediated calcium influx32. These results provide evidence that these xanthines modulate TRPC1/4/5 channels through direct molecular interaction with the channels and are consistent with the hypothesis that Pico145 and AM237 bind to a well-defined, high-affinity-binding site essential to TRPC1/4/5 channel gating. Targeting this binding site may be a promising strategy to develop further generations of potent and selective TRPC1/4/5 chemical probes and drug candidates.Currently, the most potent and selective TRPC1/4/5 inhibitor is the xanthine Pico145, which inhibits the channels with IC33, mouse TRPC434 and mouse TRPC535. To date, TRPC6 is the only TRPC channel for which small-molecule-bound structures have been published37. Here, we report the structure of a TRPC1/4/5 channel in complex with a small-molecule modulator: the homomeric TRPC5:C5 channel in complex with Pico145. We found that Pico145 binds between the trans-membrane domains of two TRPC5 subunits, where it displaces a phospholipid in a conserved lipid-binding site. Docking and mutagenesis studies support these findings and provide additional insights into the mode-of-action of xanthines as modulators of TRPC5 and TRPC4 channels. In addition, we have identified a putative intracellular zinc-binding site of TRPC5 that is conserved within the TRPC family. This work provides a rational basis for the design of new TRPC1/4/5 modulators and suggests important functional roles for the conserved TRPC1/4/5 lipid and zinc-binding sites. Therefore, our findings may help unravel physiological mechanisms of TRPC1/4/5 modulation, and pave the way for structure-based TRPC1/4/5 drug discovery efforts.Several high-resolution TRPC structures have been determined by single-particle cryo-electron microscopy (cryo-EM), including zebrafish TRPC435). Upon overexpression in HEK 293 cells, TRPC5:C5 channels formed by this construct were activated by EA  and inhibited by Pico145  , followed by a PreScission protease site (PreS) and a C-terminally truncated (Δ766–975) hTRPC5  exchange performed on-resin and ultracentrifugation used in lieu of size exclusion chromatography to remove aggregated material. The protein was pure according to SDS-PAGE analysis  and showed the typical TRPC fold: a large intracellular domain made up of the N- and C termini of each monomer, six trans-membrane helices per monomer and relatively short extracellular loops ; the next two helices form the pore domain, together with the re-entrant pore helix (E3 loop), which lines the ion permeation pathway. The C terminus (residues 626 onwards) contains the TRP domain and the coiled-coil domain, which is formed by one helix from each monomer. The maltose-binding protein (MBP), protease site (PreS) and the first 16 amino acid residues of TRPC5 were not resolved in the map, likely because of flexibility. Likewise, residues 37–60, 74–95, 118–134, 273–285, 387–391, 665–702 and 734–765 were not modelled due to them being poorly resolved. All residues that were not modelled are found in the intracellular domain or extracellular loop 1; the trans-membrane helices and the other extracellular loops were modelled into the electron density.The cryo-EM structure of a human TRPC5:C5 channel in the presence of Pico145 was determined with a global resolution of 3.0 Å using C4 symmetry 35 revealed a backbone (Cα) RMSD of 0.81 Å showing a similar overall structure, consistent with both being in the closed state. There were, however, several notable local differences between the apo and Pico145-bound structures  interact with the 3-(trifluoromethoxy)phenoxy substituent on C8 of Pico145. The 4-chlorobenzyl substituent on N-7 of Pico145 points upwards, away from the lipid site, making a non-polar interaction with L572. Pico145 also makes additional interactions with helix S6 of the monomer on the left  to a global resolution of 2.9 Å. The density around the Pico145-binding site in this structure showed features consistent with both the bound phospholipid and Pico145, suggesting partial occupancy under these conditions . The docking suggests that Pico145 adopts a similar pose in TRPC4 and TRPC5, making similar interactions with conserved residues, particularly residues equivalent to the TRPC5 residues Q573, F576 and W577  are modulated by the xanthines Pico145 and AM237, whereas TRPC3/6/7 channels are not2+]i) measurements  act through intracellular signalling, and may interfere with the composition of phospholipids bound to TRPC5, while Gd3+ has complex effects in the presence of Pico14524. Therefore, we decided to test TRPC5 responses to AM237, which is thought to occupy the same binding site as Pico145 (see above and refs. 32). Mutations that resulted in TRPC5 activation by EA, but not by AM237, were likely to reveal residues contributing to xanthine binding. Wild-type TRPC5 was activated by EA (EC50 5.7 nM) and AM237 (EC50 10.6 nM)  and AM237 (EC50 110 nM) activated the TRPC5Q573T variant  was unresponsive to 100 nM EA.The constructs were tested using fluorometric intracellular calcium  Fig. . Both EAant Fig. , but pot76A Fig.  and TRPC77A Fig.  could be237 EC50 .6 nM , revealed the presence of a similar binding site in the PHD domain of the zinc-binding E3 ubiquitin ligase UHRF1 (PDB 3zvz) where the motif coordinates a Zn2+ ion. Manual overlay of this structure with our TRPC5 structure revealed a close alignment of residues  can open neuronal TRPC5 channels through an unknown mechanism. Therefore, we solved an additional structure of TRPC5  in the presence of ZnCl2  , molecular docking, and site-directed mutagenesis. We hypothesise that Pico145 initially integrates with the lipid membrane, before entering the lipid-binding site between two TRPC5 subunits and replacing the bound phospholipid. This hypothesis is consistent with the hydrophobic nature of Pico145, and with the observation that the effect of Pico145 is not as rapid as would be expected for a pore blocker 24.The widely expressed TRPC1/4/5 channels have been implicated in various physiological and pathological mechanisms and are an emerging class of potential drug targets25. However, three closely related xanthine derivatives, AM23731 and the photoaffinity probes Pico145-DA and Pico145-DAAlk32, are partial agonists of the homomeric TRPC5:C5 channel that inhibit other TRPC1/4/5 channels. Pico145 is a competitive antagonist of AM23731 and concentration-dependently inhibits both activation and photoaffinity labelling of TRPC5:C5 by Pico145-DAAlk32. These data are consistent with the hypothesis that xanthine-based TRPC1/4/5 modulators all bind to the same lipid-binding site of TRPC1/4/5 channels (this paper and refs. 32), where they can stabilise either open or closed channel conformations depending on xanthine substituent patterns and the composition of the specific TRPC tetramer. Therefore, targeting the xanthine-binding site of TRPC1/4/5 channels may allow the development of modulators of specific TRPC channel tetramers, a major challenge in TRPC1/4/5 pharmacology.The xanthines Pico145 and HC-070 are the most potent and selective TRPC1/4/5 inhibitors to date22. Lipids may affect TRPC1/4/5 channels indirectly, e.g., the phospholipid S1P can activate channels (at least in part) through G-protein signalling39. However, more direct mechanisms are possible as well, e.g., lysophosphatidylcholine (LPC) activates TRPC5 in excised outside-out patches in the absence of GTP40. Such mechanisms may involve direct molecular interactions and/or changes to the membrane environment of TRPC1/4/5 channels. The TRPC5 lipid-binding site that Pico145 interacts with is highly conserved within the TRPC family lipids to the conserved TRPC lipid-binding sites is a dynamic, reversible process that may have a direct functional effect on channel gating, and that this site can be targeted by TRPC1/4/5 activators as well as inhibitors. However, further studies are required to fully understand the mechanisms of TRPC1/4/5 channel binding and gating by (phospho)lipids and different small-molecule TRPC1/4/5 activators such as EA.TRPC1/4/5 channels are modulated by endogenous and dietary lipids, but the mechanisms are complex and incompletely understood2+-dependent activation of TRPC5 channels was recently reported to contribute to oxidative neuronal death23, but the molecular mechanism of Zn2+ regulation of TRPC5 was not determined. We have identified a putative intracellular zinc-binding site of TRPC5, which is conserved in all TRPC channels. Cryo-EM of TRPC5 in the presence of ZnCl2 resulted in a further TRPC structure determined to a resolution of 2.8 Å. Presence of ZnCl2 did not change the overall structure compared to TRPC5:Pico145, and the density of the Zn2+ ion could not be unambiguously identified in either structure. These observations suggest flexibility of the region or partial maximum occupancy of the zinc-binding site, and may indicate that zinc is already bound in TRPC5:Pico145, or that the role of zinc in TRPC5 modulation is subtle. A subtle role of Zn2+ in TRPC5 modulation would also be consistent with the delayed Zn2+-mediated [Ca2+]i increase observed in calcium imaging experiments, and the small TRPC5 currents evoked by intracellular application of ZnCl223. Part of the putative zinc-binding site has also been implicated in TRPC5 glutathionylation: mutation of C176, C178 or C181 prevented TRPC5 opening in response to glutathionylation41. A separate report proposed that palmitoylation of C181 is required for correct trafficking of TRPC5 to the plasma membrane in striatal neurons42. These data suggest that this small domain may be an important regulatory node of TRPC channels and that further experiments are needed to fully understand its role in TRPC channel biology.Intracellular Zn2+ ions. Our data are consistent with the description of a structure of hTRPC5 in complex with the xanthine HC-070 in a non-peer-reviewed study by Song et al.43, which appeared shortly after the initial disclosure of our study on bioRxiv. These studies lay the foundations for the structure-based design of TRPC1/4/5 modulators, and may therefore support the development of new TRPC1/4/5 chemical probes and drug candidates for an increasing number of therapeutic areas.To the best of our knowledge, our data provide the first structural insight into TRPC1/4/5 channel modulation and suggest direct modulatory roles for (phospho)lipids and Zn44. Briefly, human TRPC5 was cloned as a C-terminally truncated form (Δ766–975) with an N-terminal maltose-binding protein tag followed by a PreScission protease cleavage site (as in ref. 35) using TRPC5-SYFP231 as a PCR template for TRPC5, which contains the previously described V455M mutation45. Point mutations to the xanthine-binding site were introduced into TRPC5-SYFP2 in pcDNA4. Bacmids and baculoviruses were produced according to the Bac-to-Bac protocol (Invitrogen).All plasmids were cloned using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) and mutations were made using the Q5 Site-directed mutagenesis kit (New England Biolabs). The BacMam vector was a kind gift from Professor Eric Gouaux (Vollum Institute)2. After 24 h, 5 mM sodium butyrate (Sigma Aldrich) was added and the temperature was lowered to 30 °C. After a further 48 h, cells were harvested by centrifugation and frozen. The protein purification protocol was adapted from Duan et al.35. Unless stated otherwise, all detergents (and amphipol PMAL-C8) were supplied by Generon. For a typical purification, a 200 ml cell pellet was thawed and resuspended in 20 ml of 1% DDM, 0.1% CHS, 150 mM NaCl (Sigma Aldrich), 30 mM HEPES (Sigma Aldrich) pH 7.5, 1 mM DTT (Fisher Scientific Ltd) and protease inhibitor cocktail (Sigma Aldrich), and incubated by rotating at 4 °C for 1 h. After ultracentrifugation at 100,000×g for 1 h at 4 °C, the supernatant was incubated with 400 µl bed volume of pre-washed amylose resin (New England Biolabs) for 18 h, rotating at 4 °C. The resin was washed with 20 ml of 0.1 % digitonin and 0.01% CHS, 150 mM NaCl, 30 mM HEPES pH 7.5, 1 mM DTT. The resin was resuspended in 10 ml of 0.1% PMAL-C8, 150 mM NaCl, 30 mM HEPES pH 7.5, 1 mM DTT and rotated for 4 h at 4 °C. 100 mg of Biobeads (Bio-rad) were then added and incubated for 18 h, rotating at 4 °C, to remove detergents. The resin was washed with 20 ml of buffer containing no detergent  before eluting in 5 ml of the same buffer plus 40 mM maltose (Sigma Aldrich). The eluate was subjected to ultracentrifugation at 100,000×g for 1 h at 4 °C to remove any precipitated material and the supernatant was concentrated to the required concentration with 100 kDa cut off vivaspin 500 concentrators (Sigma Aldrich). In a typical purification protocol, 50–100 µg of purified TRPC5 was produced from 200 ml of cell suspension.P2 virus was added to 2.0 million per ml of Freestyle™ 293-F Cells (ThemoFisher Scientific) in Gibco FreeStyle 293 Expression Medium (Invitrogen) at a final volume of 10% at 37 °C and 5% CO−1 was added to the grid and the sample was stained as follows46. The grid was incubated with TRPC5 at room temperature for 30 s and the excess protein solution was wicked away using filter paper (Whatman Grade 1). This was followed by two successive incubations with uranyl acetate stain, in each of which 3 μl of 1% uranyl acetate was applied to the grid, followed by 1 min incubation and wicking away of excess liquid. The grid was then left to air dry. For Pico145-containing grids, Pico145 (10 mM in DMSO) was added to 2 mg ml−1 purified TRPC5 to a final concentration of 100 µM, which was subsequently diluted 50-fold in detergent-free wash buffer, resulting in a final concentration of 2 µM Pico145 and 0.04 mg ml−1 TRPC5. Negative stain grids were imaged using a Technai T12 microscope fitted with a LaB6 filament operating at 120 kV with a nominal magnification of ×49,000 on a 2 k × 2 K Gatan CCD camera.Sample quality was assessed by negative stain electron microscopy. Briefly, carbon-coated grids were glow-discharged for 40 s in a Pelco glow discharge unit. After glow-discharging, 3 μl of purified TRPC5 at 0.04 mg ml−1. A 3 µl aliquot of the sample was applied to a Quantifoil Cu R1.2/1.3, 300 mesh holey carbon grid, which had been glow-discharged for 30 s using a Pelco glow discharge unit. An FEI Vitribot was used to blot the grids for 6 s (blot force 6) at 100% humidity and 4 °C before plunging into liquid ethane. The grids were loaded into an FEI Titan Krios transmission electron microscope  operating at 300 kV, fitted with a Gatan K2 direct electron detector operating in counting mode. Automated data collection was carried out using EPU software (2.2) and Digital Micrograph (3.23) with a defocus range of −1 to −3 µm. A total of 3,482 micrographs were collected with a pixel size of 1.07 Å. The total dose, 75 e− Å−2, was acquired by use of a dose rate of 8.64 e− pixel−1 s−1 (7.58 e− Å−2 s−1) across 40 frames for 10 s total exposure time. The parameters for the structures in the presence of ZnCl2 or 50 uM Pico145 used 1 mg ml−1 final protein concentration and used the same EPU software with a defocus range of −1 to −3 µm. Micrographs were collected with a pixel size of 1.07 Å. The total dose, 60 e− Å−2, was acquired by using a dose rate of 8.54 e− pixel−1·s−1 (7.49 e−Å−2 s−1) across 40 frames for 8 s total exposure time.Pico145  was added to purified TRPC5 in PMAL-C8 at 2.0 mg ml47. The initial drift and beam-induced motion were corrected for using MotionCor248, and CTf estimation was performed using Gctf49. Automated particle picking was performed in crYOLO (1.3.5)50 using the general model. For the Pico145-bound structure, this resulted in a particle stack containing 612,983 particles that were imported into Relion-3.0.7. An initial round of 2D classification resulted in a particle stack consisting of 519,962 particles. A 3D initial model was generated in Relion and low-pass filtered to 60 Å when used in 3D classification. The best class, consisting of 266,887 particles, was taken forward and refined with C4 symmetry imposed to give a global resolution of 3.3 Å after post-processing, with resolutions estimated by the gold standard FSC = 0.143 criterion. Three iterative rounds of CTF refinement51 and particle polishing were completed, which improved the resolution of the map to 3.1 Å. The polished particles underwent two further rounds of 3D classification resulting in a particle stack containing 158,111 particles that were refined to 3.0 Å. A local resolution map was calculated in Relion which showed that the core of the structure was at a higher resolution (2.8 Å) than the global average. For the structure in the presence of ZnCl2, crYOLO picked 552,075 particles, which was reduced to 479,810 upon 2D classification. 3D classification further reduced this to 228,615 particles, which were refined and post-processed, resulting in a 3.1 Å structure. After three iterative rounds of CTF refinement and particle polishing, the resolution of the map was improved to 2.8 Å—further 3D classification did not improve the map. For the 50 µM  Pico145 structure, micrographs were subjected to crYOLO particle picking , followed by 2D classification , 3D classification . 3D classified particles were subjected to refinement and post-processing, resulting in a 3.1 Å structure. Three rounds of CTF refinement and particle polishing improved the overall resolution to 2.9 Å—further 3D classification did not improve the map.An overview of the image processing protocol is shown in Supplementary Fig. 52 were performed before fitting Pico145 into the map in WinCoot (0.8.9.2)53. Models and maps were visualised and figures prepared in UCSF Chimera (1.13.1)54.The model of TRPC5 with 100 µM Pico145 was produced by the manual fitting of 6aei into the model. Several rounds of real-space refinement in Phenix (1.16 −3549)55. The TRPC5 pdb file was prepared using the Protein Preparation Wizard in the Maestro Graphical User Interface (GUI). This aimed to remove any steric clashes of amino acid side chains and optimise the position of hydrogen atoms to facilitate docking studies. The receptor grid was generated using Schrödinger software, allowing docking of ligands in a 36×36×36 Å grid. The Pico145 ligand was prepared using the OMEGA module56 of OpenEye software  to produce an energy-minimised 3D structure before importing into the Maestro GUI. Docking of Pico145 was carried out using the Glide module of Schrödinger software using the XP mode with flexible ligand sampling and biased sampling of torsions for all predefined functional groups. Epik state penalties were added to the docking score. A maximum of 10 poses for the ligand was requested in the output file and post-docking minimisation was carried out.The region of the TRPC5 tetrameric structure assigned for docking studies was chosen based upon residues identified to be close Pico145 in our TRPC5:Pico145 structure . A 25 Å clip of the TRPC5 structure around these residues was termed as the receptor for docking studies using Glide 2+]i) was measured using the ratiometric Ca2+ dye Fura-2. HEK 293 cells  were plated in 6-well plates at 1 million cells per well.  The following day, cells were transfected with 2 µg DNA using 6 µl of JetPrime  according to the manufacturer’s protocol. The next day cells were washed, trypsinised and plated onto black, clear-bottom poly-D-lysine coated 96-well plates with 60,000 cells per well. 24 h after plating, the cells were loaded with the Fura-2 dye by removal of media and addition of SBS  containing 2 μM Fura-2 acetoxymethyl ester  and 0.01% pluronic acid (Merck) for 1 h at 37 °C. After this incubation, cells were washed with fresh SBS and incubated at room temperature for a further 30 min. SBS was then changed to recording buffer (SBS with 0.01% pluronic acid and 0.1% DMSO to match compound buffer) immediately prior to experimentation. In the case of inhibitor studies, the buffer was replaced with SBS with 0.01% pluronic acid and the relevant inhibitor or vehicle. Cells were then incubated for 30 min prior to the experiment. Measurements were carried out using a FlexStation and SoftMax Pro 7 software , using excitation wavelengths of 340 and 380 nm, at an emission wavelength of 510 nm. ([Ca2+]i) recordings was performed at room temperature at 5 s intervals for 300 s (unless stated otherwise). EA or AM237 was added by the FlexStation from a compound plate  after recording for 60 s. Responses were calculated at 240–300 s compared to the signal at baseline (0–60 s), and an average was taken within each individual experiment. Averages from individual experiments were normalised relative to the maximum signal for each TRPC5 variant/activator combination and to the vehicle control, combined and fit  with GraphPad Prism 8 with [agonist] for EA or AM237, or [inhibitor] for Pico145 vs response.Intracellular calcium  and N  have been defined in the figure legends.For intracellular calcium recordings, 24 and AM23731 were prepared according to previously reported procedures. (−)-englerin A (EA) was obtained from PhytoLab . AM237, Pico145 and EA were made up as 10 mM stocks in 100% DMSO, aliquots of which were stored at −20 °C (AM237 and Pico145) or −80 °C (EA). Further dilutions of compounds were made in DMSO and these were dissolved 1:1000 in a compound buffer (SBS + 0.01% pluronic acid) before being added to cells. Fura-2 AM (Invitrogen UK) was dissolved at 1 mM in DMSO.Pico145Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis  and fluorescent ligand binding. Structure–activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics—Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.The adenosine A Unsurprisingly therefore, A3R is a pharmaceutical target. Interestingly, the A3R has been described as enigmatic, whereby many of the effects attributed to A3Rs are contradictory1. Despite this, A3R antagonists having been described as potential treatments of asthma, chronic obstructive pulmonary disease (COPD) and glaucoma3, continuous research into antagonists at the A3R are warranted. It has also been suggested that the A3R is over expressed in various tumour cells suggesting it may be a viable drug target against cancer proliferation6. While a number of novel potent and selective A3R antagonists have been previously described9, one of the challenges associated with the druggability of the AR family has been the targeting of individual subtypes with sufficient specificity to limit off-target side effects10.The adenosine A2AR and A2BR are predominantly Gs-coupled whereas A1R and A3R generally couple to Gi/o. This classical pathway following A3R activation and Gi/o coupling is the inhibition of adenylate cyclase (AC) results in a decrease in cAMP levels, although extracellular signal-regulated kinase 1/2 (ERK1/2) activation has also been described11.Although all AR members are activated by the endogenous agonist adenosine, the A1R and A2AR are two of the best structurally characterised G protein-coupled receptors (GPCRs), with multiple structures available for both16, although the A3R structure is yet to be resolved. The limited availability of diverse high-resolution structures of the A3R bound to pharmacologically distinct ligands has meant there is a discrepancy between the capability to predict compound binding versus pharmacological behaviour17. In silico screening of vast compound libraries against receptor structures, known as structural-based drug design, offers huge potential in the development of highly selective ligands18. With this in mind, compounds K1-K25, K28 and K35, previously discovered as A3R ligands19, and the newly identified compounds K26, K27, K29-K34 and K36-K39 were pharmacologically characterised as potential antagonists using A3R-mediated inhibition of cAMP accumulation. Here, we describe the identification of a potent and selective A3R antagonist, K18 -5-methylisoxazol-4-yl]carbonyl}-2-methyl-1,3-thiazole-4-carbohydroximamide). Using molecular dynamic (MD) simulations, Molecular Mechanics—Poisson Boltzmann Surface Area (MM-PBSA) calculations and site-directed mutagenesis, we eluded its potential binding site. Kinetic binding experiments of K18 and its congener molecules K5, and K17 using a bioluminescence resonance energy transfer (BRET) method combined with functional assays led to the identification of important structural features of K18 for binding and activity. Further evaluation of this compound  may afford a novel therapeutic benefit in pathologies such as inflammation and asthma.The A3R antagonists some of which have previously been identified to bind A1R, A3R or A2AR using radio-labelled assays19. Our screen was carried out using A3R expressing Flp-In™-Chinese hamster ovary (CHO) cells where adenosine-3',5' cyclic monophosphate (cAMP) accumulation was detected following a combined stimulation of 10 μM forskolin , 1 μM tested compound and the predetermined IC80 concentration of 2S,3S,4R,5R)-5-(6-aminopurin-9-yl)-N-ethyl-3,4-dihydroxyoxolane-2-carboxamide (NECA) (3.16 nM). Compound K1-39 were identified by unblinding  to identify selective A–39 to id3R antagonist MRS 1220  appeared to be antagonists at the tested concentration of 1 μM  to have sub-micromolar binding affinities for A3R showed no activity in our cAMP-based screen  and increasing concentrations of NECA identified K1, K10, K17, K18, K25 and K32 as A3R selective antagonists  and are summarised in Table The similarity of the different ARs has the consequence that many compounds display reduced selectivity. Using the A3R antagonists were confirmed to antagonise -3,4-dihydroxy-5-[6-[(3-iodophenyl)methylamino]purin-9-yl]-N-methyloxolane-2-carboxamide (IB-MECA) , K18 was the most potent, showed A3R specificity and higher A3R binding affinity -2-(2-hex-1-ynyl-6-methylaminopurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol (HEMADO)19. To ensure the use of NECA or IB-MECA in our study did not influence characterisation of the compounds, we assessed the ability of K18 to antagonise cAMP inhibition by HEMADO at the A3R, and compared its potency to K17 . K18 was able to antagonise A3R-mediated phosphorylation of ERK1/2 with the antagonist potency  not significantly different compared to the cAMP-inhibition assay  group is positioned close to V1695.30, M1775.40, I2496.54 and L2647.34 of the A3R orthosteric binding site forming attractive vdW interactions. The amino group of the ligand is hydrogen-bonded to the amido group of N2506.55. The isoxazole ring is engaged in an aromatic π-π stacking interaction with the phenyl group of F1685.29 .We have previously investigated the binding mode of K18 at the human A3R, the ones which showed -according to the molecular dynamic (MD) simulations- the most frequent and the most important contacts were chosen for investigation and included amino acids L903.32, F1685.29, V1695.30, M1775.40, L2466.51, I2496.54, N2506.55, L2647.34 and I2687.39  of K18 at the WT and mutant A3R were compared in order to determine the potential antagonist binding site , whereas M177A5.40 and V169A5.30 were significantly smaller. Interestingly, we found an increase in the affinity for L90A3.32 and L264A7.34 when compared to WT. As would be expected, the K18 affinity at the A3R mutants was not different between agonists, confirming agonist independence  = 2.86 ± 0.89 × 107 M−1 min−1, Koff (k2) = 0.4397 ± 0.014 min−1 with a KD of 17.92 ± 4.45 nM.  with a determined Kon (k3) and Koff (k4) rate of 3.25 ± 0.28 × 108 M−1 min−1 and 0.0248 ± 0.005 min−1, respectively. This gave a residence time (RT) (RT = 1/Koff) of 40.32 min. It was noticed in the analysis for K5, K17 and K18 that the fit in some cases was ambiguous and/or the fitted value of the compound dissociation rate constant was high . In order to determine the reliability of the fitted k4 value, data were also analysed using an equation that assumes compound dissociation is too rapid for the dissociation rate constant to be determined reliably and the fits to the two equations were compared . This model allowed estimate of the equilibrium binding affinity of the compound (Ki) but not the binding kinetics of K5, K17 and K18  display cross species (human and rat) reactivity29. Comparison of residues of the binding area between human and rat A3R show that they differ in residues 1675.28, 1695.30, 1765.37, 2536.58, 2647.34    suggested that the presence of M2647.34 most likely hampers K18 binding due to steric hindrance of the dichloro-phenyl group  is oriented deep in the binding area making contacts with L903.32, L2466.51 and W2436.48. M2647.34 forces the large lipophilic moiety of the thiophene ring (3-NHCOCH2SPh(CF3)2) of K25 to locate close to TM2 favouring contacts with A692.61, V722.64, and I2687.39  of K18 (0.1 μM) was determined (0–60 min) as 24 min and the intrinsic clearance (CLint) calculated as 287.2 μl/min/mg  remaining after 120 min as 90%, with a t1/2 of > 120 min. This is considerably higher than the reference compound propantheline (1 μM) which was determined to have a half-life of 55 min. The t1/2 of K18 (1 μM) in PBS (pH 7.4) over 240 min was determined as > 240 min, with 87% remaining at 240 min and was comparable to the reference compound propantheline (1 μM), with a determined t1/2 of > 240 min.The metabolic in vitro 30. Given that AR subtypes play distinct roles throughout the body, obtaining highly specific receptor antagonists and agonists is crucial. The virtual screening described by Lagarias et al.19 used a combination of a ligand-based and structure-based strategies based upon the experimental structure of A2AR in complex with the selective antagonist/inverse agonist ZM24138531 (PDB ID 3EML32), which has little affinity for A3R and 500- to 1000-fold selectivity for A2AR over A1R. Although, our high hit rate for A3R selective antagonist appears counter-intuitive, since the ligand-based virtual screening tool Rapid Overlay of Chemical Structures (ROCS) was used to predict structures similar to ZM241385,19.The search for an AR subtype specific compound often leads to compounds active at more than one of the AR subtypes because of the broad and similar orthosteric binding site of ARs3R antagonists identified though virtual screening. Of these eight compounds, K10, K17, K18, K20, K23, K25 and K32 were determined to be competitive. Whereas K20 and K23 are antagonists at both the A1R and A3R, K10, K17, K18, K25 and K32 were are A3R selective antagonists. Indeed, we found no functional activity, or indeed binding affinity (< 30 μM), at the other AR subtypes. K1, K20 and K23 showed weak antagonism of the A2AR with no activity at the A2BR . However, a number of compounds previously determined to have micromolar binding affinity for A3R , show no antagonistic potency in our initial functional screen. Further testing confirmed that these compounds are low potency antagonists and, although supporting the previously published radioligand binding data, confirmed the need for functional testing: not all compounds with binding affinity show high functional potency.Here, we present the pharmacological characterisation of eight A3R, when expressed in Flp-In-CHO cells, displays constitutive activity. Compounds which preferably bind to the inactive (R) state, decreasing the level of constitutive activity33 and in the case of a Gi/o-coupled GPCR leading to an elevated cAMP, are referred to as inverse agonists. In this study all the A3R antagonists identified and both A1R antagonists (K20 and K23) were found to act as inverse agonists. We also report an elevation in cAMP accumulation when cells were stimulated with DMSO, which is concentration-dependent. Given that even low concentrations of DMSO have been reported to interfere with important cellular processes34, the interpretation of these data should be made with caution.We show the A3R affinity, as following 0Cl < 1Cl < 2Cl i.e. K5 < K17 < K18. This theory is supported by both radioligand binding, NanoBRET ligand-binding and functional data. Moreover, MD simulations show that these compounds adopt a similar binding mode at the A3R orthosteric binding site, but the free-energy MM-PBSA calculations show that K18, having two chlorine atoms and an increased lipophilicity, leaves the solution state more efficiently and enters the highly lipophilic binding area. Importantly, substitution of the 1,3-thiazole ring in K17 with either a 2-pyridinyl ring (K32) or a 3-pyridinyl ring (K10) but not a 4-pyridinyl ring (K11) maintains A3R antagonistic potency. Also, substitution of 1,3-thiazole ring in K17 with the p-iodo-phenyl in K9 loses the antagonistic potency. Although we have not directly determined the effects of similar pyridinyl ring substitutions on the higher affinity antagonist K18, we suspect there would be no significant increase in the potency of K18 given the small changes we observed for K17.We show that the presence of a chloro substituent in the phenyl ring of 3-phenyl-isoxazole favoured A28 (built into Prism 8.0) for fitting fast kinetics data obtained from NanoBRET experiments and assumes the unlabelled ligand rapidly equilibrates with the free receptor. Very rapid competitor dissociation can lead to failure of the fit, eliciting either an ambiguous fit  or unrealistically large K3 and K4 values. Whereas we were able to successfully fit the MRS 1220 kinetic data with the Motulsky and Mahan model due to its slow dissociation, fitting of K5, K17 and K18 kinetic data with this model often resulting in an ambiguous fit. Our new model, assuming fast compound dissociation, successfully fits the data and allows the determination of binding affinity. In the cases where the data were able to fit the Motulsky and Mahan model, the dissociation constant was higher (of the order of 1 min−1), indicating rapid dissociation. Although we found nearly a tenfold difference in determined binding affinity for MRS 1220, K5, K17 and K18 between BRET ligand binding and radioligand binding assays, we demonstrated the order of affinity remains consistent. Indeed, this is seen across all three experimental approaches: Schild analysis, NanoBRET ligand-binding assay and radioligand binding.For the first time, we demonstrated the utilisation of a new model which expands on the ‘Kinetic of competitive binding’ model3R compounds35. We previously reported22 no detectable Gi/o response following co-stimulation with forskolin and NECA or IB-MECA for A3R mutants F168A5.29, L246A6.51, N250A6.55 and I268A7.39 and our findings are in line with previous mutagenesis studies investigating residues important for agonist and antagonist binding at the human A3R37. Through performing Schild analysis  we experimentally determined the effect of receptor mutation on antagonist affinity for L90A3.32, V169A/E5.30, M177A5.40, I249A6.54 and L264A7.34 A3R. The pA2 value for I249A6.54 A3R is similar to WT, whereas M177A5.40 and V169A5.30 are significantly smaller suggesting these residues appear to be involved in K18 binding. Interestingly we found an increase in K18 affinity at L90A3.32 and L264A7.34 when compared to WT. Our detailed MD simulations, results published elsewhere21 have investigated the selectivity profile of K18 and have demonstrated that K18 failed to bind A1R and A2AR due to a more polar area close to TM5, TM6 when compared to A3R.Combining MD simulations with mutagenesis data, we presented a final binding pose of K18 which appears to be within the orthosteric binding site, involving residues previously described to be involved in binding of AWe have also performed a preliminary pharmacokinetic assessment of K18 to assess its potential as a lead compound for future use in drug design. Based upon our initial findings K18 has a metabolic half-life and intrinsic clearance equivalent to verapamil and terfenadine. K18 is highly stable in human plasma. As such these studies suggest K18 is an ideal candidate for future investigations although a more detailed pharmacokinetic assessment is required.3R-selective across species or at rat A3R alone are useful pharmacological tools to define the role of these receptors. The human and rat A3R display 72% homology38. The lack of rat A3R selective antagonists prompted us to investigate if our characterised A3R antagonists are potential antagonists at the rat A3R. We reported no binding of our lead A3R antagonist, K18, at the rat A3R and MD simulations suggest this is due to steric hinderance by M2647.34. This finding suggests that K18 may not only be A3R specific within the human ARs but may also be selective across species. Of the compounds that showed rat A3R binding , K25 show the highest binding affinity and represents an interesting candidate for further investigation. MD simulations show K25 forms a stable complex with rat A3R and we suggest a potential binding pose.Antagonists that are A3R antagonist which also warrants further investigation.In conclusion, we present findings of a unique scaffold (K18) which is both chemically and metabolically stable and as such can be used as a starting point for detailed structure–activity relationships and represents a useful tool that warrants further assessment. Furthermore, we introduce K25 as a potential rat A2, in a humidified atmosphere. This study used CHO cell lines as a model due to the lack of endogenous AR subtype expression . CHO-K1-A1R, CHO-K1-A2AR, CHO-K1-A2BR and CHO-K1-A3R cells were routinely cultured in Hams F-12 nutrient mix  supplemented with 10% Foetal bovine serum (FBS) . Flp-In-CHO cells purchased from Thermo Fisher Scientific (R75807) were maintained in Hams F-12 nutrient mix supplemented with 10% FBS containing 100 μg/mL Zeocin Selection Antibiotic (Thermo Fisher Scientific).Cell lines were maintained using standard subculturing routines as guided by the European Collection of Cell Culture (ECACC) and checked annually for mycoplasma infection using an EZ-PCR mycoplasma test kit from Biological Industries . All procedures were performed in a sterile tissue culture hood using aseptic technique and solutions used in the propagation of each cell line were sterile and pre-warmed to 37 °C. All cells were maintained at 37 °C with 5% COStable Flp-In-CHO cell lines were generated through co-transfection of the pcDNA5/FRT expression vector (Thermo Fisher Scientific) containing the gene of interest and the Flp recombinase expressing plasmid, pOG44 (Thermo Fisher Scientific). Transfection of cells seeded in a T25 flask at a confluency of ≥ 80% was performed using TransIT-CHO Transfection Kit , in accordance with the manufacturer’s instructions. Here, a total of 6 μg of DNA (receptor to pOG44 ratio of 1:9) was transfected per flask at a DNA:Mirus reagent ratio of 1:3 (w/v). 48 h post-transfection, selection using 600 μg/mL hygromycin B (Thermo Fisher Scientific) (concentration determined through preforming a kill curve) was performed for two days prior to transferring the cells into a fresh T25 flask. Stable Flp-In-CHO cell lines expressing the receptor of interest were selected using 600 μg/mL hygromycin B whereby the media was changed every two days. Successful mutant cell line generation for non-signalling mutants were confirmed by Zeocin sensitivity (100 μg/mL).3R expressing HEK 293 cell line along with the Nluc-tagged rat A3R pcDNA3.1 + construct for the generation of stable Nluc-tagged rat A3R expressing HEK 293 cells were kindly gifted to us by Stephen Hill and Stephen Briddon (University of Nottingham). HEK 293 cells in a single well of 6-well plate (confluency ≥ 80%) were transfected with 2 μg of DNA using polyethyleneimine  (Polysciences Inc) at a DNA:PEI ratio of 1:6 (w/v). Briefly, DNA and PEI were added to separate sterile tubes containing 150 mM sodium chloride (NaCl) , allowed to incubate at room temperature for 5 min, mixing together and incubating for a further 10 min prior to adding the combined mix dropwise to the cells. 48 h post-transfection, stable Nluc-rat A3R expressing HEK 293 cell were selected using 600 μg/mL Geneticin (Thermo Fisher Scientific) whereby the media was changed every two days. HEK 293 cell lines were routinely cultured in DMEM/F-12 GlutaMAX (Thermo Fisher Scientific) supplemented with 10% FBS .The Nluc-tagged human A3R originally in pcDNA3.1 +  was cloned into the pcDNA5/FRT expression vector and co-transfected with pOG44 to generate a stable Flp-In-CHO cell line. Mutations within the A3R were made using the QuikChange Lightening Site-Directed Mutagenesis Kit (Agilent Technologies) in accordance with the manufacturer’s instructions. The Nluc-tagged rat A3R pcDNA3.1 + construct, used in the generation of the stable Nluc-tagged rat A3R expressing HEK 293 cell line was kindly gifted to us by Stephen Hill and Stephen Briddon (University of Nottingham). All oligonucleotides used for mutagenesis were designed using the online Agilent Genomics ‘QuikChange Primer Design’ tool  and pur3R based on the 1,2,4-Triazoloquinoxalin-1-one linked to BY63039, was kindly gifted to us by Stephen Hill and Stephen Briddon (University of Nottingham). PMA was purchased from Sigma-Aldrich. Compounds under investigation were purchased from e-molecules and dissolved in DMSO. The concentration of DMSO was maintained to < 1.5% across all experiments .Adenosine, NECA, IB-MECA, HEMADO, DPCPX  and MRS 1220 were purchased from Sigma-Aldrich and dissolved in dimethyl-sulphoxide (DMSO). CA200645, a high affinity AR xanthine amine congener (XAC) derivative containing a polyamide linker connected to the BY630 fluorophore, was purchased from HelloBio  and dissolved in DMSO. AV039, a highly potent and selective fluorescent antagonist of the human A2AR and A2BR) or inhibition (A1R or A3R) experiments, cells were harvested and re-suspended in stimulation buffer (PBS containing 0.1% BSA and 25 μM rolipram) and seeded at a density of 2000 cells per well of a white 384-well Optiplate and stimulated for 30 min with a range of agonist concentrations. In order to allow the A1R/A3R mediated Gi/o response to be determined, co-stimulation with forskolin, an activator of AC40, at the indicated concentration (depending on cell line) was performed. Testing of potential antagonists was performed in a competition experiment where cells received a co-stimulation with forskolin, agonist and compound/DMSO control, without test compound pre-incubation. cAMP levels were then determined using a LANCE cAMP kit as described previously41. In order to reduce evaporation of small volumes, the plate was sealed with a ThermalSeal film (Excel Scientific) at all stages.For cAMP accumulation (A3R expressing Flp-In-CHO were seeded at a density of 2000 cells per well of a white 384-well Optiplate and stimulated with agonist and test compounds for 5 min at 37 °C. Plate reading was conducted using a Mithras LB 940 (Berthold technology). All results were normalised to 5 min stimulation with 1 μM PMA, a direct protein kinase C (PKC). To determine if the measured pERK1/2 level was Gi-mediated, we treated cells with Pertussis toxin (PTX) (Tocris Biosciences) for 16 h at 100 ng/mL prior to pERK assay.ERK1/2 phosphorylation was measured using the homogeneous time resolved fluorescence (HTRF) Phospho-ERK (T202/Y204) Cellular Assay Kit  two-plate format in accordance with the manufacturer’s instructions. A42. In brief, membranes for radioligand binding were prepared from CHO cells stably transfected with hAR subtypes in a two-step procedure. In the first step, cell fragments and nuclei were removed at 1000×g and then the crude membrane fraction was sedimented from the supernatant at 100,000×g. The membrane pellet was resuspended in the buffer used for the respective binding experiments and it was frozen in liquid nitrogen and stored at – 80 °C. For radioligand binding at the A1R, 1 nM [3H]CCPA was used, for A2AR 10 nM [3H]NECA and for A3R 1 nM [3H]HEMADO. Non-specific binding of [3H]CCPA was determined in the presence of 1 mM theophylline and in the case of [3H]NECA and [3H]HEMADO 100 μM R-PIA was used. Ki values from competition experiments were calculated using Prism  assuming competitive interaction with a single binding site. The curve fitting results  showed R2 values ≥ 0.99 for all compounds and receptors, indicating that the used one-site competition model assuming a Hill slope of n = 1 was appropriate.All pharmacological methods followed the procedures as described in the literature1R and A3R with a slow off-rate43. Using an N-terminally NanoLuc (Nluc)-tagged A3R expressing cell line, competition binding assays were conducted. The kinetic data was fitted with the ‘kinetic of competitive binding’ model28  to determine affinity (pKi) values and the association rate constant (Kon) and dissociation rates (Koff) for unlabelled A3R antagonists. In several cases this model resulted in an ambiguous fit . We developed a new model which expands on the ‘kinetic of competitive binding’ model to accommodate very rapid competitor dissociation, assuming the unlabelled ligand rapidly equilibrates with the free receptor. This method allows determination of compound affinity (pKi) from the kinetic data.Through the use of NanoBRET, real-time quantitative pharmacology of ligand-receptor interactions can be investigated in living cells. CA200645, acts as a fluorescent antagonist at both A3R, we conducted competition binding assays using Nluc-tagged rat A3R expressing HEK 293 cells and the fluorescent compound AV03939 rather than xanthine based CA200645, which have previously been reported as inactive at rat A3R44. For both human and rat A3R experiments, filtered light emission at 450 nm and > 610 nm (640–685 nm band pass filter) was measured using a Mithras LB 940 and the raw BRET ratio calculated by dividing the 610 nm emission with the 450 nm emission. Here, Nluc on the N-terminus of A3R acted as the BRET donor (luciferase oxidizing its substrate) and CA200645/AV039 acted as the fluorescent acceptor. CA200645 was used at 25 nM, as previously reported26 and AV039 was used at 100 nM . BRET was measured following the addition of the Nluc substrate, furimazine (0.1 μM). Nonspecific binding was determined using a high concentration of unlabelled antagonist, MRS 1220 at 10 nM or 10 μM, for human and rat A3R, respectively.In order to identify if the characterised compounds also bound the rat A28 (built into Prism 8.0) to determine the test compound association rate constant and dissociation rate constant. Data were fit to the “Kinetics of competitive binding” equation in Prism 8.0 :RL]t is specific binding at time t, N the Bmax, [L] the tracer concentration, [I] the unlabelled competitor compound concentration, k1 the tracer association rate constant, k2 the tracer dissociation rate constant, k3 the compound association rate constant and k4 the compound dissociation rate constant.Specific binding of tracer vs time data was analysed using the Motulsky and Mahan methodKi) but not the binding kinetics of the compound. The equation is,ρI is fractional occupancy of receptors not bound by L:kobs,+ I is the observed association rate of tracer in the presence of competitor, defined as,Data were also analysed using an equation that assumes compound dissociation is too rapid for the dissociation rate constant to be determined reliably and the fits to the two equations compared . This equation assumes rapid equilibration between compound and receptor and consequently provides an estimate of the equilibrium binding affinity of the compound  was estimated from the slope (k) of percentage compound remaining (In(%K18 remaining)) versus time (t1/2 =—In(2)/k), assuming first order kinetics. The intrinsic clearance  was calculated according to the following formula:Preliminary pharmacokinetic assessments of K18 was out-sourced to Eurofins Panlabs  and including tests for intrinsic clearance (human liver microsomes), plasma (human) stability and half-life in PBS. These tests were conducted in duplicate using a single concentration of K18 (0.1 μM or 1 μM) using the substrate depletion method. Here, the percentage of K18 remaining at various incubation times was detected using high-performance liquid chromatography mass spectrometry (HPLC–MS). Reference compounds  were supplied and tested alongside K18. The half-life , with all dose-inhibition or response curves being fitted using a 3-parameter logistic equation to calculate response range or Ei-coupled A1R and A3R  or stimulation for A2AR and A2BR , relative to NECA/IB-MECA . For cAMP experiments on A3R mutants, data was normalised to 100 μM forskolin, representing the maximum cAMP accumulation possible for each cell line. In the case of pERK1/2 response, normalisation was performed to PMA, a direct PKC activator providing the maximum pERK1/2 level of the system.In the rare cases where significant outliers were identified through the ROUT method ) , these were excluded from data analysis and presentation. Dose-inhibition/dose–response curves were normalised to forskolin, expressed as percentage forskolin inhibition for G2 values  for the potential antagonists. In cases where the Schild slope did not differ significantly from unity, the slope was constrained to unity giving an estimate of antagonist affinity (pKB). pA2 and pKB coincide when the slope is exactly unity. Through performing Schild analysis, whereby the pA2 is independent of agonist, we were able to experimentally determine the effect of receptor mutation on antagonist binding. The pA2 values obtained through conducting Schild analysis of K18 at WT and mutant A3R were compared in order to indicate important residues involved in K18 binding. Whereas an increase in the pA2 for a particular mutant when compared to WT suggested the antagonist was more potent, a decrease indicated a reduced potency.Schild analysis was performed to obtain pA46. Statistical analysis, performed using Prism 8.0, was undertaken for experiments where the group size was at least n = 5 and these independent values used to calculate statistical significance  using a one-way ANOVA with a Dunnett’s post-test for multiple comparisons or Students’ t-test, as appropriate. Any experiments conducted n < 5 should be considered preliminary. Compounds taken forward for further investigation after our preliminary screening (n = 3) were selected based on a high mean cAMP accumulation (> 80%).All experiments were conducted in duplicate  to ensure the reliability of single values. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology3R with K5, K17, K18 or MRS 1220 and rat A3R with K18 or K25 was based on a homology model of A2AR  and are detailed in Lagarias et al.21. Each ligand−protein complex was embedded in hydrated POPE bilayers. A simulation box of the protein–ligand complexes in POPE lipids, water and ions was built using the System Builder utility of Desmond . A buffered orthorhombic system in 10 Å distance from the solute atoms with periodic boundary conditions was constructed for all the complexes. The MD simulations were performed with Amber14 and each complex-bilayer system was processed by the LEaP module in AmberTools14 under the AMBER14 software package47. Amber ff14SB force field parameters48 were applied to the protein, lipid14 to the lipids, GAFF to the ligands49 and TIP3P50 to the water molecules for the calculation of bonded, vdW parameters and electrostatic interactions. Atomic charges were computed according to the RESP procedure51 using Gaussian0352 and antechamber of AmberTools1447. The temperature of 310 K was used in MD simulations in order to ensure that the membrane state is above the main phase transition temperature of 298 K for POPE bilayers53. In the production phase, the relaxed systems were simulated in the NPT ensemble conditions for 100 ns. The visualization of produced trajectories and structures was performed using the programs Chimera54 and VMD55. All the MD simulations were run on GTX 1060 GPUs in lab workstations or on the ARIS Supercomputer.Preparation of the complexes between human A3R was estimated by the 1-trajectory MM-PBSA approach56. Effective binding energies (ΔGeff) were computed considering the gas phase energy and solvation free energy contributions to binding—see appendix II22.Relative binding free energies of the complexes between K5, K17, K18, MRS 1220 and Ahttps://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to Pharmacology57, and are permanently archived in the Concise Guide to Pharmacology 2017/1858.Key protein targets and ligands in this article are hyperlinked to corresponding entries in Supplementary file 1