Pyrazolo-triazolo-pyrimidine Scaffold as a Molecular Passepartout for the Pan-Recognition of Human Adenosine Receptors

Adenosine receptors are largely distributed in our organism and are promising therapeutic targets for the treatment of many pathologies. In this perspective, investigating the structural features of the ligands leading to affinity and/or selectivity is of great interest. In this work, we have focused on a small series of pyrazolo-triazolo-pyrimidine antagonists substituted in positions 2, 5, and N8, where bulky acyl moieties at the N5 position and small alkyl groups at the N8 position are associated with affinity and selectivity at the A3 adenosine receptor even if a good affinity toward the A2B adenosine receptor has also been observed. Conversely, a free amino function at the 5 position induces high affinity at the A2A and A1 receptors with selectivity vs. the A3 subtype. A molecular modeling study suggests that differences in affinity toward A1, A2A, and A3 receptors could be ascribed to two residues: one in the EL2, E168 in human A2A/E172 in human A1, that is occupied by the hydrophobic residue V169 in the human A3 receptor; and the other in TM6, occupied by H250/H251 in human A2A and A1 receptors and by a less bulky S247 in the A3 receptor. In the end, these findings could help to design new subtype-selective adenosine receptor ligands.


Introduction
The effects of adenosine are mediated through a family of cell-surface G-proteincoupled receptors, which are currently classified into four adenosine receptor subtypes: A 1 , A 2A , A 2B , and A 3 .While the A 1 and A 3 receptors interact with G i and G o proteins, the mechanism of the A 2A and A 2B subtypes is the stimulation of adenylyl cyclase via G s proteins.The consequences of these interactions are a reduction, in the case of A 1 and A 3 , or an increase, in the case of A 2A and A 2B , in the cAMP levels as the second messenger.In addition, all four subtypes may positively couple to phospholipase C via different Gprotein subunits [1][2][3][4].It has also been demonstrated that adenosine receptors can activate different signal pathways not related to G proteins, like the β-arrestin one, which induces different responses with respect to the G-protein signals [3].
Considering the large distribution of adenosine in the organism, it is quite evident that adenosine receptors could be considered an important target for the treatment of several pathologies.In fact, in the last decades, different classes of potent and selective agonists and antagonists have been reported with the aim of characterizing and better understanding the pathophysiological role of adenosine receptor subtypes and their possible involvement in several disorders [5][6][7][8][9][10][11].In particular, A 1 antagonists are investigated for their effect on both cardiovascular and metabolic diseases [7]; A 2A and A 2B antagonists are attracting increasing attention as cancer immunotherapy agents, while A 2A is also under study for the treatment of neurodegenerative disorders (i.e., istradefylline is already approved as adjunctive therapy to levodopa in Parkinson's disease) [9,10]; and, finally, A 3 antagonists proved effective in preclinical animal models of brain ischemia and oxygen-glucose deprivation in hippocampal slices [6][7][8][9][10][11].Despite the undoubtedly therapeutic potential of these ligands, only istradefylline has been approved in the last years.The biggest problem of adenosine receptors is that adenosine signaling is almost ubiquitous in our body, leading to a very broad spectrum of effects that, at a therapeutic level, could lead to several unpleasant side effects [6].The pyrazolo-triazolo-pyrimidine nucleus substituted in positions 2, 5, N7, and N8 has already been detected as one of the representative scaffolds among human adenosine receptor antagonists [12][13][14].
In fact, it has been widely reported that by modulating the pattern of substitutions, in particular, at positions 5, N7, and N8, various compounds with different profiles of affinity and selectivity toward the four adenosine receptor subtypes have been obtained [15].The aim of this work is to try to better explain with the help of computational methodologies the influence of these patterns of substitutions on both affinity and selectivity vs. adenosine receptor subtypes.In particular, highlighting the specific residues in the four adenosine receptors that are probably responsible for the observed compounds' affinity profile could not only help rationalize the obtained results of these compounds and others already reported in the literature, but it could also aid the design of new potent and selective ligands.

Computational Methodologies
The molecular modeling studies have been accomplished on a workstation equipped with a 20-core Intel Core i9-9820X 3.3 GHz processor and by running Ubuntu 20.04 as operating system.

Protein Preparation
X-ray structures with PDB IDs 5UEN [16] and 4EIY [17] were retrieved for hA 1 AR and hA 2A AR.The structures were pre-processed by removing apocytochrome b(562)RIL, replacing IL3, and reverting mutations of the crystal construct into wild type.The structures were then prepared with the Structure Preparation tool of the Molecular Operating Environment (MOE) 2022.02suite [18] by adding missing atoms/residues/loops and capping N/C-terminals (which were not reconstructed) with acetyl and N-methyl group.Protomeric and tautomeric states were optimized with the Protonate3D tool, and hydrogens were then minimized with Amber 14:EHT force field.Non-protein atoms were removed.The Na + ion, acting as negative allosteric modulator [19], and three coordination water molecules, co-crystallized to hA 2A AR on structure 4EIY, were kept for docking studies.Na + and water molecules were also aligned to hA 1 AR structure, minimized, and kept for further modeling.The influence of Na+ on antagonists' docking was assessed before, showing how the posing accuracy is affected by the presence of Na + [20].
Structures for hA 2B and hA 3 AR were built by homology modeling using the prepared structures of hA 2A and hA 1 AR, respectively.Extracellular Loop 2 (EL2) (from residue N145 to residue G160) was removed from the A 2A AR structure template (4EIY).The models were generated using Prime of the Schrödinger suite [21,22] with knowledge-based setting and keeping ligands, Na + , and the three selected water molecules as modeling environment.Non-conserved residues were minimized with OPLS4 force field.
The Ballesteros-Weinstein GPCR numbering scheme, based on counting residues from the most conserved positions in each transmembrane helix (TM), has been employed throughout the manuscript and extracted from the GPCRdb website [23,24].
In order to enable docking of bulky compounds (1-7), an induced-fit docking procedure was employed, using compound 4 as reference (the bulkiest compound with reported binding affinity for all AR subtypes).In particular, the induced-fit docking tool of the Schrödinger package was used [26], optimizing side chains within 5 Å of the docking poses and using SP as scoring function for Glide redocking.The trimming option was included for side chains of residues occluding the binding pocket such as E172 EL2 , M177 5.35 , and T270 7.35 in the case of hA 1 AR; E169 EL2 , M174 5.35 , H264 EL3 , and M270 7.35 in the case of hA 2A AR; E174 EL2 , M179 5.35 , N266 EL3 , and M272 7.35 in the case of hA 2B AR; and V169 EL2 , M174 5.35 , I253 6.58 , and L264 7.35 in the case of hA 3 AR.A refined structure was selected for each receptor with visual inspection (bidentate H-bond with N6.55) and used for Glide-SP docking of compounds 1-7.
Docking results were filtered by excluding poses with positive ligand-receptor van der Waals and electrostatic interaction potential (computed in MOE with Amber 14:EHT force field and assigning PM3 partial charged to ligands).Poses were ranked on the basis of electrostatic interaction potential, and one or two poses per compound were selected, prioritizing diverse conformations showing a bidentate hydrogen bond (or approximate one) with N6.55 using visual inspection.
The selected poses are reported in Videos S1-S4 generated using a Python script and the following tools: UCSF Chimera for 3D structure representation [27], MOE for per-residue electrostatic and hydrophobic interaction computation, Gnuplot for heat-map plotting, MEncoder for video assembly.

Chemistry
Synthesis and characterization of compounds 1-7 have already been reported in the literature [28,29]; for new experiments on A 2B receptor, the same batch of compounds 1-4 synthetized before have been used.Briefly, as reported in Scheme 1, the 8-alkyl- The Ballesteros-Weinstein GPCR numbering scheme, based on counting residues from the most conserved positions in each transmembrane helix (TM), has been employed throughout the manuscript and extracted from the GPCRdb website [23,24].
In order to enable docking of bulky compounds (1-7), an induced-fit docking procedure was employed, using compound 4 as reference (the bulkiest compound with reported binding affinity for all AR subtypes).In particular, the induced-fit docking tool of the Schrödinger package was used [26], optimizing side chains within 5 Å of the docking poses and using SP as scoring function for Glide redocking.The trimming option was included for side chains of residues occluding the binding pocket such as E172 EL2 , M177 5.35 , and T270 7.35 in the case of hA1 AR; E169 EL2 , M174 5.35 , H264 EL3 , and M270 7.35 in the case of hA2A AR; E174 EL2 , M179 5.35 , N266 EL3 , and M272 7.35 in the case of hA2B AR; and V169 EL2 , M174 5.35 , I253 6.58 , and L264 7.35 in the case of hA3 AR.A refined structure was selected for each receptor with visual inspection (bidentate H-bond with N6.55) and used for Glide-SP docking of compounds 1-7.
Docking results were filtered by excluding poses with positive ligand-receptor van der Waals and electrostatic interaction potential (computed in MOE with Amber 14:EHT force field and assigning PM3 partial charged to ligands).Poses were ranked on the basis of electrostatic interaction potential, and one or two poses per compound were selected, prioritizing diverse conformations showing a bidentate hydrogen bond (or approximate one) with N6.55 using visual inspection.
The selected poses are reported in Videos S1-S4 generated using a Python script and the following tools: UCSF Chimera for 3D structure representation [27], MOE for perresidue electrostatic and hydrophobic interaction computation, Gnuplot for heat-map plotting, MEncoder for video assembly.

Chemistry
Synthesis and characterization of compounds 1-7 have already been reported in the literature [28,29]; for new experiments on A2B receptor, the same batch of compounds 1-4 synthetized before have been used.Briefly, as reported in Scheme 1, the 8-alkyl

Biology 2.3.1. Binding at Human A 1 , A 2A , A 2B , and A 3 Adenosine Receptors
All pharmacological methods followed the procedures described earlier [30].In brief, membranes for radioligand binding were prepared from CHO cells stably transfected with human adenosine receptor subtype (obtained as reported in reference [30]) in a two-step procedure.In a first low-speed step (1000× g), cell fragments and nuclei were removed.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, frozen in liquid nitrogen, and stored at −80 • C.
Compounds were dissolved in DMSO and then diluted to the desired concentration in buffer, and at least 6 different concentrations were tested.DMSO in the final solution never exceeded 2%.For radioligand binding at A 1 adenosine receptors, 1 nM [ 3 H]CCPA was used, whereas 30 and 10 nM [ 3 H]NECA were used for A 2A and A 3 receptors, respectively.Non-specific binding of [ 3 H]CCPA was determined in the presence of 1 mM theophylline; in the case of [ 3 H]NECA, 100 µM R-PIA was used [30,31].For radioligand binding assay of compounds 1-4 at A 2B receptors, membranes (20 µg membrane protein) were incubated in a total volume of 200 µL (assay buffer: Tris/HCl pH 7.4, 10 mM MgCl 2 , 0.1% bovine serum albumin) with 10 nM [ 3 H]ZM241385.Non-specific binding of [ 3 H]ZM241385 was determined in the presence of 0.3 mM NECA.After 3 h at room temperature, samples were filtered and washed as described [31].K i values from competition experiments were calculated with the program SCTFIT [32] and represented the mean of 3-6 replicates with 95% confidence limits.Binding data for compounds 1-12 at the A 1 , A 2A , and A 3 adenosine receptors were taken from our previous works [28,29,33].Binding data toward A 2B receptors for compounds 8-12 were already reported in a previous work following a different procedure [33].

Adenylyl Cyclase Activity in CHO Cells Expressing hA 2B Receptors
Functional studies for the hA 2B adenosine receptors were performed using adenylyl cyclase experiments.Minor modifications were carried out on the previously reported procedures [30,34].In this experimental procedure, the homogenate of hA 2B -CHO cells was subjected to high-speed centrifugation, and the sedimented membrane pellet was then resuspended in buffer (50 mMTris/HCl pH 7.4) and directly used for the assay.IC 50 values of antagonists were determined by their concentration-dependent inhibition of NECAstimulated adenylyl cyclase activity (NECA, 5 µM).About 150,000 cpm of [α-32 P]ATP was incubated with membranes and the incubation mixture for 20 min without EGTA and NaCl [34].Hill equation was used to calculate IC 50 which was the mean of 3-6 replicates.Hill coefficients were near unity.1, all the compounds showed affinities in the nanomolar range at the four adenosine receptor subtypes with different levels of selectivity.An appropriate discussion regarding the structure-activity relationship profiles of these compounds has been extensively reported [13][14][15]33]; nevertheless, a brief summary may be necessary to better understand the computational studies.

As depicted in Table
It is quite evident that the simultaneous introduction of bulky acyl moiety at the N5 position and small alkyl groups at the N8 position led to derivatives (e.g., compounds 1, 2) with high affinity at the A 3 adenosine receptor (AR) with good levels of selectivity vs. the other receptor subtypes.The increasing size of the N8 substituent (e.g., compound 5) led to a significant reduction in affinity at the A 3 subtype with a reduction in the levels of selectivity.It should be otherwise noted that this pattern of substitution led also to a good affinity at the A 2B ARs (compounds 1-4), but the highest affinity was still retained at the A 3 subtype.Instead, the introduction of thienyl moiety at the N5 position (e.g., compound 6) induced a reduction in both affinity and selectivity for the A 3 AR subtype.  1 Displacement of specific [ 3 H]CCPA binding at human A 1 receptors expressed in CHO cells. 2 Displacement of specific [ 3 H]NECA binding at human A 2A receptors expressed in CHO cells. 3Displacement of specific [ 3 H]ZM241385 binding at human A 2B receptors expressed in CHO cells. 4Displacement of specific [ 3 H]DPCPX binding at human A 2B receptors expressed in HEK-293 cells. 5IC 50 values of the inhibition of NECA-stimulated adenylyl cyclase activity in CHO cells expressing hA 2B receptors. 6Displacement of specific [ 3 H]NECA binding at human A 3 receptors expressed in CHO cells.Data are expressed as geometric means with 95% confidence limits; n.d.= not determined.
In contrast, the derivatives with the free amino function at the 5 position induced a different pattern of affinity.In fact, all the compounds showed high affinity (0.8-2.8 nM) at the A 2A AR without selectivity vs. the A 1 and A 2B subtypes while possessing affinity in the high nanomolar range (300-700 nM) at the human A 3 AR, confirming the fundamental role of the bulky substituent at the N5 position for A 3 AR recognition and the free amino function for A 2A /A 1 interaction.
With the aim of rationalizing this behavior, we performed molecular modeling studies investigating the capability of these compounds to assume a reasonable bound state at the orthosteric binding pocket of the hA 1 , hA 2A , hA 2B , and hA 3 ARs.
The four AR subtypes share a high sequence identity, with values ranging around 40% (Table S1).In particular, hA 1 AR shares 46% residues with hA 3 AR, 43% with hA 2B AR, and 38% with hA 2A AR, with sequence similarity values above 48% in all cases (see Table S1).Moreover, hA 2A AR has a 45% sequence identity with hA 2B AR and 30% with hA 3 AR, which shares 35% residues with hA 2B AR.Most of the dissimilarities are located in the extracellular and intracellular loop regions, with transmembrane (TM) helices having even higher similarities (the TM sequence identity being between a minimum of 45% (A 2B /A 3 ) and a maximum of 66% (A 2A /A 2B ), see Table S1).
The availability of X-ray tridimensional (3D) structures for the antagonist-bound A 2A and A 1 ARs in the inactive state provided a starting point for the modeling studies.In particular, structures with PDB code 4EIY [17] and 5UEN [16] were used for the hA 2A and hA 1 ARs, respectively.The choice was driven by prioritizing higher-resolution structures with a low number of mutations in the crystallographic construct.Moreover, the choice of structure 4EIY for A 2A AR was made for a receptor co-crystallized with the inverse agonist ZM-241385, characterized by a 7-amino-2-(furan-2-yl)- [1,2,4]triazolo[1,5-a][1,3,5]triazine scaffold highly similar to that of the 5-amino-2-(furan-2-yl)- [1,2,4]triazolo [1,5-c]pyrimidin of the series of compounds reported here, suggesting the PDB structure, affected by inducedfit phenomena, to be a good candidate for docking studies.
No experimental structure is available for A 3 AR, and only agonist-bound G-proteinbound cryo-Electron Microscopy structures are available for A 2B AR [35,36].To be consistent throughout the AR subtype comparison and avoid activation-state-dependent differences, the 3D structures of the A 2B and A 3 ARs were modeled using A 2A AR's (see alignment in Figure S1) and A 1 AR's (see alignment in Figure S2) inactive-state structures as templates, respectively, which provided the highest similarity to the couples A 1 /A 3 (46% sequence identity, 65% sequence similarity) and A 2A /A 2B (45% sequence identity, 56% similarity).
Compounds 1-12 were docked at the four AR structures using a semi-rigid docking approach (ligand flexible/protein rigid), and one or more reasonable binding modes could be predicted just for compounds 8-12 at all receptor subtypes.Two reasonable binding modes were selected for almost all the compounds, prioritizing poses with a bidentate hydrogen bond with N6.55 considering the importance of this residue for antagonist and agonist binding [37,38].
In the case of hA 2A AR (Figure 1B, Video S2), the poses with the best electrostatic interaction potential resembled the X-ray conformation of the 7-amino-2-(furan-2-yl)- [1,2,4]triazolo [1,5-a] [1,3,5]triazine portion of ZM-241385 (Figure S3).The exocyclic 5-amino group and nitrogen N3 are involved in a bidentate hydrogen bond with N253 6.55 , and the aromatic pyrazolo-triazolo-pyrimidine scaffold participates in a pi-pi stacking with F168 on EL2.The furane ring points deep into the binding pocket, facing H250 6.52 , where a T-shaped pi-pi interaction could occur.The alkyl group at position 8 points toward an area delimited by TM7/1/2/3 and EL2.The 5-amino group is further stabilized by a hydrogen bond with a glutamate on EL2 (E169), which is solvent exposed, but held in proximity to the top of the orthosteric pocket by an ionic interaction with H264 EL3 in most of the A 2A AR X-ray structures.A favorable electrostatic interaction potential is reported for E169 and compounds 8-12 (Video S2).An analogue situation was observed in the case of hA 2B AR (Figure 1D and Video S3).
Also, in the case of hA 1 AR, hA 2B AR, and hA 3 AR, a ZM-241385-like pose was generated for almost all compounds 8-12 (except compound 10 in the case of hA 1 AR and hA 3 AR) (Figure 1A,D,C and Videos S1, S3, and S4).In the case of hA 1 AR and hA 2B AR (Videos S1 and S3, Figure 1A,D), the key residues mentioned above for hA 2A AR are all conserved, with F171 EL2 , E172 EL2 , H251 6.52 , and N254 6.55 of hA 1 AR and F173 EL2 , E174 EL2 , H251 6.52 , and N254 6.55 of hA 2B AR acting like F168 EL2 , E169 EL2 , H250 6.52 , and N253 6.55  of hA 2A AR.The electrostatic interaction between E172 EL2 of hA 1 AR and compounds 8-12 is not as evident as that in the case of hA 2A AR (Video S1), and no direct hydrogen bond was observed between the residues and the 5-amino group.However, this is due to the conformation of E172 EL2 in the X-ray structure, and alternative conformation might be expected in a dynamic solvent-exposed environment.Differently, in the case of hA 3 AR, the position of E169(hA 2A )/E172(hA 1 )/E174(hA 2B ) is occupied by a hydrophobic residue, such as V169 (Figure S4).The favorable interaction between negatively charged and polar residues at this position with the free amino group at position 5 is, thus, missing.Moreover, a further difference can be noted: at position 6.52, instead of the Histidine that characterizes all the other subtypes, a less bulky Serine group (S247 6.52 ) is present that is incapable of making a T-shaped pi-pi interaction with the furane ring of the compounds.Together with this, a Serine residue (S181 5.42 ) also replaces an asparagine at position 5.42, which is conserved in all the other AR subtypes, creating more space at the bottom of the binding pocket.
in the X-ray structure, and alternative conformation might be expected in a dynamic solvent-exposed environment.Differently, in the case of hA3 AR, the position of E169(hA2A)/E172(hA1)/E174(hA2B) is occupied by a hydrophobic residue, such as V169 (Figure S4).The favorable interaction between negatively charged and polar residues at this position with the free amino group at position 5 is, thus, missing.Moreover, a further difference can be noted: at position 6.52, instead of the Histidine that characterizes all the other subtypes, a less bulky Serine group (S247 6.52 ) is present that is incapable of making a T-shaped pi-pi interaction with the furane ring of the compounds.Together with this, a Serine residue (S181 5.42 ) also replaces an asparagine at position 5.42, which is conserved in all the other AR subtypes, creating more space at the bottom of the binding pocket.An alternative reasonable binding mode can be observed for most of compounds 8-12 (Figure S5) which is highly ranked in terms of electrostatic interaction potential for the hA1 and hA3 ARs.The alternative conformation maintains the pi-pi stacking interaction with the phenylalanine on EL2 and the bidentate hydrogen bond with N6.55 on TM6.These poses engage N6.55 through the exocyclic amino group at position 5 and through N6 instead of N3.The 8-alkyl group faces the bottom of the binding pocket, while the furanyl group points toward the extracellular tip of TM2.This pose is also observed in the case of the hA2A and hA2B ARs but seems unfavored compared to the ZM-241385-like one because of a major inward position and consequent steric hindrance of TM2 as compared to the hA1 and hA3 ARs.
For compounds 1-7, it was not possible to observe the aforementioned binding modes by conducting docking studies.A different approach has been adopted to relieve possible clashes of the receptors with the bulky groups at position N5.The bulkiest compound showing binding affinity at all four receptor subtypes, compound 4, was selected to optimize the conformation of the binding pocket using induced-fit docking.The refined structures were then used for docking all compounds 1-7.With this procedure, a ZM-241385-like pose was obtained for all the compounds at all four AR subtypes.The exocyclic 5-amino group and nitrogen N3 are engaged in a bidentate hydrogen bond (or approximate one) with N6.55, occupied by N254, N253, N254, and N250 in the hA1, hA2A, hA2B, and hA3 ARs, respectively, and the aromatic scaffold is involved in pi-pi interactions with F171, F168, F173, and F168 on EL2 (Figure 2A-D and Videos S1-S4).The N5 An alternative reasonable binding mode can be observed for most of compounds 8-12 (Figure S5) which is highly ranked in terms of electrostatic interaction potential for the hA 1 and hA 3 ARs.The alternative conformation maintains the pi-pi stacking interaction with the phenylalanine on EL2 and the bidentate hydrogen bond with N6.55 on TM6.These poses engage N6.55 through the exocyclic amino group at position 5 and through N6 instead of N3.The 8-alkyl group faces the bottom of the binding pocket, while the furanyl group points toward the extracellular tip of TM2.This pose is also observed in the case of the hA 2A and hA 2B ARs but seems unfavored compared to the ZM-241385-like one because of a major inward position and consequent steric hindrance of TM2 as compared to the hA 1 and hA 3 ARs.
For compounds 1-7, it was not possible to observe the aforementioned binding modes by conducting docking studies.A different approach has been adopted to relieve possible clashes of the receptors with the bulky groups at position N5.The bulkiest compound showing binding affinity at all four receptor subtypes, compound 4, was selected to optimize the conformation of the binding pocket using induced-fit docking.The refined structures were then used for docking all compounds 1-7.With this procedure, a ZM-241385-like pose was obtained for all the compounds at all four AR subtypes.The exocyclic 5-amino group and nitrogen N3 are engaged in a bidentate hydrogen bond (or approximate one) with N6.55, occupied by N254, N253, N254, and N250 in the hA 1 , hA 2A , hA 2B , and hA 3 ARs, respectively, and the aromatic scaffold is involved in pi-pi interactions with F171, F168, F173, and F168 on EL2 (Figure 2A-D and Videos S1-S4).The N5 substituent increases the contacts with the hydrophobic residues in the binding site in all cases (Videos S1-S4).A hydrogen bond with E172/E169/E174 cannot occur in the case of these compounds with a consequent minor stabilization of the bound state in the hA 1 , hA 2A , and hA 2B ARs.Moreover, the E172 EL2 -H264 EL3 ionic interaction observed in hA 2A AR would also be disrupted, contributing to a loss of ligand stabilization.
substituent increases the contacts with the hydrophobic residues in the binding site in all cases (Videos S1-S4).A hydrogen bond with E172/E169/E174 cannot occur in the case of these compounds with a consequent minor stabilization of the bound state in the hA1, hA2A, and hA2B ARs.Moreover, the E172 EL2 -H264 EL3 ionic interaction observed in hA2A AR would also be disrupted, contributing to a loss of ligand stabilization.

Discussion
In this work, twelve already-known pyrazolo-triazolo-pyrimidines were used to try to explain their profile of affinity and selectivity toward the four adenosine receptor subtypes through the identification of differences and/or similarities in the interaction patterns with the residues of the single subtypes.The four AR subtypes are characterized by high sequence identity in the orthosteric binding pocket; thus, the rationalization of ligand binding selectivity at different AR subtypes is not an easy task.It is reported that extracellular loops, especially EL2, might play a role in the ligand recognition process, especially in determining metastable binding sites along the ligand-receptor binding pathway [39][40][41].In the current study, the contribution of EL2 or other ELs in the binding process has not been investigated, and, consequently, its role in affecting affinity and selectivity remains to be elucidated.The usage of docking, maintaining full rigidity on the protein structure (regardless of the use of an induced-fit docking technique), does not allow the investigation of the binding kinetics or metastable binding sites along the binding root.Molecular Dynamics (MD) simulations and Supervised Molecular Dynamics (SuMD) [42] could aid in this perspective, but a major limitation still takes place.For A3 AR, there is still no availability of 3D experimental structures, and for hA2B AR, the newly released cryo-EM structures in the active state have still not solved the coordinates of EL2 [35,36].Considering that the reliability of loop modeling is poor, as long as an experimental 3D structure is not released to the scientific community, selectivity studies will still be limited.

Discussion
In this work, twelve already-known pyrazolo-triazolo-pyrimidines were used to try to explain their profile of affinity and selectivity toward the four adenosine receptor subtypes through the identification of differences and/or similarities in the interaction patterns with the residues of the single subtypes.The four AR subtypes are characterized by high sequence identity in the orthosteric binding pocket; thus, the rationalization of ligand binding selectivity at different AR subtypes is not an easy task.It is reported that extracellular loops, especially EL2, might play a role in the ligand recognition process, especially in determining metastable binding sites along the ligand-receptor binding pathway [39][40][41].In the current study, the contribution of EL2 or other ELs in the binding process has not been investigated, and, consequently, its role in affecting affinity and selectivity remains to be elucidated.The usage of docking, maintaining full rigidity on the protein structure (regardless of the use of an induced-fit docking technique), does not allow the investigation of the binding kinetics or metastable binding sites along the binding root.Molecular Dynamics (MD) simulations and Supervised Molecular Dynamics (SuMD) [42] could aid in this perspective, but a major limitation still takes place.For A 3 AR, there is still no availability of 3D experimental structures, and for hA 2B AR, the newly released cryo-EM structures in the active state have still not solved the coordinates of EL2 [35,36].Considering that the reliability of loop modeling is poor, as long as an experimental 3D structure is not released to the scientific community, selectivity studies will still be limited.
Nevertheless, comparing possible binding final states can still be informative, at least to assess the capability of the competitive antagonists to be hosted in the orthosteric sites of ARs.
As mentioned before, the pyrazolo-triazolo-pyrimidines reported in this paper show binding capacity at all AR subtypes.Compounds 8-12, characterized by a free amino group at position 5, show a binding affinity in the nanomolar range in the case of the hA 2A , hA 2B , and hA 1 ARs, while they reach an affinity of hundreds of nanomolar in the case of hA 3 AR.This trend is inverted for compounds acylated at position N5, with increased

Table 1 .
[28,29]cal profile at the four human adenosine receptor subtypes of the pyrazolo-triazolopyrimidine compounds 1-12.R and R 1 refer to general structure depicted in Scheme 1.All data were already reported by[33]and Michielan et al. (1-7)[28,29], except for binding data at hA 2B receptor for compounds 1-4 which are reported for the first time in this work.