The signaling and selectivity of α‐adrenoceptor agonists for the human α2A, α2B and α2C‐adrenoceptors and comparison with human α1 and β‐adrenoceptors

Abstract α2‐adrenoceptors, (α2A, α2B and α2C‐subtypes), are Gi‐coupled receptors. Central activation of brain α2A and α2C‐adrenoceptors is the main site for α2‐agonist mediated clinical responses in hypertension, ADHD, muscle spasm and ITU management of sedation, reduction in opiate requirements, nausea and delirium. However, despite having the same Gi‐potency in functional assays, some α2‐agonists also stimulate Gs‐responses whilst others do not. This was investigated. Agonist responses to 49 different α‐agonists were studied (CRE‐gene transcription, cAMP, ERK1/2‐phosphorylation and binding affinity) in CHO cells stably expressing the human α2A, α2B or α2C‐adrenoceptor, enabling ligand intrinsic efficacy to be determined (binding KD/Gi‐IC50). Ligands with high intrinsic efficacy (e.g., brimonidine and moxonidine at α2A) stimulated biphasic (Gi‐Gs) concentration responses, however for ligands with low intrinsic efficacy (e.g., naphazoline), responses were monophasic (Gi‐only). ERK1/2‐phosphorylation responses appeared to be Gi‐mediated. For Gs‐mediated responses to be observed, both a system with high receptor reserve and high agonist intrinsic efficacy were required. From the Gi‐mediated efficacy ratio, the degree of Gs‐coupling could be predicted. The clinical relevance and precise receptor conformational changes that occur, given the structural diversity of compounds with high intrinsic efficacy, remains to be determined. Comparison with α1 and β1/β2‐adrenoceptors demonstrated subclass affinity selectivity for some compounds (e.g., α2:dexmedetomidine, α1:A61603) whilst e.g., oxymetazoline had high affinity for both α2A and α1A‐subtypes, compared to all others. Some compounds had subclass selectivity due to selective intrinsic efficacy (e.g., α2:brimonidine, α1:methoxamine/etilefrine). A detailed knowledge of these agonist characteristics is vital for improving computer‐based deep‐learning and drug design.


| INTRODUC TI ON
α2-adrenoceptors, comprising α2A, α2B and α2C-subtypes, are Gicoupled G-protein coupled receptors (GPCRs) expressed in heart, blood vessels and kidney (important for blood pressure 1 ), but also on platelets and in brain. 2,3 Clonidine, the prototypical α2-agonist developed in 1962 as a nasal decongestant/topical vasoconstrictor, caused unexpected bradycardia, hypotension and sedation (as noted by the trial physician who allowed his secretary to administer herself a few drops of nasal clonidine as she had a cold: she unexpectedly fell asleep for 24 h, and became bradycardic and hypotensive, but fully recovered), leading to the development of centrally-acting α2agonist drugs. 3,4 Now, central activation of α2-adrenoceptors is the main target for α2-agonist antihypertensive drugs along with more recent α2-adrenoceptor neurological and psychiatric modulation. 3,[5][6][7] Central α2-adrenoceptors include presynaptic autoreceptors, where noradrenaline activation inhibits further noradrenaline release from the same neuron, pre-synaptic heteroreceptors where noradrenaline activation inhibits the release of other neurotransmitters, and postsynaptic receptors. 3,[5][6][7][8][9] After clonidine, further α2-agonists were developed with different properties, such as less lipophilic brimonidine (UK14304) aiming to reduce blood brain barrier transmission and sedation. 10,11 Brimonidine was also more efficacious, similar to adrenaline and noradrenaline, while clonidine had partial agonist activity. 12,13 In the brain, 90% of α2-adrenoceptors are α2A-adrenoceptors (as measured by receptor number not mRNA) and are highly expressed throughout, including the prefrontal cortex and locus coeruleus. 6,14,15 Many physiological and pharmacological functions, and therefore targets for clinical α2-agonists, are through activation of these α2A-adrenoceptors. 2,5,15 As well as antihypertensive properties, α2-agonists are now used for sedation, to improve delirium, for ADHD, help with panic and pain, and to minimse withdrawal symptoms from opioids, benzodiazepines, alcohol and nicotine. 16 A broad range of α2-agonists exist with different pharmacological and physicochemical properties and clinical uses. Dexmedetomidine is one of the most potent α2-agonists to date 17 and is increasingly used in intensive care. It is used to sedate people requiring prolonged ventilation, induce short-term sedation for procedures, as an adjunct to reduce doses of other sedatives (where a particular benefit is its lack of respiratory depression), reduce opiate consumption, reduce nausea and reduce delirium often seen post-operatively and in intensive care patients. 16,18,19 It also has potential to help with delirium, agitation and induce sedation in the palliative care setting. 19 Furthermore, dexmedetomidine acts through endogenous sleep pathways, 20 mimicking natural sleep and has a unique window for inducing "arousal" or "cooperative" sedation, enabling neurosurgery to be undertaken in awake patients. 18,21 Clonidine and guanfacine are used in ADHD patients and avoid the hypertensive and cardiovascular risks of the traditional stimulants methylphenidate and amphetamine. 7 Tizanidine helps spasticity, muscle spasm and muscle cramps. 16 Bromonidine and oxymetazoline are still used as topical vasoconstrictors in rosacea 22 and brimonidine for glaucoma where it reduces aqueous humor production whilst increasing its outflow. 11 The remaining 10% of brain α2-adrenoceptors are α2Cadrenoceptors and appear particularly prevalent in the striatum and hippocampus. 14 The expression and effects of the α2B-adren oceptors appear very minor in brain. 6 α2-adrenoceptors have been extensively studied. The original studies were restricted to using different tissue preparations -human platelet, colonic adenocarcinoma or rat cortex for α2A, neonatal rat lung for α2B and opossum kidney for α2C; e.g., [23][24][25] introducing problems of species variation. Other studies have shown that α2-adrenoceptors couple to both Gi and Gs-proteins and thus have a biphasic agonist concentration response -cAMP inhibition at low agonist concentrations followed by cAMP stimulation at high agonist concentrations. 17,[26][27][28][29][30][31][32] However, for reasons unknown, only some compounds activate Gs-stimulated cAMP while other com-| 3 of 23 PROUDMAN et al.

| Cell lines and cell culture
CHO-K1 (RIDD: CVCL_0214) stably transfected with a CRE-SPAP reporter gene and the human α2A-adrenoceptor (CHO-α2A), human α2B-adrenoceptor (CHO-α2B) or human α2C-adrenoceptor (CHO-α2C) were used 40 as were lines expressing the same CRE-SPAP reporter and human β1-adrenoceptor (CHO-β1) or human β2adrenoceptor (CHO-β2, 38 ). The parental cell line, which expresses the CRE-SPAP reporter but no transfected receptor, and from which these lines were generated, was also used. All cells were grown in Dulbecco's modified Eagle's medium nutrient mix F12 (DMEM/F12) containing 10% foetal calf serum and 2 mM L-glutamine in a 37°C humidified 5% CO 2 : 95% air atmosphere. Cells were always grown in the absence of any antibiotics. Mycoplasma contamination has intermittently been monitored within the laboratory (negative) but cell lines were not tested routinely with each experiment.

| CRE-SPAP gene transcription
CRE-SPAP production was measured as in. 41 Briefly, cells were grown to confluence in clear 96-well plates in 100 μL DMEM/F12 containing 10% fetal calf serum and 2 mM L-glutamine, and serumstarved with serum free media (sfm, DMEM/F12 containing 2 mM Lglutamine) 24 h before experimentation. Where used, pertussis toxin (PTX 100 ng/mL) was added to this sfm and thus the cells received 24 h treatment with PTX. On the experiment day, the sfm was removed and replaced with 100 μL sfm or 100 μL sfm containing antagonist at the final required concentration. Agonist in 10 μL (diluted in sfm) was then added to each well and the plates incubated at 37°C for 10 min, followed by 10 μM addition of forskolin (final well concentration 3 μM) and cells incubated for 5 h at 37°C (5% CO 2 ). After 5 h, all drugs and media were removed, 40 μL sfm was added to each well and the cells incubated for a further hour at 37°C before being incubated at 65°C for 30 min (to destroy any endogenous phosphatases), cooled to 37°C, 100 μL 5 mM pNPP in diethanolamine buffer added to each well and incubated at 37°C until the yellow color developed before being read on a Dynatech MRX plate reader at 405 nm.

| 3 H-cAMP accumulation
Cells were grown to confluence in 48-well clear plates. Cells were pre-labeled by incubation with 2 μCi/mL 3 H-adenine (0.5 mL per well) for 2 h at 37°C (5% CO 2 ). The 3 H-adenine was removed, each well washed by the addition and removal of 1 mL sfm, then 0.5 mL sfm containing 100 μM IBMX added to each well. Agonist in 5 μL (diluted in sfm) was added to triplicate wells and incubated for 10 min at 37°C. Where used, forskolin (10 μM) was then added to the wells, and plates incubated for 5 h at 37°C (5% CO 2 ). The reaction was terminated by the addition of 50 μL concentrated HCl per well, the plates were then frozen, thawed and 3 H-cAMP separated from other 3 H-nucleotides by Dowex and alumina column chromatography, with each column being corrected for efficiency by comparison with 14 C-cAMP recovery as previously described. 38

| ERK1/2-phosphorylation
Extracellular-signal-regulated kinases (ERK1/2) activation was measured using a Surefire Alphascreen pERK1/2 kit. Cells were grown to confluence in 96-well clear plates and double serum starved by washing the cells twice with 100 μL sfm before incubating in a further (third) 100 μL sfm for 24 h. Agonists in 20 μL sfm were added to the well (wells contained about 80 μL after some evaporation over 24 h, thus approximately a 1:5 dilution) and incubated for 2-4 min (at 37°C). Reagents were then removed, 20 μL lysis buffer added to each well and ERK1/2-phosphorylation measured using the Alphascreen kit as per manufacturer's instructions. After a minimum of 2 h in the dark, the plates were read on an EnVision plate reader using standard Alphascreen settings. Basal and maximum ERK1/2-phosphorylation (as determined by 10 μM PDBu, Phorbol 12,13-dibutyrate) was measured in each plate.

| 3 H-rauwolscine (yohimbine) whole cell binding
The affinity of the agonists was assessed using the whole cell binding and is identical to that used to determine the affinity of agonists at the α1-adrenoceptors 39 and β-adrenoceptors. 38 Cells were grown to confluence in white-sided 96-well plates. Media was removed from each well and 100 μL ligand (diluted in sfm to twice their final concentration) added to triplicate wells, followed immediately by the addition of 100 μL 3 H-rauwolscine (diluted in sfm) and incubated for 2 h at 37°C (5% CO 2 , humidified atmosphere). The media and all drugs were then removed from the wells, the cells washed twice by the addition and removed of 2 × 200 μL 4°C PBS. Cells were inspected under a light microscope to ensure they were still adherent after the wash, and 100 μL Microscint 20 was then added to each well. Total binding and non-specific binding (determined by the presence of 10 μM RX821002) was defined in every plate. Radioligand concentrations were determined from taking the average of triplicate 50 μL samples of each 3 H-rauwolscine concentration used and counted on a PerkinElmer TriCarb Scintillation counter.

| Functional experiments-One-site concentration responses curves
Many agonist responses were best described by a one-site sigmoidal agonist concentration-response curve. These were fitted to the data using the following equation with Graphpad Prism 7: where Emax is the maximal response, [A] is the agonist concentration and EC 50 is the concentration of agonist that produces 50% of the maximal response.

| Functional experiments-Two-site concentration responses curves
Many concentration response curves clearly contained two components -an inhibitory response followed by a stimulatory response, thus a two-site analysis was performed using the following equation: where basal is the response in the absence of agonist, FK is the response to a fixed concentration of forskolin, [A] is the concentration of agonist, IC 50 is the concentration of agonist that inhibits 50% of the response to forskolin (Gi-coupled response), EC 50 is the concentration of agonist that caused a half maximal stimulation (Gs-coupled response) and S MAX is the maximum stimulation of this Gs-coupled-component. In experiments where three different fixed concentrations of the same antagonist were used, Schild plots were constructed using the following equation:

| Functional experiments-Calculation of antagonist K D values from a parallel shift
A straight line was fitted to the points and a slope of 1 indicates competitive antagonism. 42

| Calculation of agonist K D from 3 Hrauwolscine whole cell competition binding
In all cases where a K D value is stated, increasing concentrations of agonist fully inhibited the specific binding of 3 H-rauwolscine (unless otherwise annotated in the tables). The following equation was then fitted to the data using Graphpad Prism 7 and the IC 50 was determined as the concentration required to inhibit 50% of the specific binding.
where [A] is the concentration of the competing agonist and IC 50 is the concentration at which half of the specific binding of 3 H-rauwolscine has been inhibited. In some cases the maximum concentration of competing ligand was not able to inhibit all of the specific 3 H-rauwolscine binding.
Where no inhibition of radioligand binding was seen, even with maximum concentration of competing ligand possible, "no binding" is given in the tables. Where the inhibition produced by the maximum concentration of the competing ligand was 50% or less, an IC 50 could not be determined and thus a K D value not calculated. This is shown in the tables as IC 50 > top concentration used (i.e. IC 50 > 100 μM means that 100 μM inhibited some but less than 50% of the specific binding).
In cases where the competing ligand caused a substantial (greater than 50%, but not 100%) inhibition of specific binding, an IC 50 value was determined by extrapolating the curve to non-specific levels and assuming that a greater concentration would have resulted in 100% inhibition. These values are given as apparent K D values in the tables.
All data are presented as mean ± SEM of triplicate determinations and n in the text refers to the number of separate experiments.
Affinity selectivity ratios are given as a ratio of the K D values for the different receptors, and intrinsic efficacy is given as efficacy ratios determined from K D /IC 50 . 34,36,37,43 Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy.
org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 44 and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20. 45
To confirm that CRE-SPAP production was an accurate reflection of cAMP responses, direct cAMP measurements were made.

| Brimonidine response in α 2A cells lines with different levels of receptor expression
To examine this biphasic response further, two other cell lines stably expressing the human α2A-adrenoceptor at lower receptor expression levels were examined. As expected, lower receptor expression in cell line 2 (56.1%) and no response was seen in cell line 3. Thus the ability to stimulate a Gs-coupled response at the α2A-adrenoceptor is directly related to the receptor reserve within that system.

| CHOα 2A cells-Other α 2-agonists
Not all agonists stimulated a biphasic response. Moxonidine stimulated a clear biphasic CRE-SPAP production response, whilst naphazoline, despite a similar potency for the Gi-component, did not ( Figure 3A). In the absence of forskolin, moxonidine stimulated an agonist response whereas naphazoline did not ( Figure 3B).
Furthermore, examining many ligands showed that the ability to stimulate the Gs-response was not an all or nothing event, but compounds exist with a graded range in the size of Gs-mediated responses ( Table 1). For example, dexmedetomidine, used increasingly in ITU, was able to simulate Gs-coupling, however this was significantly less than that seen for brimonidine and the endogenous catecholamines (Supplementary Figure S2), whereas the Gs-coupled response for clonidine was barely measureable.

| 3 H-rauwolscine whole cell binding and intrinsic efficacy ratio
Affinity measurements were made from 3 H-rauwolscine binding using the same media and conditions as for the functional assays (living cells). From the K D values obtained and the IC 50 value from the Giinhibition of CRE-SPAP production, an efficacy ratio (K D /IC 50 ) 34,36,37,43 was obtained as a measure of the intrinsic efficacy of the agonist. This is the same analysis as 13 's visual comparison in human fat cells where the clonidine concentration response from binding and lipolysis were superimposable, but the lipolysis response to adrenaline and brimonidine were left-shifted with respect to binding, demonstrating greater intrinsic efficacy for adrenaline and brimonidine than clonidine. Thus efficacy ratios allow a numerical comparison and is a more accurate measure of true ligand intrinsic efficacy than either potency or maximal response. 48 The affinity of brimonidine was relatively low (log K D −6.37 ± 0.07, n = 5, Figure 2C; Table 1), compared to its IC 50 (−8.94) giving an intrinsic efficacy ratio of 2.57. This was similar for moxonidine (2.49). However, the efficacy ratio for naphazoline was only 0.78. Table 1 (CHO-α2A cells) are presented in order of decreasing efficacy ratio, as determined from Gi-inhibition of CRE-SPAP production and K D from binding. However given the close correlation between IC 50 and ERK1/2-phosphorylation EC 50 , similar results would have occurred from using efficacy ratio calculated using the ERK1/2phosphorylation as the functional response.

| CHOα 2B cells
Brimonidine also stimulated a biphasic response in CHO-α2B cells (Table 2). Both inhibitory and stimulatory parts of the response  Table 4 for mean ± sem and n numbers); biphasic log IC 50 and EC 50 values from CRE-SPAP production in presence of forskolin, or in the cases of inhibition only, log IC 50 and % inhibition from the 3 μM forskolin control; log efficacy ratio (K D /IC 50 ); log EC 50 and % maximum response compared to 3 μM forskolin from CRE-SPAP production in the absence of forskolin; and log EC 50 and % maximum response compared to 10 μM PDBU from ERK1/2-phosphorylation.
Affinity was also assessed, and compounds ranked in order of intrinsic efficacy ( Table 2).

| CHO-CRE-SPAP cells
There were no CRE-SPAP responses to any of the agonist ligands Of note, some Gi-coupled receptors have been found to stimulate calcium responses (e.g., muscarinic M2 receptor 49 ). Calcium/ Gq-coupling was not assessed as part of this study.

| DISCUSS ION
Certain α2-agonists stimulate biphasic cAMP responses at α2adrenoceptors, with Gi-cAMP inhibition at low concentrations followed by Gs-mediated stimulation at higher concentrations.
However, other ligands, of equal Gi-mediated potency do not stimulate Gs. This study aimed to investigate this.
Brimonidine stimulated biphasic α2A-adrenoceptor responses for both CRE-SPAP production and 3 H-cAMP accumulation as previously observed. 17,[26][27][28][29][30][31][32]47 This Gi and Gs-protein coupling is through third intracellular loop residues, 31 and is similar to adenosine A1 receptor agonist responses. 41 However, whilst moxonidine and naphazoline have similar Gi-potency, only moxonidine stimulated a Gs-response. This is similar to 33 Table 4 for mean ± SEM and n numbers); biphasic log IC 50 and EC 50 values from CRE-SPAP production in presence of forskolin, or in the cases of inhibition only, log IC 50 and % inhibition from the 3 μM forskolin control; log efficacy ratio (K D /IC 50 ); log EC 50 and % maximum response compared to 3 μM forskolin from CRE-SPAP production in the absence of forskolin; and log EC 50 and % maximum response compared to 10 μM PDBU from ERK1/2-phosphorylation.
The ligands are arranged in order of     Table 4 for mean ± SEM and n numbers); inhibition log IC 50 and % inhibition from the 3 μM forskolin control; log efficacy ratio (K D /IC 50 ); log EC 50 and % maximum response compared to 3 μM forskolin from CRE-SPAP production in the absence of forskolin; and log EC 50 and % maximum response compared to 10 μM PDBU from ERK1/2-phosphorylation. The ligands are arranged in order of

TA B L E 3 (Continued)
with similar Gi-responses (including full agonists) had different Gsresponses. When extended to other α2-agonists, a graded spectrum was seen from agonists with large Gs-stimulatory components, through to those with none.
As CRE-SPAP responses can involve ERK1/2-phosphorylation separately from the Gs-cAMP pathway (biased signaling at β2- Ligand affinity was examined to enable the two properties of agonist ligands (affinity and intrinsic efficacy) to be studied separately and a measure of intrinsic efficacy (efficacy ratio) obtained.
For brimonidine and moxonidine, the efficacy ratio was high (log 2.57 and 2.48 respectively), suggesting few receptors need occupying to stimulate agonist responses (i.e. the compounds had high intrinsic efficacy). Naphazoline had a lower efficacy ratio at 0.78 (lower intrinsic efficacy). This has been attributed to a loss of dexmedetomidine selectivity at higher doses, 16 however it is tempting to consider it may, in part, be due to α2-Gs-activation. α2-agonists used systemically in clinical practice (e.g., clonidine for hypertension, dexmedetomidine for sedation, guanfacine for ADHD, tizanidine for spasticity) are midrange, partial agonists.
The As affinity and intrinsic efficacy measurements were made in all α2-adrenoceptor subtypes under identical conditions, ligand affinity and rank orders of intrinsic efficacy can be directly compared.
Furthermore, as identical conditions were used for α1-adrenceptor measurements, 39 comparison across all human α-and β1 and β2-adrenoceptors is possible.
Oxymetazoline was the most affinity-selective α2-agonist (α2A affinity 200-fold higher than α2B and 28-fold higher than α2Cadrenoceptors) similar to comparisons from human colonic adenocarcinoma cells (α2A), neonatal rat lung (α2B) and opossum kidney cells (α2C) 23,24 and in rat, 25 guinea pig 28 and pig. 56 Other similarities exist -guanfacine and guanabenz had 10-fold higher α2A than α2B affinity similar to. 25 Although precise values vary, not least because of species differences, the pattern of higher affinity for dexmedetomidine and medetomidine, followed by clonidine and guanabenz and lower affinity for catecholamines and xylazine is common across studies. 17,25,28,[57][58][59] However, there was little α2-selective affinity for the other α-agonists, also noted by 17 and no α2B-selective agonists.
As expected, catecholamines had high intrinsic efficacy.
Medetomidine, and stereoisomer dexmedetomidine, were the most potent agonists for all α2-subtypes, but also had the highest affinities (as in 28 ). Thus, the intrinsic efficacy of these is only mid-range.
This high potency has been reported before. 17

TA B L E 4 (Continued)
dexmedetomidine was their most potent α2-agonist compound, more than catecholamines, is absolutely correct but only part of the story. Dexmedetomidine did not have the highest intrinsic efficacy (i.e. not the most efficacious agonist) either in terms of maximum response or if efficacy ratios are calculated using their data (again mid-ranking). As higher intrinsic efficacy determines the Gs-coupling, this explains why, despite being the most potent agonists, medetomidine and dexmedetomidine did not elicit the largest Gs-stimulation.
There is some correlation between the intrinsic efficacy of compounds at the different α2-subtypes with some agonists being more efficacious at all three subtypes (e.g., catecholamines) and others having lower efficacy (e.g., clonidine and rilmenidine).
However, there are some differences ( Figure 6D-F). Brimonidine/ UK14304 are highly efficacious α2A and α2C-agonists (both present in brain), with medetomidine and dexmedetomidine being less efficacious. However, the rank order of compounds is reversed at α2B-adrenoceptors with medetomidine and dexmedetomidine being more efficacious than brimonidine/UK14304. This rank order is different for other compounds -oxymetazoline and xylometazoline are higher up the rank order in α2B and lower in α2A and α2Csubtypes. This suggests there may be some subtype selectivity for intrinsic efficacy.
A61603 was a very efficacious ligand at all α-adrenoceptors (although not β1/β2-adrenoceptors). However, it has 1000-fold higher α1A-affinity than for any other α-adrenoceptor, giving rise to more potent α1A functional responses. A61603 is an affinity-selective α1A-agonist. Interestingly at α2A-adrenoceptors, A61603 was the only compound where the Gs-response was lower than predicted from Gi-potency and intrinsic efficacy. The reason is unknown, although the binding was so poor that affinity (and efficacy ratio) could not be accurately established. The line is that of best fit and the slope is not 1 and does not necessarily go through the origin as this represents a function of efficacy (i.e. differences in cell line which include receptor number, receptor-effector coupling etc.). The data for oxymetazoline, xylometazoline and dihydroergotamine are not included in these plots as the compounds generated agonist ERK1/2phosphorylation responses in nontransfected cells and are therefore non-α2-mediated responses. Compounds with the greatest perpendicular distance from the line represent compounds with the greatest degree of selective intrinsic efficacy. Perhaps more interesting is the comparison between α1 and α2-subtypes. Dexmedetomidine has 100-fold higher affinity for α2 than α1-adrenoceptor subtypes with mid-range efficacy at all six αsubtypes, suggesting that affinity is largely driving the higher α2 vs α1-potency of dexmedetomidine responses. However, brimonidine only has a 10-fold higher α2 than α1-affinity but very high α2-intrinsic efficacy (giving potent responses) and low α1 intrinsic efficacy. The α2-selectivity of brimonidine appears to be driven more by α2selective intrinsic efficacy with less reliance on selective affinity.
There are examples of the reverse. R-phenylephrine, etilefrine, metaraminol and methoxamine have similar affinity across all αsubtypes but are highly efficacious at α1-adrenoceptors with low efficacy at α2A and α2C-subtypes (interestingly α2B is once again a little different). These compounds α1-selective functional responses are being driven by α1-selective intrinsic activity, whilst A61603, above, has α1A-selective affinity.
In conclusion, both (1) system high receptor reserve and (2) agonist high intrinsic efficacy are required for α2-Gs-mediated responses to be observed. From the Gi-mediated efficacy ratio (binding K D /Gi-IC 50 ), the degree of Gs-stimulation observed within a given system can be predicted. It remains to be determined whether this Gs-coupling is clinically relevant and the precise receptor conformational changes that occur, given the structural diversity of compounds with high intrinsic efficacy.
This study also shows the importance of separating affinity and intrinsic efficacy to understand agonist ligand responses. Some

JGB has been on the Scientific Advisory Board for CuraSen
Therapeutics since 2019.

DATA AVA I L A B I L I T Y S TAT E M E N T
Further information and requests for data and reagents should be directed to and will be fulfilled by the corresponding author, Jillian Baker. Please contact jillian.baker@nottingham.ac.uk

E TH I C A L S TATEM ENT
No animals, human tissue, human volunteers or patients were used in this study.