The selectivity of α-adrenoceptor agonists for the human α1A, α1B, and α1D-adrenoceptors

Highly selective drugs offer a way to minimize side-effects. For agonist ligands, this could be through highly selective affinity or highly selective efficacy, but this requires careful measurements of intrinsic efficacy. The α1-adrenoceptors are important clinical targets, and α1-agonists are used to manage hypotension, sedation, attention deficit hypersensitivity disorder (ADHD), and nasal decongestion. With 100years of drug development, there are many structurally different compounds with which to study agonist selectivity. This study examined 62 α-agonists at the three human α1-adrenoceptor (α1A, α1B, and α1D) stably expressed in CHO cells. Affinity was measured using whole-cell 3H-prazosin binding, while functional responses were measured for calcium mobilization, ERK1/2-phosphorylation, and cAMP accumulation. Efficacy ratios were used to rank compounds in order of intrinsic efficacy. Adrenaline, noradrenaline, and phenylephrine were highly efficacious α1-agonists at all three receptor subtypes. A61603 was the most selective agonist and its very high α1A-selectivity was due to selective α1A-affinity (>660-fold). There was no evidence of Gq-calcium versus ERK-phosphorylation biased signaling at the α1A, α1B, or α1D-adrenoceptors. There was little evidence for α1A calcium versus cAMP biased signaling, although there were suggestions of calcium versus cAMP bias the α1B-adrenoceptor. Comparisons of the rank order of ligand intrinsic efficacy suggest little evidence for selective intrinsic efficacy between the compounds, with perhaps the exception of dobutamine which may have some α1D-selective efficacy. There seems plenty of scope to develop affinity selective and intrinsic efficacy selective drugs for the α1-adrenoceptors in future

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

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

The affinity and selectivity of α‐adrenoceptor antagonists, antidepressants and antipsychotics for the human α2A, α2B, and α2C‐adrenoceptors and comparison with human α1 and β‐adrenoceptors

α2-adrenoceptors, subdivided into α2A, α2B and α2C subtypes and expressed in heart, blood vessels, kidney, platelets and brain, are important for blood pressure, sedation, analgesia and platelet aggregation. Brain α2C-adrenoceptor blockade has also been suggested to be beneficial for antipsychotic action. However, comparing α2-adrenoceptor subtype affinity is difficult due to significant species and methodology differences in published studies. Here, 3H-rauwolscine whole cell binding was used to determine the affinity and selectivity of 99 α-antagonists (including antidepressants and antipsychotics) in CHO cells expressing human α2A, α2B or α2C-adrenoceptors, using an identical method to β and α1-adrenoceptor measurements, thus allowing direct human receptor comparisons. Yohimbine, RX821002, RS79948 and atipamezole are high affinity non-selective α2-antagonists. BRL44408 was the most α2A-selective antagonist, although its α1A-affinity (81nM) is only 9-fold greater than its α2C-affinity. MK-912 is the highest-affinity, most α2C-selective antagonist (0.15nM α2C-affinity) although its α2C-selectivity is only 13-fold greater than at α2A. There are no α2B-selective antagonists. A few α-ligands with significant β-affinity were detected, e.g. naftopidil where its clinical α1A-affinity is only 3-fold greater than off-target β2-affinity. Antidepressants (except mirtazapine) and first generation antipsychotics have higher α1A than α2-adrenoceptor affinity but poor β-affinity. Second generation antipsychotics varied widely in their α2-adrenoceptor affinity. Risperidone (9nM) and paliperidone (14nM) have the highest α2C-adrenoceptor affinity however this is only 5-fold selective over α2A, and both have higher affinity for α1A (2nM and 4nM respectively). So, despite a century of yohimbine use, and decades of α2-subtype studies, there remains plenty of scope to develop α2-subtype selective antagonists

Salmeterol's Extreme b2 Selectivity Is Due to Residues in Both Extracellular Loops and Transmembrane Domains

ABSTRACT Salmeterol is a long-acting b2-agonist, widely used as an inhaled treatment of asthma and chronic obstructive pulmonary disease. It has very high b2-affinity (log K D 28.95) and is very selective for the b2-adrenoceptor (1000-fold selectivity over the b1-adrenoceptor). This study used a mutagenesis approach to determine the exact amino acids in the human b2-adrenoceptor responsible for this very high selectivity. Wild-type b2-and b1-adrenoceptors, chimeric b2/b1-adrenoceptors, and receptors with single-point mutations were transfected into Chinese hamster ovary-K1 cells, and affinity and function were studied using [ ]cAMP accumulation. Extracellular loop 3 (and specifically amino acid K305) had the largest single effect by reducing salmeterol's affinity for the b2-adrenoceptor by 31-fold. H296 in transmembrane 6 also had a major effect (18-fold reduction in salmeterol affinity). Combining these, in the double mutant b2-H296K-K305D, reduced salmeterol's affinity by 275-fold, to within 4-fold of that of the b1-adrenoceptor, without affecting the affinity or selectivity of other b2-agonists (salbutamol, formoterol, fenoterol, clenbuterol, or adrenaline). Another important amino acid was Y308 in transmembrane 7, although this also affected the affinity and selectivity of other agonists. F194 in extracellular loop 2 and R304 in extracellular loop 3 also had minor effects. None of these mutations (including the double mutant b2-H296K-K305D) affected the efficacy or duration of action of salmeterol. This suggests that the high affinity and selectivity of salmeterol are due to specific amino acids within the receptor itself, but that the duration of action is at least in part due to other factors, for example lipophilicity

Randomized controlled trial of an internet-based self-guided hand exercise program to improve hand function in people with systemic sclerosis: the Scleroderma Patient-centered Intervention Network Hand Exercise Program (SPIN-HAND) trial

BACKGROUND: Systemic sclerosis (scleroderma; SSc) is a rare autoimmune connective tissue disease. Functional impairment of hands is common. The Scleroderma Patient-centered Intervention Network (SPIN)-HAND trial compared effects of offering access to an online self-guided hand exercise program to usual care on hand function (primary) and functional health outcomes (secondary) in people with SSc with at least mild hand function limitations. METHODS: The pragmatic, two-arm, parallel-group cohort multiple randomized controlled trial was embedded in the SPIN Cohort. Cohort participants with Cochin Hand Function Scale (CHFS) scores ≥ 3 and who indicated interest in using the SPIN-HAND Program were randomized (3:2 ratio) to an offer of program access or to usual care (targeted N = 586). The SPIN-HAND program consists of 4 modules that address (1) thumb flexibility and strength; (2) finger bending; (3) finger extension; and (4) wrist flexibility and strength. The primary outcome analysis compared CHFS scores 3 months post-randomization between participants offered versus not offered the program. Secondary outcomes were CHFS scores 6 months post-randomization and functional health outcomes (Patient-Reported Outcomes Measurement Information System profile version 2.0 domain scores) 3 and 6 months post-randomization. RESULTS: In total, 466 participants were randomized to intervention offer (N = 280) or usual care (N = 186). Of 280 participants offered the intervention, 170 (61%) consented to access the program. Of these, 117 (69%) viewed at least one hand exercise instruction video and 77 (45%) logged into the program website at least 3 times. In intent-to-treat analyses, CHFS scores were 1.2 points lower (95% CI − 2.8 to 0.3) for intervention compared to usual care 3 months post-randomization and 0.1 points lower (95% CI − 1.8 to 1.6 points) 6 months post-randomization. There were no statistically significant differences in other outcomes. CONCLUSION: The offer to use the SPIN-HAND Program did not improve hand function. Low offer uptake, program access, and minimal usage among those who accessed the program limited our ability to determine if using the program would improve function. To improve engagement, the program could be tested in a group format or as a resource to support care provided by a physical or occupational therapist. TRIAL REGISTRATION: NCT03419208. Registered on February 1, 2018

Protocol for a partially nested randomised controlled trial to evaluate the effectiveness of the scleroderma patient-centered intervention network COVID-19 home-isolation activities together (SPIN-CHAT) program to reduce anxiety among at-risk scleroderma patients

Objective: Contagious disease outbreaks and related restrictions can lead to negative psychological outcomes, particularly in vulnerable populations at risk due to pre-existing medical conditions. No randomised controlled trials (RCTs) have tested interventions to reduce mental health consequences of contagious disease outbreaks. The primary objective of the Scleroderma Patient-centered Intervention Network COVID-19 Home-isolation Activities Together (SPIN-CHAT) Trial is to evaluate the effect of a videoconference-based program on symptoms of anxiety. Secondary objectives include evaluating effects on symptoms of depression, stress, loneliness, boredom, physical activity, and social interaction.Methods: The SPIN-CHAT Trial is a pragmatic RCT that will be conducted using the SPIN-COVID-19 Cohort, a sub-cohort of the SPIN Cohort. Eligible participants will be SPIN-COVID-19 Cohort participants without a positive COVID-19 test, with at least mild anxiety (PROMIS Anxiety 4a v1.0 T-score >= 55), not working from home, and not receiving current counselling or psychotherapy. We will randomly assign 162 participants to intervention groups of 7 to 10 participants each or waitlist control. We will use a partially nested RCT design to reflect dependence between individuals in training groups but not in the waitlist control. The SPIN-CHAT Program includes activity engagement, education on strategies to support mental health, and mutual participant support. Intervention participants will receive the 4-week (3 sessions per week) SPIN-CHAT Program via video-conference. The primary outcome is PROMIS Anxiety 4a score immediately post-intervention.Ethics and dissemination: The SPIN-CHAT Trial will test whether a brief videoconference-based intervention will improve mental health outcomes among at-risk individuals during contagious disease outbreak

³H-cAMP accumulation in response to pindolol at the wildtype β1-adrenoceptor and polymorphic variants.

<p>³H-cAMP accumulation in response to pindolol in <b>A</b> wildtype cells, <b>B</b> Gly49/Gly389 cells and <b>C</b> Ser49/Arg389 cells. Bars represent basal ³H-cAMP accumulation and that in response to 10 µM isoprenaline. Data points are mean ± s.e.mean of triplicate determinations. These single experiments are representative of 5 separate experiments in each case and demonstrate agonist actions at both agonist conformations of the β1-adrenoceptor in the wildtype and polymorphic variants.</p

Inhibition of ³H-CGP 12177 binding at the wildtype receptor and polymorphic variants.

<p>Inhibition of ³H-CGP 12177 specific binding by <b>A</b> metoprolol and <b>B</b> carvedilol in wildtype (WT), Gly49/Gly389 cells and Ser49/Arg389 cells. Non-specific binding was determined by 10 µM propranolol. The concentration of ³H-CGP 12177 present in each case was 0.96 nM. Data points are mean ± s.e.mean of triplicate determinations and these single experiments are representative of 6 separate experiments in each case. <b>C</b> Correlation plot for the affinity of all the ligands from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077582#pone-0077582-t001" target="_blank">Table 1</a> for the wildtype (x-axis) and polymorphic variants (y-axis). There is a strong correlation between the affinity measurements made in the wildtype and those measured in either the Gly49/Gly389 receptor (R² = 1.00, slope 0.99±0.01) and the Ser49/Arg389 receptor (R² = 0.99, slope = 0.92±0.02). This demonstrates that ligands had very similar affinity for all three receptors.</p

Affinity of antagonists for the two agonist conformations of the wildtype and polymorphic variants of the β1-adrenoceptor.

<p>Log K_D values for bisoprolol, CGP 20712A, carvedilol and CGP 12177 obtained from ³H-cAMP accumulation the human wildtype β1-adrenoceptor or the polymorphic variant. Values are mean ± s.e.mean. of n separate determinations. The concentrations of antagonist use were: bisoprolol 30 nM when cimaterol was the agonist and 10 µM when CGP 12177 was the agonist; CGP 20712A 3 nM and 30 nM with cimaterol and 1 µM and 10 µM with CGP 12177; carvedilol 3 nM with cimaterol and 1 µM with CGP 12177; and CGP 12177 was at 1 nM and 10 nM when cimaterol was the agonist.</p

Agonist responses at the wildtype and polymorphic variants of the β1-adrenoceptor.

<p>Log EC₅₀ values and % isoprenaline maximal responses obtained from ³H-cAMP accumulation in cells expressing either the human wildtype β1-adrenoceptor or the polymorphic variants. Values are mean ± s.e.mean of n separate determinations.</p><p>One-way ANOVA with post hoc Neuman-Keuls was performed comparing the log EC₅₀ values and % maximum isoprenaline responses obtained for the wildtype receptor with those obtained for each polymorphic variant in turn.</p>#<p> = p<0.05.</p><p>The log EC₅₀ values and % maximum isoprenaline responses for the partial agonists cimaterol, CGP 12177, nebivolol and xamoterol are significantly different for the Ser49/Arg389 receptor compared with wildtype. This could be due to either the higher receptor expression level of the Ser49/Arg389 cell line or more efficient receptor-effector coupling of the Ser49/Arg389 receptors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077582#s3" target="_blank">Discussion</a> for further detail).</p