The Isoleucine at Position 118 in Transmembrane 2 Is Responsible for the Selectivity of Xamoterol, Nebivolol, and ICI89406 for the Human ß1-Adrenoceptor.

Known off-target interactions frequently cause predictable drug side-effects (e.g., ß1-antagonists used for heart disease, risk ß2-mediated bronchospasm). Computer-aided drug design would improve if the structural basis of existing drug selectivity was understood. A mutagenesis approach determined the ligand-amino acid interactions required for ß1-selective affinity of xamoterol and nebivolol, followed by computer-based modeling to provide possible structural explanations. 3H-CGP12177 whole cell binding was conducted in Chinese hamster ovary cells stably expressing human ß1, ß2, and chimeric ß1/ß2-adrenoceptors (ARs). Single point mutations were investigated in transiently transfected cells. Modeling studies involved docking ligands into three-dimensional receptor structures and performing molecular dynamics simulations, comparing interaction frequencies between apo and holo structures of ß1 and ß2-ARs. From these observations, an ICI89406 derivative was investigated that gave further insights into selectivity. Stable cell line studies determined that transmembrane 2 was crucial for the ß1-selective affinity of xamoterol and nebivolol. Single point mutations determined that the ß1-AR isoleucine (I118) rather than the ß2 histidine (H93) explained selectivity. Studies of other ß1-ligands found I118 was important for ICI89406 selective affinity but not that for betaxolol, bisoprolol, or esmolol. Modeling studies suggested that the interaction energies and solvation of ß1-I118 and ß2-H93 are factors determining selectivity of xamoterol and ICI89406. ICI89406 without its phenyl group loses its high ß1-AR affinity, resulting in the same affinity as for the ß2-AR. The human ß1-AR residue I118 is crucial for the ß1-selective affinity of xamoterol, nebivolol, and ICI89406 but not all ß1-selective compounds. SIGNIFICANCE STATEMENT: Some ligands have selective binding affinity for the human ß1 versus the ß2-adrenoceptor; however, the molecular/structural reason for this is not known. The transmembrane 2 residue isoleucine I118 is responsible for the selective ß1-binding of xamoterol, nebivolol, and ICI89406 but does not explain the selective ß1-binding of betaxolol, bisoprolol, or esmolol. Understanding the structural basis of selectivity is important to improve computer-aided ligand design, and targeting I118 in ß1-adrenoceptors is likely to increase ß1-selectivity of drugs.

The signaling and selectivity of α-adrenoceptor agonists for the human α2A, α2B and α2C-adrenoceptors and comparison with human α1 and ß-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 ß1/ß2-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 ß-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, 3 H-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 ß 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 (81 nM) is only 9-fold greater than its α2C-affinity. MK-912 is the highest-affinity, most α2C-selective antagonist (0.15 nM α2C-affinity) although its α2C-selectivity is only 13-fold greater than at α2A. There are no truely α2B-selective antagonists. A few α-ligands with significant ß-affinity were detected, for example, naftopidil where its clinical α1A-affinity is only 3-fold greater than off-target ß2-affinity. Antidepressants (except mirtazapine) and first-generation antipsychotics have higher α1A than α2-adrenoceptor affinity but poor ß-affinity. Second-generation antipsychotics varied widely in their α2-adrenoceptor affinity. Risperidone (9 nM) and paliperidone (14 nM) have the highest α2C-adrenoceptor affinity however this is only 5-fold selective over α2A, and both have a higher affinity for α1A (2 nM and 4 nM, 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.

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 100 years 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 3 H-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 affinity and selectivity of α-adrenoceptor antagonists, antidepressants, and antipsychotics for the human α1A, α1B, and α1D-adrenoceptors.

α1-adrenoceptor antagonists are widely used for hypertension (eg, doxazosin) and benign prostatic hypertrophy (BPH, eg, tamsulosin). Some antidepressants and antipsychotics have been reported to have α1 affinity. This study examined 101 clinical drugs and laboratory compounds to build a comprehensive understanding of α1-adrenoceptor subtype affinity and selectivity. [3H]prazosin whole-cell binding was conducted in CHO cells stably expressing either the full-length human α1A, α1B, or α1D-adrenoceptor. As expected, doxazosin was a high-affinity nonselective α1-antagonist although other compounds (eg, cyclazosin, 3-MPPI, and ARC239) had higher affinities. Several highly α1A-selective antagonists were confirmed (SNAP5089 had over 1700-fold α1A selectivity). Despite all compounds demonstrating α1 affinity, only BMY7378 had α1D selectivity and no α1B-selective compounds were identified. Phenoxybenzamine (used in pheochromocytoma) and dibenamine had two-component-binding inhibition curves at all three receptors. Incubation with sodium thiosulfate abolished the high-affinity component suggesting this part is receptor mediated. Drugs used for hypertension and BPH had very similar α1A/α1B/α1D-adrenoceptor pharmacological profiles. Selective serotonin reuptake inhibitors (antidepressants) had poor α1-adrenoceptor affinity. Several tricyclic antidepressants (eg, amitriptyline) and antipsychotics (eg, chlorpromazine and risperidone) had high α1-adrenoceptor affinities, similar to, or higher than, α blockers prescribed for hypertension and BPH, whereas others had poor α1 affinity (eg, protriptyline, sulpiride, amisulpiride, and olanzapine). The addition of α blockers for the management of hypertension or BPH in people already taking tricyclic antidepressants and certain antipsychotics may not be beneficial. Awareness of the α-blocking potential of different antipsychotics may affect the choice of drug for those with delirium where additional hypotension (eg, in sepsis) may be detrimental.

Salmeterol's extreme ß2 selectivity is due to residues in both extracellular loops and transmembrane domains.

Salmeterol is a long-acting ß2-agonist, widely used as an inhaled treatment of asthma and chronic obstructive pulmonary disease. It has very high ß2-affinity (log KD -8.95) and is very selective for the ß2-adrenoceptor (1000-fold selectivity over the ß1-adrenoceptor). This study used a mutagenesis approach to determine the exact amino acids in the human ß2-adrenoceptor responsible for this very high selectivity. Wild-type ß2- and ß1-adrenoceptors, chimeric ß2/ß1-adrenoceptors, and receptors with single-point mutations were transfected into Chinese hamster ovary-K1 cells, and affinity and function were studied using [(3)H]CGP 12177 [(-)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one] whole-cell binding and [(3)H]cAMP accumulation. Extracellular loop 3 (and specifically amino acid K305) had the largest single effect by reducing salmeterol's affinity for the ß2-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 ß2-H296K-K305D, reduced salmeterol's affinity by 275-fold, to within 4-fold of that of the ß1-adrenoceptor, without affecting the affinity or selectivity of other ß2-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 ß2-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.

Identification of key residues in transmembrane 4 responsible for the secondary, low-affinity conformation of the human ß1-adrenoceptor.

The ß1-adrenoceptor exists in two agonist conformations/states: 1) a high-affinity state where responses to catecholamines and other agonists (e.g., cimaterol) are potently inhibited by ß1-adrenoceptor antagonists, and 2) a low-affinity secondary conformation where agonist responses, particularly CGP12177 [(-)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one] are relatively resistant to inhibition by ß1-adrenoceptor antagonists. Although both states have been demonstrated in many species (including human), the precise nature of the secondary state is unknown and does not occur in the closely related ß2-adrenoceptor. Here, using site-directed mutagenesis and functional measurements of production of a cyclic AMP response element upstream of a secreted placental alkaline phosphatase reporter gene and accumulation of (3)H-cAMP, we examined the pharmacological consequences of swapping transmembrane (TM) regions of the human ß1- and ß2-adrenoceptors, followed by single point mutations, to determine the key residues involved in the ß1-adrenoceptor secondary conformation. We found that TM4 (particularly amino acids L195 and W199) had a major role in the generation of the secondary ß1-adrenoceptor conformation. Thus, unlike at the human ß1-wild-type adrenoceptor, at ß1-TM4 mutant receptors, cimaterol and CGP12177 responses were both potently inhibited by antagonists. CGP12177 acted as a simple partial agonist with similar KB and EC50 values in the ß1-TM4 but not ß1-wild-type receptors. Furthermore pindolol switched from a biphasic concentration response at human ß1-wild-type adrenoceptors to a monophasic concentration response in the ß1-TM4 mutant receptors. Mutation of these amino acids to those found in the ß2-adrenoceptor (L195Q and W199Y), or mutation of a single residue (W199D) in the human ß1-adrenoceptor thus abolished this secondary conformation and created a ß1-adrenoceptor with only one high-affinity agonist conformation.

Impact of polymorphic variants on the molecular pharmacology of the two-agonist conformations of the human ß1-adrenoceptor.

ß-blockers are widely used to improve symptoms and prolong life in heart disease primarily by inhibiting the actions of endogenous catecholamines at the ß1-adrenoceptor. There are two common naturally occurring polymorphisms within the human ß1-adrenoceptor sequence: Ser or Gly at position 49 in the N-terminus and Gly or Arg at position 389 in the C-terminus and some clinical studies have suggested that expression of certain variants may be associated with disease and affect response to treatment with ß-blockers. The ß1-adrenoceptor also exists in two agonist conformations - a high affinity catecholamine conformation and a low affinity secondary agonist conformation. Receptor-effector coupling and intracellular signalling from the different conformations may be affected by the polymorphic variants. Here, we examine in detail the molecular pharmacology of the ß1-adrenoceptor polymorphic variants with respect to ligand affinity, efficacy, activation of the different agonist conformations and signal transduction and determine whether the polymorphic variants do indeed affect this secondary conformation. Stable cell lines expressing the wildtype and polymorphic variants were constructed and receptor pharmacology examined using whole cell binding and intracellular secondary messenger techniques. There was no difference in affinity for agonists and antagonists at the human wildtype ß1-adrenoceptor (Ser49/Gly389) and the polymorphic variants Gly49/Gly389 and Ser49/Arg389. Furthermore, the polymorphic variant receptors both have two active agonist conformations with pharmacological properties similar to the wildtype receptor. Although the polymorphism at position 389 is thought to occur in an intracellular domain important for Gs-coupling, the two agonist conformations of the polymorphic variants stimulate intracellular signalling pathways, including Gs-cAMP intracellular signalling, in a manner very similar to that of the wildtype receptor.

The pharmacological effects of the thermostabilising (m23) mutations and intra and extracellular (ß36) deletions essential for crystallisation of the turkey ß-adrenoceptor.

The X-ray crystal structure of the turkey ß-adrenoceptor has recently been determined. However, mutations were introduced into the native receptor that was essential for structure determination. These may cause alterations to the receptor pharmacology. It is therefore essential to understand the effects of these mutations on the pharmacological characteristics of the receptor. This study examined the pharmacological effects of both the m23 mutations and the ß36 deletions, both alone and then in combination in the ß36-m23 mutant used in the crystallisation and structure determination of the turkey ß-adrenoceptor. Stable CHO-K1 cell lines were made of each of the receptor mutants and the affinity and efficacy of ligands assessed by (3)H-CGP 12177 whole cell ligand binding, (3)H-cAMP accumulation, and CRE-SPAP gene transcription assays. The m23 mutations reduced affinity for agonists, partial agonists and neutral antagonists by about tenfold whilst the ß36 deletions alone had no effect on ligand affinity. Both sets of changes appeared to reduce the agonist activation of the receptor. Both the m23 and the ß36 receptors retained two active agonist-induced receptor conformations similar to that of the original tßtrunc receptor. The combined ß36-m23 receptor bound ligands with similar affinity to the m23 receptor; however, agonist activation was only observed with a few agonists including the catecholamines. Although the combination of mutations severely reduced the activation ability, the final crystallised receptor (ß36-m23) was still a fully functional receptor capable of binding agonist and antagonist ligands and activating intracellular agonist responses.

Role of key transmembrane residues in agonist and antagonist actions at the two conformations of the human beta1-adrenoceptor.

Studies with 4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP 12177) at the human beta1-adrenoceptor have provided evidence for two binding modes or conformations that have markedly different pharmacological properties. Here, key transmembrane residues (Asp104, Asp138, Ser228, Ser229, Ser232, Phe341, Asn344 and Asn363) have been mutated to provide structural insights into the nature of these conformations. [(3)H]CGP 12177 binding and cAMP response element-mediated reporter gene studies confirmed that CGP 12177 was a neutral antagonist (log K(D) = -9.18) at the "catecholamine site" and an agonist at the "CGP 12177 site" (log EC(50) = -8.12). Agonist responses to isoprenaline and CGP 12177 had different sensitivities to beta1-antagonists (e.g., CGP 20712A; log K(D) = -8.65 and -7.26, respectively). Site-directed mutagenesis showed that Asn363 and Asp138 were key residues for binding of agonists and antagonists, and they were also essential for the agonist actions of CGP 12177. S228A and S229A in transmembrane-spanning region (TM) 5 reduced the binding of CGP 12177 and had an identical effect on its agonist and antagonist actions. Both N344A and F341A in TM6 abolished the ability of CGP 20712A to discriminate between responses elicited by isoprenaline and CGP 12177. The fact that both Asp138 and Asn363 are absolutely required for CGP 12117 binding in both agonist and antagonist modes leads to the conclusion that the secondary agonist binding site for CGP 12117 must overlap with the catecholamine binding site. Modeling studies provide a basis for these overlapping sites with either the tert-butylamino group or the hydroxyethyloxy and imidazolone portions of CGP 12177 capable of forming polar interactions with Asp138 and Asn363.