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Conopeptide ?-TIA defines a new allosteric site on the extracellular surface of the ?1B-adrenoceptor.

ABSTRACT: The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey ?(1)-adrenergic receptor crystal structure, we modeled the ?(1B)-adrenoceptor (?(1B)-AR) to help identify the allosteric site for ?-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ?-TIA, we identified 14 residues on the ECS of the ?(1B)-AR that influenced ?-TIA binding. Double mutant cycle analysis and docking confirmed that ?-TIA binding was dominated by a salt bridge and cation-? between Arg-4-?-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-? interaction between Trp-3-?-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-?-TIA and Val-197, Trp-3-?-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for ?(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

SUBMITTER: Ragnarsson L

PROVIDER: S-EPMC3548491 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Conopeptide ρ-TIA defines a new allosteric site on the extracellular surface of the α1B-adrenoceptor.

Ragnarsson Lotten L Wang Ching-I Anderson CI Andersson Åsa Å Fajarningsih Dewi D Monks Thea T Brust Andreas A Rosengren K Johan KJ Lewis Richard J RJ

The Journal of biological chemistry 20121126 3

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to ...[more]

PMID: 23184947

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Project description: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.

Dataset Information

Conopeptide ?-TIA defines a new allosteric site on the extracellular surface of the ?1B-adrenoceptor.

Publications

Conopeptide ρ-TIA defines a new allosteric site on the extracellular surface of the α1B-adrenoceptor.

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