Determinants of Ligand Subtype-Selectivity at α_1A-Adrenoceptor Revealed Using Saturation Transfer Difference (STD) NMR

Abstract

α- and α-adrenoceptors (α-AR and α-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene superfamily is made up of numerous subfamilies that, like α-AR and α-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes, and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents, however, makes the application of NMR to study ligand binding challenging. Here, stabilized α-AR and α-AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization, and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for α-AR over α-AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design.

Diazepam is not a direct allosteric modulator of α1‐adrenoceptors, but modulates receptor signaling by inhibiting phosphodiesterase‐4

“…α 1A ‐AR variant A4 and α 1B ‐AR variant #15 were expressed in Escherichia coli and purified as described previously . Stabilized rat neurotensin receptor 1 (enNTS 1 ) was expressed and purified as described by Bumbak et al .…”

“…Saturation transfer difference (STD) NMR samples constituted 5 μmol/L α 1A ‐AR A4, or 5 μmol/L enNTS1, and 500 μmol/L test ligands with and without competition of 10 μmol/L prazosin in 500 μL of DDM buffer (0.05% [or 1 mmol/L] DDM, 50 mmol/L potassium phosphate, 100 mmol/L NaCl, 10% 2 H 2 O, pH 7.4) in 5 mm NMR tubes. STD NMR was performed as described previously on a 700 MHz Bruker Avance IIIHD spectrometer using a cryogenically cooled triple resonance probe and the resultant spectra were analyzed using Mnova NMR 10 (Mestrelab, Santiago de Compostela, Spain).…”

“…Saturation binding of QAPB in the absence or presence of 50 μmol/L diazepam was measured using COS‐7 cells stably expressing WT human α 1A ‐AR and α 1B ‐AR as previously described . Cells were gently suspended with pipetting into 1.4 mL of phenol red‐free Dulbecco's modified Eagle medium (DMEM) at 20°C, resulting in a concentration of approximately 6 000 00 cells/mL.…”

“…cAMP response element (CRE) response assays were performed as previously described by transfecting WT α 1A ‐ and α 1B ‐AR stably expressing COS‐7 cells, parental COS‐7 cells or parental HEK293T cells, with a β‐galactosidase expression plasmid under the control of the CRE promoter . CRE reporter‐transfected cells were seeded into 96‐well culture plates for 24 hours before treatment with various compounds for 6 hours at 37°C 5% CO 2 .…”

“…However, no further studies have been reported on the mechanism of benzodiazepine modulation of α 1 ‐ARs, or the receptor binding site. We recently engineered thermostabilized mutants of α 1A ‐AR and α 1B ‐AR, which enabled binding epitope determination of orthosteric ligands with nuclear magnetic resonance (NMR) spectroscopy . In this work we sought to use these thermostabilized receptors and our NMR approach to further understand how benzodiazepines bind to and modulate α 1 ‐ARs so as to apply this information as a starting point for developing more selective modulators targeting this allosteric site.…”