Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library

Abstract

The β₂-adrenergic receptor (β₂AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β₂AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β₂AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β₂AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β₂AR. In cell-signaling studies, 15 inhibits cAMP production through the β₂AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β₂AR. This study presents an allosteric small-molecule ligand for the β₂AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

Keywords: DNA-encoded small-molecule library; G-protein–coupled receptor; allosteric modulator; drug discovery; β2-adrenergic receptor.

Fig. 1.

Screening of the DEL and the chemical structure of 15. (A) Schematic illustration of the screening of a DNA-encoded compound library. DNA-encoded library molecules, synthesized using a DNA-tagged, split-and-pool combinatorial chemical synthesis approach, were mixed with a target (purified β₂AR) immobilized on a matrix. Target binding (active) molecules were collected through affinity-based selection, and the encoding DNA tags were sequenced to identify the binding molecules. (B) Compound 15 is composed of three building blocks: methybenzamide (green), bromo-benzyl (red), and cyclohexylmethyl-benzene (blue). The amide backbone is shown in black.

Fig. S1.

Schematic illustration of the DEL selection procedure for isolation of β₂AR binders. DNA-encoded small molecules in a library were incubated with either 25 μg of detergent (DDM)-solubilized, Flag-tagged β₂AR immobilized on M1 anti-Flag beads, or a free-beads control for 1 h at room temperature (RT), while vigorously agitated. After the beads were repeatedly washed (five times), target-binding molecules were eluted by incubation at 72 °C for 30 min, as described in SI Materials and Methods, Affinity Selection. Denatured proteins were removed from the eluted sample, which was subsequently desalted. After the molecule number in the eluted and cleaned sample was determined by Q-PCR as described in SI Materials and Methods, the sample was subjected to the next round of selection for five times and subsequently one round of negative selection by incubating with free beads. The flow-through sample from the negative selection was subjected to the next-generation DNA sequencing to identify the binding molecules by their DNA tags.

Fig. 2.

Characterization of 15 for its binding to the β₂AR. (A) Dose–response curves of isoproterenol (ISO) competition binding to the β₂AR reconstituted in nanodiscs with ¹²⁵I-CYP were obtained in the presence of various concentrations of 15 as indicated. Values were expressed as percentages of the maximal ¹²⁵I-CYP binding level obtained from a one-site competition binding-log IC₅₀ curve fit of the vehicle [0.9% dimethyl sulfoxide (DMSO)] control data. Points on curves represent mean ± SEM obtained from at least three independent experiments done in duplicate. (B) The half-maximal concentration of 15 in the changes of isoproterenol competition binding was obtained from a dose–response curve replotted with the data set at 0.1 μM isoproterenol with various doses of 15 in A. (C) Dose-dependent increases in inverse agonist ³H-ICI-118,551 (ICI) binding to the β₂AR in nanodiscs. Points on the curve represent normalized values as percentages of the ³H-ICI-118,551 binding amount in the absence of 15 and mean ± SEM obtained from at least four independent experiments done in duplicate. (D) Characteristics of 15 for its physical interaction with the β₂AR were determined by the isothermal titration calorimetry (ITC) analysis with the detergent-solubilized, purified receptor. The thermogram (Top) and binding isotherm with the best titration curve fit (Bottom) shown are representatives of three independent experiments. Values represent mean ± SEM. (E) Extent of nanobody-60 (Nb60) binding to the β₂AR determined by ELISA in the presence of different ligands including 15. Values were expressed as ratios of the level of Nb60 binding in the vehicle (0.5% DMSO) control sample and represent mean ± SEM obtained from three independent experiments done in duplicate. BI, BI-167107. (F) The levels of ³H-Fen binding to the β₂AR upon treatment with the vehicle control (0.5% DMSO) or 15 at 50 μM in the absence or presence of transducers, either trimeric G_αβγ protein or β-arrestin1 (β-arr1) together with Fab30. Values were expressed as fold changes of the level of ³H-Fen binding in the vehicle (DMSO) control sample without the transducer and represent mean ± SEM obtained from three independent experiments done in duplicate. (G) Compound 15 dose-dependent decreases in the level of ³H-Fen high-affinity binding to the β₂AR promoted by either G_αβγ protein or β-arr1 together with Fab30. Values were expressed as percentages of the maximal ³H-Fen binding level promoted by each transducer in the vehicle control (0.5% DMSO) and represent mean ± SEM obtained from at least three independent experiments done in duplicate. All of the statistical analyses in the figure were performed, as described in Materials and Methods.

Fig. 3.

The effect of 15 on β₂AR-mediated functional activities. After pretreatment with 15 for 20 min at various concentrations as indicated, the β₂AR-mediated activities in cells were measured upon stimulation with isoproterenol (ISO) in a dose-dependent manner: (A) cAMP production by the endogenously expressed β₂AR and (B) β-arrestin recruitment to the exogenously expressed β₂V₂R. Values were expressed as percentages of the maximal level of the isoproterenol-induced activity in the vehicle (0.5% DMSO) control. Points on curves represent mean ± SEM obtained from four independent experiments done in duplicate. All of the statistical analyses in the figure were performed as described in Materials and Methods.

Fig. S2.

Alternative functional assays confirming the inhibitory activity of 15. After pretreatment with 15 at different concentrations as indicated for 20 min, several dose-dependent isoproterenol (ISO)-induced activities were monitored. (A) The level of cAMP was monitored in cells stably expressing the FRET-based bio-sensor (ICUE2) and the β₂AR. (B) The extent of β-arrestin recruitment was determined by FRET signals between stably expressed CFP-β-arrestin2 and β₂AR-YFP in HEK-293 cells. (C) The Gs-protein activation was measured by GTP hydrolysis in vitro, with the β₂AR reconstituted in HDL particles and purified Gs proteins. Values were expressed as percentages of the maximal level of the isoproterenol-induced activity in the vehicle (0.5% DMSO) control. Points on curves represent mean ± SEM obtained from at least three independent experiments done in duplicate. Statistical analyses were performed as described in SI Materials and Methods.

Fig. 4.

Specificity of 15 inhibition for β₂AR-mediated activity. Various receptors were transiently expressed to monitor β-arrestin recruitment, including (A) β₂AR, (B) β₂V₂R, (C) β₁AR, (D) V₂R, (E) VIPR, and (F) AT₁R. After pretreatment with 15 at different concentrations as indicated for 20 min, the extent of agonist-induced β-arrestin recruitment to these receptors was determined in a dose-dependent manner. Values were expressed as percentages of the maximal level of the activity induced by the agonist of each receptor in the vehicle (0.5% DMSO) control. Points on graphs represent mean ± SEM obtained from at least three independent experiments done in duplicate. AngII, angiotensin II; AVP, arginine vasopressin; ISO, isoproterenol; VIP, vasoactive intestinal peptide.

Fig. S3.

No inhibitory activity of 15 in cAMP production mediated by other receptors. After pretreatment with 15 at different concentrations for 20 min, cAMP production was monitored upon stimulation of endogenously expressed (A) prostaglandin E2 (PGE₂) and (B) vasoactive intestinal peptide (VIP) receptors. Values were expressed as percentages of the maximal level of the activity induced by the agonist of each receptor in the vehicle (0.5% DMSO) control. Points on curves represent mean ± SEM obtained from at least three independent experiments done in duplicate. Statistical analyses were performed as described in SI Materials and Methods.

Fig. S4.

The inhibitory activity of 15 in agonist-induced β-arrestin endocytosis. After pretreatment with 15 at different concentrations for 20 min, the level of β-arrestin endocytosis (Endo), upon stimulation of (A) β₂V₂R, (B) V₂R, and (C) AT₁R with respective agonists in a dose-dependent manner, was monitored. Values were expressed as percentages of the maximal level of the activity induced by the agonist of each receptor in the vehicle (0.5% DMSO) control. Points on curves represent mean ± SEM obtained from at least three independent experiments done in duplicate. AngII, angiotensin II; AVP, arginine vasopressin; ISO, isoproterenol.

Fig. S5.

Compound 15-mediated inhibition in agonist-induced signals upon stimulation of the β₂AR with a range of agonists. The level of 15-induced inhibition of β₂AR-mediated signals upon stimulation with (A and B) epinephrine (Epi); (D and E) fenoterol (Fen); and (G and H) clenbuterol (Clen) was determined by monitoring cAMP production (A, D, and G) and β-arrestin recruitment (B, E, and H) as described for Fig. 3. Values were expressed as percentages of the maximal level of the isoproterenol (ISO)-induced activity in the vehicle (0.5% DMSO) control. (C, F, and I) The extent of 15-induced curve shifts in ¹²⁵I-CYP competition binding with Epi (C), Fen (F), and Clen (I) was also determined as essentially described for Fig. 2A. Points on curves represent mean ± SEM obtained from at least three independent experiments done in duplicate.

Scheme S1.

Synthesis of compound 15.