Abstract
Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs.
SIGNIFICANCE STATEMENT We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.
Introduction
Smoothened (SMO) is a G protein-coupled receptor (GPCR) that, alongside 10 paralogs of Frizzleds, forms the Class F of GPCRs (Schulte, 2010). SMO signaling is of utmost importance during embryonic patterning and development, and dysfunction of SMO signaling is causative in the development of diverse tumors including basal cell carcinoma (Ingham and McMahon, 2001). Therefore, pharmacological targeting of SMO and SMO signaling evolved as an attractive antitumor treatment strategy established in clinical practice (Wu et al., 2017). On a structural level, this seven-transmembrane domain spanning receptor is characterized by a large, extracellular cysteine-rich domain (CRD) and a long C-terminal domain (Schulte, 2010). While SMO is essential for transmitting transcriptional responses via heterotrimeric G proteins and Glioma-associated oncogene (Gli) signaling induced by hedgehog proteins (three mammalian homologs: Desert, Indian, and Sonic hedgehog), the nature and mode of action of the endogenous ligand and the mechanisms of receptor activation are not fully understood (Byrne et al., 2016; Kong et al., 2019; Schulte and Kozielewicz, 2019). It is known that hedgehog proteins bind Patched, a cholesterol transporter, which in turn regulates SMO activation by postulated regulation of cholesterol levels (Zhang et al., 2018b). Cholesterol and other naturally occurring sterols are positive allosteric modulators and agonists of SMO in Gli and G protein-dependent signaling (Nachtergaele et al., 2012; Sever et al., 2016; Raleigh et al., 2018; Kowatsch et al., 2019; Qi et al., 2019). Moreover, recently solved structures of SMO in its active conformation have provided valuable insight into ligand-induced activation mechanism of the Class F receptor (Deshpande et al., 2019; Qi et al., 2019).
Due to the distinct link to human cancer and occurrence of several cancer-associated SMO mutations [e.g., D473H6.54 or W535L7.55; superscript numbering refers to Ballesteros Weinstein nomenclature of GPCRs (Ballesteros and Weinstein, 1995)], a plethora of small ligands, antagonists and inverse agonists, has been developed to target this receptor (Wu et al., 2017). Three of these compounds, vismodegib, sonidegib, and glasdegib, are approved as drugs for the treatment of basal-cell carcinoma (vismodegib and sonidegib) and acute myeloid leukemia (glasdegib) (Chen, 2016; Hoy, 2019). In addition, nature provides an effective SMO antagonist, the plant alkaloid cyclopamine (Incardona et al., 1998; Taipale et al., 2000). These and other ligands, such as SMO agonists (e.g., SAG series of analogs, purmorphamine), neutral antagonists (e.g., SANT-1), or inverse agonists (e.g., cyclopamine-KAAD), are frequently used to explore SMO pharmacology (Chen et al., 2002b; Rominger et al., 2009; Chen, 2016). There are two binding pockets on the transmembrane core of SMO (Wang et al., 2014). The upper binding pocket can accommodate ligands, such as SAG or vismodegib, whereas the lower binding pocket binds, e.g., SANT-1. To date, binding affinities of the SMO ligands were determined using classic radioligand binding methods (Frank-Kamenetsky et al., 2002; Rominger et al., 2009; Wang et al., 2014) and, more often, fluorescence-based assays using the green-yellow fluorescent BODIPY-cyclopamine (Chen et al., 2002a,b; Manetti et al., 2010; Gorojankina et al., 2013; Huang et al., 2016, 2018). The fluorescently labeled cyclopamine has been used in three separate experimental approaches: assessment of ligand-receptor interaction based on detection of fluorescence using confocal microscopy, flow cytometry, or fluorescence polarization (Chen et al., 2002a,b; Bee et al., 2012; Gorojankina et al., 2013; Huang et al., 2016, 2018; Lu et al., 2018). While these methods offer valuable insight into ligand-receptor binding in live cells, they suffer from several shortcomings, including 1) laborious protocols that require long ligand incubation times (up to 10 hours); 2) extensive cell washing due to lipophilicity of BODIPY-cyclopamine; 3) high levels of non-specific binding in untransfected cells or in the presence of saturating concentrations of unlabeled competitors; 4) the need for data normalization of BODIPY-fluorescence values to receptor expression values; and 5) in the case of radioligand binding experiments, health risks, need for well-controlled designated areas, and waste disposal.
To overcome these experimental limitations, we established and validated a live-cell, nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based binding assay to assess the binding properties of BODIPY-cyclopamine and unlabeled SMO ligands to an N terminally nanoluciferase (Nluc)-tagged SMO (Nluc-SMO) and SMO lacking the CRD (ΔCRD Nluc-SMO) in HEK293 cells devoid of endogenous SMO (ΔSMO HEK293). This proximity-based ligand-binding assay was developed recently to assess ligand binding to Class A GPCRs and receptor tyrosine kinases (Stoddart et al., 2015, 2018a,b; Mocking et al., 2018; Bosma et al., 2019; Bouzo-Lorenzo et al., 2019; Sykes et al., 2019). It relies on the high specificity of BRET between Nluc-tagged protein (BRET donor) and fluorescently tagged ligand (BRET acceptor) that can only occur when both BRET partners are within a distance of 10 nm (100 Å). Thus, the interference of non-specifically bound probe, outside of the BRET radius to the receptor is, in contrast to detecting solely ligand fluorescence, minimal. Along these lines, no washing steps are required. In the present study, we employ NanoBRET to monitor binding of commercially available SMO ligands. Furthermore, the sensitivity of the assay enabled us to dissect the pharmacological properties of separate BODIPY-cyclopamine binding pockets in the transmembrane-spanning receptor core of the Class F receptor SMO. Thus, this NanoBRET-based binding assay provides a valuable complement to the toolbox of high-throughput compatible screening assays for Class F GPCRs.
Materials and Methods
DNA Cloning and Mutagenesis.
Nluc-A3 was from Stephen Hill, University of Nottingham, UK (Stoddart et al., 2015). SMO-Rluc8 coding for mouse Smoothened was from Nevin A. Lambert, Augusta University, Georgia (Wright et al., 2019). The mouse SMO sequence was subcloned into an empty N terminally tagged Nluc vector containing 5-HT3A signal peptide using BamHI and XbaI restriction sites. First, the BamHI site present in mouse SMO was removed using site-directed mutagenesis (GeneArt; Thermo Fisher Scientific) with the following primers: 5′-CCTCCAGGGGCTGGGGTCCATTCATTCCCGC-3′ (forward primer) and 5′-GCGGGAATGAATGGACCCCAGCCCCTGGAGG-3′ (reverse primer). Next, the mouse SMO sequence was cloned in-frame into the Nluc vector using forward primer: 5′-GACGGATCCGCGGCCTTGAGCGGGAACGTG-3′ and reverse primer: 5′-CGTTCTAGATCAGAAGTCCGAGTCTGCATC-3′. ΔCRD Nluc-SMO was generated using the mouse SMO lacking the BamHI site by cloning it into N terminally tagged Nluc vector between BamHI and XbaI using forward primer: 5′-GACGGATCCGAGGTACAAAACATCAAGTTC-3; and reverse primer: 5′-CGTTCTAGATCAGAAGTCCGAGTCTGCATC-3′. ΔCRD and full-length Nluc-SMO D477G6.54/E522K7.38 were generated with site-directed mutagenesis (GeneArt; Thermo Fisher Scientific) by first obtaining D477G6.54 mutation with the following primers: 5′-GCTGCCACTTCTATGGCTTCTTCAACCAGGC-3′ (forward primer) and 5′-GCCTGGTTGAAGAAGCCATAGAAGTGGCAGC-3′ (reverse primer). Subsequently the E522K7.38 mutation was introduced with 5′-CCCAGCCTCCTGGTGAAGAAGATCAATCTAT-3′ (forward primer) and 5′-ATAGATTGATCTTCTTCACCAGGAGGCTGGG-3 (reverse primer). All constructs were validated by sequencing (Eurofins GATC, Konstanz, Germany).
Cell Culture.
ΔSMO HEK293 cells were generated with CRISPR/Cas9 genome editing using SMO-targeting sgRNA 5′-CAACCCCAAGAGCTGGTACGAGG-3′. The cells were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% l-glutamine (all Thermo Fisher Scientific) in a humidified CO2 incubator at 37°C. To generate cell lines stably expressing Nluc-SMO and ΔCRD Nluc-SMO, ΔSMO HEK293 cells were transfected with Nluc-SMO and ΔCRD Nluc-SMO constructs using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions. About 24 hours post transfection, cells were passaged at 1:10, and 48 hours post transfection medium was supplemented with 2000 µg/ml geneticin (Thermo Fisher Scientific). The medium was replaced every 2 days to select the cells transfected with the plasmids. The cells were maintained in the presence of the antibiotic for a period of 3 weeks until the stable culture was established. Absence of mycoplasma contamination was routinely confirmed by PCR using 5′-GGCGAATGGGTG AGTAACACG-3′ and 5′-CGGATAACGCTTGCGACTATG-3′ primers detecting 16S ribosomal RNA of mycoplasma in the media after 2 to 3 days of cell exposure. All cell culture plastics were from Sarstedt, unless otherwise specified.
Live-Cell ELISA.
For quantification of cell surface receptor expression by labeling with anti-Nluc antibody, ΔSMO HEK293 cells at the density of 4·× 105 cells/ml were transfected in suspension using Lipofectamine 2000 with 50–500 ng of the indicated receptor plasmid DNA with 500–950 ng of pcDNA plasmid DNA. The cells (100 µl) were seeded onto a PDL (poly-D-lysine)-coated transparent 96-well plate with flat bottom and grown overnight. Twenty-four hours later, the cells were washed twice with 0.5% BSA in PBS and incubated with a mouse anti-Nluc (2 µg/ml, #MAB10026; RnD Systems) in 1% BSA/PBS for 1 hour at 4°C. Following incubation, the cells were washed four times with 0.5% BSA/PBS and incubated with a horseradish peroxidase-conjugated goat anti-mouse antibody (1:3000, #31430; Thermo Fisher Scientific) in 1% BSA/PBS for 1 hour at 4°C. The cells were washed three times with 0.5% BSA/PBS, and 50 µl of the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (#T8665; Sigma-Aldrich, St. Louis, MO, USA) were added. The cells were incubated further for 20 minutes, and upon development of a blue product, 50 µl of 2 M HCl was added and the absorbance was read at 450 nm using a BMG Ω POLARstar plate reader. The data were analyzed in GraphPad Prism 6.
Immunoblotting.
ΔSMO HEK293 cells were transfected in suspension using Lipofectamine 2000 (50–500 ng of receptor plasmid DNA with 500–950 ng of pcDNA plasmid DNA per 4·× 105 cells/ml) and seeded (700 µl) onto wells of a 24-well plate. Protein lysates were obtained using Laemmli buffer with 0.5% NP-40 and 5% β-mercaptoethanol. Lysates were sonicated and analyzed on 4%–20% Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad) and transferred to PVDF membranes using the Trans-Blot Turbo system (Bio-Rad). After blocking with 5% milk in TBS-T, membranes were incubated with primary antibodies in blocking buffer: rabbit anti-GAPDH (1:5000, #2118; Cell Signaling Technology) and mouse anti-Nluc (0.5 µg/ml, #MAB10026; RnD Systems) overnight at 4°C. Proteins were detected with horseradish peroxidase-conjugated secondary antibody [1:5000; goat anti-rabbit (#31460; Thermo Fisher Scientific)or 1:3000; goat anti-mouse (#31430; Thermo Fisher Scientific), and Clarity Western ECL Blotting Substrate (Bio-Rad)]. All uncropped blots can be found in the Supplemental Fig. 1.
NanoBRET Binding Assay.
ΔSMO HEK293 cells were transiently transfected in suspension using Lipofectamine 2000 (Thermo Fisher Scientific). Cells (4 × 105 cells/ml) were transfected with 50–500 ng of receptor plasmid DNA and 500–950 ng of pcDNA. The cells (100 µl) were seeded onto a PDL-coated black 96-well cell culture plate with solid flat bottom (Greiner Bio-One). Twenty-four hours post-transfection, cells were washed once with HBSS (HyClone) and maintained in the same buffer. In the saturation experiments, the cells were incubated with different concentrations of BODIPY-cyclopamine (80 µl) for 60 minutes at 37°C before the addition of the luciferase substrate coelenterazine h (5 µM final concentration, 10 µl) for 6 minutes prior to the BRET measurement. In the competition experiments, the cells were preincubated with different concentrations of unlabeled ligands (70 µl) for 30 minutes at 37°C. Fixed concentration of BODIPY-cyclopamine was then added (10 µl) and the cells were incubated for an additional 60 minutes at 37°C before the addition of the luciferase substrate coelenterazine-h (5 µM final concentration, 10 µl) for 6 minutes prior to the BRET measurement. In the association experiments, the cells were preincubated with 10 µM SANT-1 (30 minutes), followed by coelenterazine-h h (5 µM final concentration) at 37°C prior to the addition of different BODIPY-cyclopamine concentrations. The BRET signal was measured every minute for 90 minutes at 37°C. The BRET ratio was determined as the ratio of light emitted by BODIPY-cyclopamine (energy acceptor) and light emitted by Nluc-tagged biosensors (energy donors). The BRET acceptor (bandpass filter 535–30 nm) and BRET donor (bandpass filter 475–30 nm) emission signals were measured using a CLARIOstar microplate reader (BMG). ΔBRET ratio was calculated as the difference in BRET ratio of cells treated with ligands and cells treated with vehicle. BODIPY fluorescence was measured prior to reading luminescence (excitation: 477–14 nm, emission: 525–30 nm). To calculate Z-factors (Zhang et al., 1999), ΔSMO HEK293 stably overexpressing Nluc-SMO or ΔCRD Nluc-SMO were plated onto PDL-coated 96-well plates. On the next day, the cells were pre-incubated either with vehicle (0.1% dimethylsulfoxide, 48 wells) or 10 µM SANT-1 (48 wells) for 30 minutes at 37°C prior to the addition of BODIPY-cyclopamine (200 nM for Nluc-SMO stable cells and 10 nM for ΔCRD Nluc-SMO stable cells). The following equation was used to calculate Z-factor:
Data were analyzed using GraphPad Prism 6.
Computational Studies.
Molecular docking was conducted with Glide SP (2018-4; Schrödinger LLC, New York, 2018) using default parameters. BODIPY-cyclopamine was docked to a 25 × 25 × 25 Å3 box located either on the mass center of SAG21k (i.e., the upper binding pocket) or to the mass center of both SAG21k and the 7TM-bound cholesterol (i.e., the lower binding pocket) of SMO [PDB ID: 6O3C (Deshpande et al., 2019)]. Prior to docking, the SMO structure was prepared with protein preparation wizard of Schrödinger Maestro and BODIPY-cyclopamine conformations generated using LigPrep with Epik in pH 7 ± 2 (Shelley et al., 2007). The protocol was tested by docking cyclopamine to the same SMO structure (PDB ID: 6O3C) and comparing it to the cyclopamine-SMO complex (PDB ID: 4O9R, Supplemental Fig. 2). It reproduced a similar cyclopamine binding pose to that in the crystal structure (Supplemental Fig. 2). The active SMO has a larger binding site compared with the inactive SMO; this may contribute to the ligand RMSD = 2.55 Å (the highest scoring pose, Glide DockingScore).
Solvent-accessible surface areas were calculated and solvent-accessible surfaces visualized with Biovia DiscoverStudio Visualizer 2017 R2 (Dassault Systèmes SE) using 960 grid points per atom and probe radius of 1.40 Å. Active SMO structure (PDB ID: 6O3C) was used as a representative of a full-length SMO, whereas active ΔCRD-SMO structure [PDB ID: 6OT0 (Qi et al., 2019)] was used as a representative of a ΔCRD-SMO. 6O3C and 6OT0 were selected for the calculation as they represent the same conformational state (ligand-bound, active) of the receptor and offer thus the best comparability between the currently solved SMO and ΔCRD SMO structures.
Ligands.
BODIPY-cyclopamine was from BioVision Inc. Purmorphamine (9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine) was from Abcam. SAG1.3 (3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide) was from Sigma. Cyclopamine-KAAD and SANT-1 ((4-benzyl-piperazin-1-yl)-(3,5-dimethyl-1-phenyl-1H-pyrazol-4-ylmethylene)-amine) were from Abcam. All ligands were dissolved in dimethylsulfoxide and stored in aliquots at −20°C. The ligands underwent a maximum of two freeze-thaw cycles. Coelenterazine-h was from Biosynth and it was stored as 2.4 mM aliquots in acidified ethanol at −80°C. Protein-low binding tubes (Eppendorf) were used to make serial dilutions of BODIPY-cyclopamine.
Data and Statistical Analysis.
Live-cell ELISA data were analyzed using GraphPad Prism 6 and represent mean ± S.E.M. of n individual experiments (biological replicates) performed at least in duplicates (technical replicates). Live-cell ELISA data were analyzed for differences with one-way ANOVA with Fisher’s least significant difference post hoc analysis. Significance levels are given as *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Please refer to the figure legends for more details on the displayed data.
BODIPY-cyclopamine saturation curves were fit using three-parameter or biphasic nonlinear regression models (logarithmic scale for BODIPY-cyclopamine concentrations) or one- or two-site saturation nonlinear regression models (linear scale for BODIPY-cyclopamine concentrations). Error bars on the binding curves represent mean ± S.E.M. from n independent experiments for each tested concentration. Affinity values obtained from logarithmic scale data are presented as a best-fit pKd (pKi for unlabeled ligands) ± S.D. Maximal binding values obtained from linear data are presented as a best-fit Bmax with 95% confidence interval (CI). NanoBRET binding models were selected based on an extra-sum-of square F-test (P < 0.05).
Competition binding curves were analyzed using a one-site competitive binding model to obtain equilibrium dissociation constants values (Ki) of unlabeled ligands as per the modified Cheng-Prusoff equation (Cheng and Prusuff, 1973):
where Ki is the searched dissociation constant of an unlabeled ligand, IC50 is the inhibitory constant 50 of an unlabeled ligand obtained from the competition curve, [LL] is the concentration of a labeled ligand used in the competition experiment, and Kd is the equilibrium dissociation constant of a labeled ligand obtained from the saturation studies.
To analyze the labeled ligand binding kinetics data, one-phase association or two-phase association models were selected based on an extra-sum-of square F-test:
One-phase association:Two-phase association:where Y0 is Y value at time x = 0, plateau is the Y value at infinite times, and kobs is the association constant expressed in , t1/2 = ln(2)/kobs.
kon (Association rate) and koff (dissociation rate) are calculated from the following linear equation:“Kinetic” Kd is calculated using kon and koff and represented with ±S.D.:
Results
N Terminally Nluc Tagged SMO Constructs Are Expressed at the Cell Surface.
To establish a NanoBRET-based binding assay for the Class F receptor SMO, we adopted the cloning strategy of previously presented Class A GPCR including a 5-HT3A receptor-derived signal sequence and an extracellular, N terminally Nluc fused to either the full-length mouse SMO or ΔCRD SMO. Subsequently, these constructs are referred to as Nluc-SMO and ΔCRD Nluc-SMO, respectively (Fig. 1A). Both receptor constructs are expressed in the cells and at the cell surface upon transient transfection of ΔSMO HEK293 cells as shown by immunoblotting of whole cell lysates and a live-cell surface ELISA (Fig. 1, B and C).
BODIPY-cyclopamine Binding to Nluc-SMO Can Be Monitored by NanoBRET.
The commercially available BODIPY-labeled derivative of the plant alkaloid cyclopamine (BODIPY-cyclopamine; Fig. 2A) associates with Nluc-SMO transiently expressed in ΔSMO HEK293 cells in a concentration-dependent manner, reaching saturation at ∼1000 nM (pKd = 6.8 ± 0.1, Fig. 2, B and C), which is consistent with recently published data (Lu et al., 2018). Similarly, BODIPY-cyclopamine binds the ΔCRD SMO construct (biphasic fit pKd1 = 8.4 ± 0.2, Fig. 2, D and E) with higher affinity and importantly the NanoBRET produced by BODIPY-cyclopamine binding was larger in the ΔCRD Nluc-SMO compared with Nluc-SMO (two-sites fit ΔBRET Bmax1 ΔCRD receptor = 0.080, 95% CI [0.07–0.09] and one-site fit ΔBRET Bmax full-length receptor = 0.034, 95% CI [0.031–0.036]). Furthermore, BODIPY-cyclopamine binding to ΔCRD Nluc-SMO was more complex than binding to Nluc-SMO as indicated by the biphasic binding curve for ΔCRD Nluc-SMO especially at higher concentrations of the ligand (biphasic fit pKd2 = 6.8 ± 0.7 and ΔBRET Bmax2 = 0.033, 95% CI [0.021–0.045]).
While quantification of BODIPY-cyclopamine binding on living cells was assessed 60 minutes after ligand addition, we were also interested in the binding kinetics of BODIPY-cyclopamine. Therefore, we followed BODIPY-cyclopamine association at different ligand concentrations using Nluc-SMO and ΔCRD Nluc-SMO expressing ΔSMO HEK293 cells (Fig. 3). The kinetic analysis results are summarized in Tables 1 and 2 and Supplemental Fig. 3. The kinetic binding analysis underlined that the affinity of BODIPY-cyclopamine to the ΔCRD Nluc-SMO was higher and BRET counts were larger compared with Nluc-SMO. The kinetic Kd values (105 ± 25 and 23 ± 16 nM for the full-length and ΔCRD receptors, respectively) were in fair agreement with the values obtained from saturation binding experiments. Moreover, association of BODIPY-cyclopamine (1000 nM) to ΔCRD Nluc-SMO followed a two-phase curve arguing for the involvement of another binding site (Table 2), which is consistent with the saturation binding results. To further dissect BODIPY-cyclopamine association to SMO we employed the SMO antagonist SANT-1 at 10 µM, which interacts with the 7TM core of the receptor (Chen et al., 2002b). For both receptor constructs SANT-1 reduced association of BODIPY-cyclopamine at the lower concentrations, but did not completely abrogate binding of 1000 nM BODIPY-cyclopamine.
NanoBRET-Based Ligand Binding Is Superior to Fluorescence-Based Quantification of BODIPY-cyclopamine Binding to SMO.
Cyclopamine is chemically similar to cholesterol, rendering it cell permeable and lipophilic resulting in detectable nonspecific binding to cells and particularly membranes. When comparing the increase in NanoBRET between BODIPY-cyclopamine and Nluc-SMO (or ΔCRD Nluc-SMO) with the increase in the fluorescence signal emerging from BODIPY-cyclopamine, specific and saturable binding can be detected by NanoBRET already in the lower nanomolar range, especially with the ΔCRD Nluc-SMO construct. On the other hand, the nonsaturable increase in fluorescence is detectable only at higher concentrations of BODIPY-cyclopamine. More importantly, the NanoBRET signal saturates at ligand concentrations that produce an unreliable increase in fluorescence, especially in the case of the ΔCRD Nluc-SMO. At higher BODIPY-cyclopamine concentrations, beyond those required to saturate the NanoBRET signal, a linear increase of fluorescence was detectable, indicating that under these experimental conditions, fluorescence includes a substantial component of unspecific ligand binding (Fig. 4, A and B). This was particularly obvious when comparing the fluorescence signal at a BODIPY-concentration producing maximal binding in the absence and presence of 10 µM SANT-1 for the Nluc-SMO and the ΔCRD Nluc-SMO constructs (Fig. 4C). At this concentration the NanoBRET signal was blocked by SANT-1, whereas fluorescence was not affected (compare Fig. 3; Fig. 4C). After having established the superiority of the NanoBRET-based binding assay over fluorescence-based detection of ligand binding, we aimed to investigate if this assay is high-throughput compatible by Z-factor analysis. As expected from the BODIPY-cyclopamine binding parameters, the Z-factor for Nluc-SMO with coelenterazine-h as Nluc substrate was poor comparing basal BODIPY-cyclopamine BRET in the absence and presence of 10 µM SANT-1. However, this could be improved by changing to furimazine as Nluc substrate. Furthermore, the Z-factor analysis with the ΔCRD SMO construct provided an excellent assay window already with coelenterazine-h (Supplemental Fig. 4).
BODIPY-cyclopamine Binding to SMO Is Surmountable.
To explore the competitive nature of BODIPY-cyclopamine binding to SMO in more detail, we combined BODIPY-cyclopamine with increasing concentrations of commercially available SMO ligands (Fig. 5A): agonists (purmorphamine and SAG1.3), antagonist (SANT-1), and inverse agonist (cyclopamine-KAAD), employing both the full-length Nluc-SMO (competition with 200 nM BODIPY-cyclopamine) as well as the ΔCRD Nluc-SMO (competition with 10 nM BODIPY-cyclopamine). While cyclopamine-KAAD and SANT-1 presented the highest affinity to Nluc-SMO, the agonist SAG1.3 was intermediate and purmorphamine showed the lowest affinity (Fig. 5B; Table 3). A similar rank order was obtained in the ΔCRD Nluc-SMO-transfected cells (Fig. 5C; Table 4). Interestingly, residual BODIPY-cyclopamine binding produced NanoBRET, at competitor concentrations sufficiently high to reach saturation, that were substantially higher in the full-length Nluc-SMO compared (Fig. 5B) to ΔCRD Nluc-SMO (Fig. 5C). At maximal competition SANT-1 reduced BODIPY-cyclopamine (200 nM) binding to 45.2% of maximal binding, whereas it completely abolished BODIPY-cyclopamine (10 nM) binding at the ΔCRD Nluc-SMO (∼0.1% binding left). These findings indicate that, at the tested concentrations, BODIPY-cyclopamine binding is surmountable to a higher degree at the ΔCRD Nluc-SMO compared with Nluc-SMO. Additionally, cyclopamine-KAAD competition with BODIPY-cyclopamine at the full-length receptor did not reach the plateau, indicating further displacement of the fluorescent ligand, presumably at a different binding pocket.
Differential Competition of BODIPY-cyclopamine Binding Allows Pharmacological Separation of Two Binding Sites.
The successful targeting of SMO with vismodegib in the therapy of basal cell carcinoma has also led to the discovery of therapy-resistant point mutations in SMO (Zhang et al., 2018a). Here, we introduced the double mutant D477G6.54/E522K7.38 into Nluc-SMO and ΔCRD Nluc-SMO (corresponding to human D4736.54 and E5187.38 mutants) to further dissect the contribution of different binding sites to BODIPY-cyclopamine-SMO interaction. The mutant versions are expressed on the cell surface upon transient transfection in ΔSMO HEK293 cells (Fig. 6A). To further define the binding characteristics of the two separate BODIPY-cyclopamine binding sites in the 7TM core of the receptor, we made use of a saturating concentration of SANT-1 (10 µM) and probed the wild-type and D477G6.54/E522K7.38 of Nluc-SMO and ΔCRD Nluc-SMO with increasing concentrations of BODIPY-cyclopamine (Fig. 6, B and C). In line with the competition data using a fixed BODIPY-cyclopamine concentration, we found that SANT-1, which solely binds to the 7TM ligand binding site of SMO (Wang et al., 2014), reduces the maximal binding of BODIPY-cyclopamine at the Nluc-SMO by one third with maintained affinity. At the ΔCRD Nluc-SMO, however, SANT-1 virtually prevents BODIPY-cyclopamine interaction with SMO up to a concentration of 100 nM. At 100 nM and above, BODIPY-cyclopamine reliably showed saturable, SANT-1 (10 µM)-insensitive binding in cells transfected with ΔCRD Nluc-SMO. The SANT-1-insensitive fraction of BODIPY-cyclopamine shows a reduced Bmax and a lower affinity. In the full-length Nluc-SMO, the double mutant did not affect Bmax of BODIPY-cyclopamine but there was a statistically significant, ca. fourfold decrease in affinity (one-site fit Bmax = 0.035, 95% CI [0.032–0.039], P = 0.8193; pKd = 6.2 ± 0.1; P < 0.0001). In ΔCRD Nluc-SMO D477G6.54/E522K7.38, the BODIPY-cyclopamine binding followed a one-site curve, as opposed to the wild-type receptor. Furthermore, the affinity was dramatically reduced by ca. 125-fold (pKd = 6.3 ± 0.1) and the maximal binding also decreased (one-site fit Bmax = 0.074, 95% CI [0.071–0.077], P < 0.0001).
In both cases, SANT-1 (10 µM), which targets the lower binding pocket, maintains its effect on BODIPY-cyclopamine at both the wild-type and the D477G6.54/E522K7.38 for full-length and ΔCRD SMO. Consistently with the SANT-1 binding mode, the double mutant in the upper site does not affect the SANT-1-insensitive fraction of BODIPY-cyclopamine binding in ΔCRD Nluc-SMO (Fig. 6C). In addition, we provide a kinetics analysis of BODIPY-cyclopamine association to D477G6.54/E522K7.38 ΔCRD Nluc-SMO (Fig. 6D; Table 5). The association of BODIPY-cyclopamine to ΔCRD Nluc-SMO D477G6.54/E522K7.38 follows a one-phase association curve with a “kinetic” Kd = 1403 ± 701 nM, which is in fair agreement (ca. threefold decrease) with the saturation binding data.
Molecular Docking of BODIPY-cyclopamine Supports the Two-binding Site Model.
To obtain more detailed insights into the BODIPY-cyclopamine binding at the atomistic level, we set up a molecular docking study. We selected the recent SMO structure [PDB ID: 6O3C (Deshpande et al., 2019)] as a target for our docking, as it manifests the two 7TM binding sites (i.e., they are occupied with small molecular ligands; Fig. 7A). At the upper binding pocket, BODIPY-cyclopamine occupies hook-like conformations, wherein the cyclopamine-moiety of the molecule is buried within the 7TM core of the receptor and the BODIPY-moiety is exposed to the solvent. The main polar interactions at the upper pocket are with E5187.38 (note: the crystal structure is of the human SMO) and K395 at ECL2. At the lower binding pocket, the whole BODIPY-cyclopamine molecule is bound within the 7TM core of the receptor, the main polar interaction counterparts being with T5287.48, E5187.38, and N219 at the N terminus. Furthermore, in silico analysis of solvent-accessible surfaces revealed a better ligand-accessibility of ΔCRD SMO compared with the wild-type receptor (Fig. 7B) as expected from the difference in binding parameters between full-length and ΔCRD SMO.
Discussion
The development of a NanoBRET-based ligand binding assay provides an interesting complement to GPCR pharmacology, enabling ligand binding studies on living cells in real time with simplified protocols (Stoddart et al., 2015, 2018a). Here, we optimize this assay for the Class F receptor SMO improving sensitivity and performance of previously used fluorescence-based approaches (Chen et al., 2002a,b; Manetti et al., 2010; Tao et al., 2011; Gorojankina et al., 2013; Huang et al., 2016, 2018). Due to its large assay window for specific binding and the low influence of unspecific binding this NanoBRET-based assay is particularly suitable for lipophilic ligands such as cyclopamine and potentially other cholesterol-like molecules, which target SMO and generally show unspecific interactions with the membrane. Furthermore, this high-throughput compatible assay should be adaptable to any fluorescently tagged molecule acting as SMO ligand and could, provided small molecules become available to target Frizzleds, also be employed for other Class F receptors.
Recent insight into the molecular mechanisms of drug action on SMO by crystallography and CryoEM provide somewhat controversial yet intriguing information regarding cyclopamine and cholesterol interaction with the 7TM ligand-binding site and the CRD (Weierstall et al., 2014; Huang et al., 2016, 2018; Deshpande et al., 2019; Qi et al., 2019). Here, we have been able to pharmacologically separate two BODIPY-cyclopamine binding sites on the 7TM core of SMO. It has been reported that total BODIPY-cyclopamine binding to full-length SMO is composed of at least two components: ligand binding to the CRD (Huang et al., 2016, 2018), for which we did not find evidence for in our experiments, and the 7TM core (Weierstall et al., 2014; Huang et al., 2018). Most importantly, SANT-1, which solely binds to the receptor core in the lower pocket of the SMO binding site (Wang et al., 2014), competes with BODIPY-cyclopamine more efficiently in the ΔCRD Nluc-SMO compared with the full length receptor. This large increase in affinity and in NanoBRET signal (Bmax), suggests that the CRD exerts a negative allosteric modulation on BODIPY-cyclopamine binding to the SMO 7TM core. The residual saturable binding of BODIPY-cyclopamine to ΔCRD Nluc-SMO above 10−7 M identifies a SANT-1-insensitive fraction, suggesting an additional binding pocket for BODIPY-cyclopamine. While BODIPY-cyclopamine binding to ΔCRD Nluc-SMO clearly follows a two-site (biphasic) regression fit, the ligand coupling to the full-length receptor follows a typical one-site model. Therefore, we assume that this SANT-1-insensitive binding site cannot be solely explained by a potential non-specific BODIPY-cyclopamine association to membranes as this second pocket would also become more apparent at the full-length receptor. Moreover, non-specific binding would most likely increase linearly. Given the simultaneous binding of SAG21k and cholesterol to SMO in the recent crystal structure (PDB ID: 6O3C) of active, nanobody NbSmo8-bound SMO (Deshpande et al., 2019) and the fact that SANT-1 and cyclopamine occupy two different parts of the small molecule binding space in SMO (Wang et al., 2014; Weierstall et al., 2014; Huang et al., 2018) it could also be possible that BODIPY-cyclopamine and SANT-1 bind the 7TM core simultaneously. It cannot be excluded that the binding modes of BODIPY-cyclopamine and cyclopamine are different as it remains to be verified by structural studies. Importantly, a recent study on ALLO-1, a small molecule ligand targeting the lower pocket of SMO, showed that this compound competes with BODIPY-cyclopamine but not with [3H]cyclopamine, further supporting our two sites hypothesis (Zhou et al., 2019). Along these lines, distinct binding poses of cyclopamine and related sterols were reported in several crystal structures using SMO from various species. In summary, there is a cyclopamine/sterol binding site in the lipid groove of the CRD (Byrne et al., 2016; Huang et al., 2016, 2018; Deshpande et al., 2019), one in the upper pocket of the 7TM core (Huang et al., 2018; Qi et al., 2019) and one in the lower position (Deshpande et al., 2019), which overlaps with the SANT-1 binding pocket (Wang et al., 2014) (Fig. 7A). Moreover, it has been demonstrated that SAG1.3-induced full-length SMO-mediated Gli transcriptional activity still reaches saturation, albeit at lower efficacy following treatment with SANT-1.This further provides functional evidence that both the upper (SAG binding site) and the lower pocket (SANT-1 binding site) in the 7TM core of SMO can be occupied by ligands simultaneously and that these pockets may allosterically regulate each other (Chen et al., 2002b). Similar conclusions were drawn from radioligand binding studies (Frank-Kamenetsky et al., 2002; Tao et al., 2011). Furthermore, the phenomenon of allosteric regulation in the SMO binding site was also inferred from studies on other SMO ligands (Hoch et al., 2015; Chen et al., 2016).
Interestingly, removal of the CRD of SMO increased both Bmax and the Kd of BODIPY-cyclopamine. The absence of the CRD, which obviously includes removal of the proposed CRD binding site for BODIPY-cyclopamine, most likely provides better access to the normally buried binding site (the lower binding pocket) in the core of the receptor causing a left shift and an increased Bmax (Fig. 7B). Related to that, one might also postulate an allosteric action of the CRD on the ligand-binding sites on the 7TM core, an idea that is fueled by the observation that ΔCRD SMO exerts a higher constitutive activity (Byrne et al., 2016; Raleigh et al., 2018). A part of the explanation for the efficacy shift of the BODIPY-cyclopamine binding curve could also be the altered BRET parameters, because the distance of the NanoBRET donor and the acceptor could be shorter in the ΔCRD SMO compared with the full-length receptor. However, while the different BRET efficiencies in the two receptor constructs would affect the amplitude of the NanoBRET signal originating from BODIPY-cyclopamine binding, they cannot explain the leftward shift of the binding curves, indicating a higher affinity to the ΔCRD Nluc-SMO. It should be noted that in previous publications low concentrations of BODIPY-cyclopamine (usually 5 nM) were used for SMO binding assays. Given our data regarding the different affinities of the separate BODIPY-cyclopamine binding sites, these low ligand concentrations neither allowed sampling the SANT-1-insensitive, low affinity site (the upper binding pocket) in the core of ΔCRD-Nluc SMO nor the putative site on the CRD of the full-length receptor. Application of the novel NanoBRET methodology, however, allows to reliably detect even picomolar and nanomolar amounts of BODIPY-cyclopamine bound to ΔCRD Nluc-SMO and ΔCRD Nluc-SMO D477G6.54/E522K7.38.
In summary, we dissect BODIPY-cyclopamine interaction with two allosterically linked binding sites on the SMO 7TM core with different affinities. Allosteric interaction between these pockets and BODIPY-cyclopamine binding to the lower one (the high affinity site) can also be inferred from the mutagenesis results reported recently (Deshpande et al., 2019). Here, we propose that the BODIPY-cyclopamine high-affinity site is the deep SANT-1 binding pocket (the lower binding pocket). Since the D477G6.54/E522K7.38 in TM6 and TM7 partially affect the high-affinity component of BODIPY-cyclopamine binding, it could be that interaction with D4776.54/E5227.38 provides a transition mechanism of ligand binding to the deeper pocket (the high affinity site). Low-affinity binding to the D477G6.54/E522K7.38 mutant in the upper 7TM site (the low affinity site) is still possible and that binding is insensitive to allosteric modulation by SANT-1 binding to the deeper site (the high affinity site). Interestingly, the difference in Bmax between the wild-type and mutated ΔCRD Nluc-SMO [(ΔCRD Nluc-SMO Bmax1 + ΔCRD Nluc-SMO Bmax2) − ΔCRD Nluc-SMO D477G6.54/E522K7.38 Bmax ∼0.039] corresponds well to the ΔCRD Nluc-SMO Bmax2 (∼0.033) and Nluc-SMO Bmax (∼0.034). Assuming no alterations in BRET transfer efficiency between different constructs, it could be that binding of BODIPY-cyclopamine to the upper pocket (the low affinity site) of a ligand-free Nluc-SMO is virtually nondetectable. Moreover, BODIPY-cyclopamine/SMO interactions in living cells surely depend also on the conformational states of the receptor as well as the presence of endogenous SMO ligand – cholesterol. These factors further add to the complexity of this binding mechanism.
It needs to be noted that the predicted conformational space of BODIPY-cyclopamine is large; one has to be careful when interpreting the docking results. However, the in silico studies indicate that the ligand can interact with E5187.38 (corresponds to E5227.38 in mouse SMO) in both binding pockets. In the upper pocket (the low-affinity site), the interaction partner is a hydroxyl group of BODIPY-cyclopamine, which can also interact with K in the mutated receptor. In the lower pocket (the high-affinity site), the partner is an amide nitrogen that cannot interact with K, however, the adjacent oxygen could. In both cases, D→G6.54 renders the binding site more spacious. As a consequence, different ligand poses could be obtained compared with the wild-type SMO. Furthermore, the docking scores are in line with our two binding site model hypotheses; the average scores of all the high affinity and the low affinity sites poses are −8.3 ± 2.4 (17 poses) and −4.3 ± 1.7 (602 poses), respectively (Supplemental Fig. 5). In addition to the allosteric interaction between the two binding pockets, BODIPY-cyclopamine binding, with its cyclopamine core and the linker-BODIPY moiety occupying the lower and the upper pockets, respectively, would also be in line with the competition binding data showing that the ligands interacting with either pocket could, at least partially, displace this fluorescent ligand.
For drug discovery efforts, the ΔCRD Nluc-SMO probe presents a valuable tool with an advantageous assay window. The intrinsic caveat of the lack of the physiologically relevant CRD, however, remains, requiring thorough validation of screening hits in assays relying on full length SMO. Further work will address in which way the separate ligand-binding sites interact allosterically and what role the CRD-core contacts play for that potential communication.
Acknowledgments
We thank Stephen Hill (University of Nottingham, UK) for the Nluc-A3 construct and Anna Krook (Karolinska Institutet, Sweden) for the access to the CLARIOstar plate reader.
Authorship Contributions
Participated in research design: Kozielewicz, Schulte.
Conducted experiments: Kozielewicz, Bowin, Turku.
Contributed new analytic tools: Kozielewicz, Bowin.
Performed data analysis: Kozielewicz, Bowin, Turku, Schulte.
Wrote or contributed to the writing of the manuscript: Kozielewicz, Bowin, Turku, Schulte.
Footnotes
- Received August 28, 2019.
- Accepted November 1, 2019.
The study was supported by grants from Karolinska Institutet, the Swedish Research Council (2017-04676), the Swedish Cancer Society (CAN2017/561), the Novo Nordisk Foundation (NNF17OC0026940), Stiftelsen Olle Engkvist Byggmästare (2016/193), Emil and Wera Cornells Stiftelse, and Wenner-Gren Foundations (UPD2018-0064).
The initial version of this manuscript was deposited as a preprint (https://doi.org/10.1101/706028).
↵This article has supplemental material available at molpharm.aspetjournals.org.
Abbreviations
- BSA
- bovine serum albumin
- CI
- confidence interval
- CRD
- cysteine-rich-domain
- PDL
- poly-D-lysine
- ELISA
- enzyme-linked immunosorbent assay
- Gli
- glioma-associated oncogene
- GPCR
- G protein-coupled receptor
- Hh
- hedgehog
- NanoBRET
- nanoluciferase-based bioluminescence resonance energy transfer
- Nluc
- nanoluciferase
- PBS
- phosphate-buffered saline
- SMO
- Smoothened
- 7TM
- seven transmembrane
- Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics