Cell-Surface ReceptorâLigand Interaction Analysis with

Subscriber access provided by TUFTS UNIV

Article

Cell-surface receptor-ligand interaction analysis with homogenous time-resolved FRET and metabolic glycan engineering: application to transmembrane and GPI-anchored receptors Henning Stockmann, Viktor Todorovic, Paul L. Richardson, Violeta L. Marin, Victoria Scott, Clare Gerstein, Marc Lake, Leyu Wang, Ramkrishna Sadhukhan, and Anil Vasudevan J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09359 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 28, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

Cell-surface receptor-ligand interaction analysis with homogenous time-resolved FRET and metabolic glycan engineering: application to transmembrane and GPI-anchored receptors Henning Stockmann,∗ Viktor Todorovic, Paul L. Richardson, Violeta Marin, Victoria Scott, Clare Gerstein, Marc Lake, Leyu Wang, Ramkrishna Sadhukhan, and Anil Vasudevan AbbVie Inc., 1 North Waukegan Rd., North Chicago, IL 60064, USA Received October 24, 2017; E-mail: [email protected]

Abstract: Ligand-binding assays are the linchpin of drug discovery and medicinal chemistry. Cell-surface receptors and their ligands have traditionally been characterized by radioligandbinding assays, which have low temporal and spatial resolution and entail safety risks. Here we report a powerful alternative, GlycoFRET, where terbium-labeled fluorescent reporters are irreversibly attached to receptors by metabolic glycan engineering. For the first time, we show time-resolved fluorescence resonance energy transfer between receptor glycans and fluorescently labeled ligands. We describe GlycoFRET for a GPI-anchored receptor, a G-protein-coupled receptor, and a heterodimeric cytokine receptor in living cells with excellent sensitivity and high signal-to-background ratios. In contrast to previously described methods, GlycoFRET does not require genetic engineering or antibodies to label receptors. Given that all cell-surface receptors are glycosylated, we expect that GlycoFRET can be generalized with applications in chemical biology and biotechnology, such as target engagement, receptor pharmacology and high-throughput screening.

INTRODUCTION Membrane proteins are an important pharmaceutical target family with approximately 50 % of all approved therapies directed against cell-surface proteins (http://www.proteinatlas.org/ humanproteome/druggable). To study their pharmacology and identify high-affinity ligands, ligand-binding assays are a vital component in drug discovery. 1 Radioactive ligand-binding assays have been a standard method to assess ligand-receptor interactions. However, these assays are plagued by a number of problems related to the use of radioactivity, including disposal of radioactive material, a short shelf-life, and long signal acquisition times to achieve sufficient sensitivity. Given that filtration steps are required in radioligand assays, these assays are time-consuming and difficult to automate and to miniaturize. 2,3 Particularly challenging is the differentiation between ligand selectively bound to target and that non-specifically associated with the cell membrane. Fluorescence-based techniques have been developed as alternatives to radioligand binding assays. For instance, the DELFIA assay is based on the use of lanthanide chelates as fluorescent probes. It leverages long fluorescence lifetimes of lanthanide complexes by gating the emission signal, so that short-lifetime background and ligand fluorescence can be reduced and results in relatively high assay sensitivity. However, washing steps remain a significant drawback and

limit miniaturization. 4 Fluorescence polarization (FP) assays have also become increasingly popular for membrane proteins, given that FP does not require receptor tagging, binding assays can be carried out on wild-type receptors or on endogenous receptors if well expressed. 5 However, FP assays suffer from many limitations, including a requirement that the ligand be sufficiently smaller than the target (generally < 5 kDa), 6 have high affinity for the target, and the fluorophore must have a solution tumbling rate matched to that of the free ligand and target-bound ligand. Because the signal is derived from the ratio of bound to unbound ligand, high concentrations of target are required generally limiting sensitivity to over-expressed targets. Interference from chemical compounds or biological material and membrane partitioning of the ligand also greatly limits applicability. Binding assays must have high signal-to-noise ratios to be useful for high-throughput screening. Fluorescence resonance energy transfer (FRET)-based binding assays provide the advantage that they require the close proximity of two labels, which enhances specificity. Techniques for measuring FRET have been described in the literature. 7–10 For instance, the combination of green fluorescent protein (GFP) and near infrared fluorescent ligands allowed the identification of compounds inhibiting the interaction of GFP-tagged proteins with their ligands. 10 However, FRET-based bioassays suffer from high background signals due to scattered excitation light and significant interference from endogenous fluorescent compounds in complex biomatrices, thus making it difficult to obtain a highly sensitive measurement. 7,11 A second problem pertains to limited options to equip the protein of interest with a FRET label. Fusion with fluorescent proteins, insertion of binding sequences for labels such as the fluorescent arsenical hairpin binder FlAsH, insertion of a recognition motif for a fluorescently labeled antibody, or fusion with a protein that can be used for autocatalytic labeling with a fluorophore. 12 The necessity of using a good fluorophore for a good signal-to-noise ratio has made fluorescently labeled antibodies popular. 13 FRET assays that employ the noncovalent labeling of receptors exhibit good sensitivity, but they do not allow the determination of the affinity of the competitor since the assay equilibrium depends both on antibodies and tracer binding kinetics. Thus, receptor fusions with the DNA repair protein alkylguanine-DNA alkyltransferase, commercially available under the name SNAP-tag, are popular. Several reports on a number of GPCRs have used combinations of receptors labeled via an N-terminal SNAP-tag together with labeled ligands to investigate receptor binding. However, these FRET systems require genetic engineering of cells and are restricted to receptors with an extracellular terminus. Moreover, the

ACS Paragon Plus Environment 1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 9

Figure 1. Schematic of the live cell GlycoFRET assay. The working principle entails the metabolic incorporation of N-azidoacetyl neuraminic acid (Neu5Az) into cell-surface glycoproteins, attachment of a terbium chelate to the azidoglycans via click chemistry, treatment of cells with unlabeled receptor ligands, treatment with time-resolved fluorescence resonance energy transfer (TR-FRET) acceptor probe, and measurement of the TR-FRET signal. Energy transfer to the acceptor (e.g. Oregon Green) is measured in the silent region between the first two terbium emission peaks with an emission filter centered at 520 nm with a 25 nm bandpass (shown in green).


Page 3 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


insertion of binding sequences for labels may affect the physical properties of receptors. 14 To address these limitations, we sought to develop a versatile and background-free ligand-binding assay technology that works in intact cells without a need for genetic engineering. RESULTS AND DISCUSSION We wished to develop a generic strategy where any receptor of interest can be equipped with a FRET donor without relying on antibodies or genetic engineering. Given that every receptor is modified with complex carbohydrates (known as glycan), we explored metabolic glycan engineering of cell surface N-acetylneuraminic acid. 15 By providing cells with peracetylated N-azidoacetyl-D-mannosamine (ManNAz), they express glycans that contain N-azidoacetyl neuraminic acid (Neu5Az). In principle, the azidosugar can then serve as a convenient attachment point for a FRET reporter via click chemistry (Huisgen 1,3-cycloaddition). 16 A serious concern with a systemic glycan labeling strategy is that the FRET donor is found not only on the receptor of interest, but also on every other cell surface receptor. A high background signal would be expected with conventional FRET because of its low signal-to-noise ratio due to direct excitation of the acceptor at the excitation wavelength of the donor and also the emission of the donor and a high autofluorescence of cells at the acceptor emission wavelength. We reasoned that this problem can be addressed with a time-resolved FRET (TR-FRET) strategy using a chelated lanthanide as a fluorescence donor, for the following reasons: First, lanthanides exhibit a large Stokes shift and have atomic-like emission spectra which allow measurement of the emission from the acceptor with very low interference from the donor. Secondly, conventional fluorophores have a fluorescence lifetime of less than 20 ns. In contrast, lanthanides have long luminescence lifetimes of greater than 1 ms, which allows the measurement of fluorescence after a time delay when shortlived fluorescence signals from cells have ceased. In turn, this allows specific detection of fluorescence emission resulting exclusively from the FRET process. Finally, since luminescence from lanthanides is nonpolarized, time-resolved FRET is far less sensitive to the fluorophore’s relative orientation. 17,18 Given that signal-to-noise ratios in TR-FRET are up to 100 times greater than conventional FRET, 18 labeling glycans with a lanthanide seemed highly attractive for our strategy. TR-FRET typically employs long lifetime fluorescent FRET donors such as europium or terbium cryptates. Terbium cryptate is of particular interest because of the negligible emission around 520 nm and 665 nm and its compatibility with both green and red fluorescent acceptor dyes. Flexibility in the choice of acceptor facilitates the development of highly active acceptor probes for a given receptor that maintain binding affinity and pharmacological properties of the ligand. With the advantages of TR-FRET over conventional FRET, we sought to test if TR-FRET can eliminate the background signal even when the entire cell-surface glycoproteome is labeled with terbium. First, we required a terbium cryptate that can be conjugated to glycans via clickchemistry. Given that no such donor has been described yet, we generated a conjugate of a commercially available, amine-reactive terbium chelate (LanthaScreen, Invitrogen) and an azadibenzocyclooctyne (ADIBO) equipped with a

terminal amine for functionalization (Supplementary Figure S1). The conjugation reaction was carried out in aqueous buffer and went to completion within about 1 hour (Supplementary Figure S2). Following the introduction of azidosugar into cell-surface glycans (Fig. 1, step 1), the highly emissive ADIBO-terbium chelate should allow labeling of receptors with a FRET donor via click chemistry (Fig. 1, step 2). Upon binding of a TR-FRET probe to its receptor, a TR-FRET signal from the sensitized acceptor should occur when the donor and the acceptor are in close proximity (Fig. 1, step 3). Inhibition of the TR-FRET signal after competitive binding with a non-labeled compound may then be used to identify and characterize new receptor ligands. To test the GlycoFRET principle, we chose to use as a test system the folate receptor, which is a glycophosphatidylinositol-anchored protein and the molecular target for cancer and inflammatory disease therapeutics. 19,20 The human folate receptor has three asparaginelinked glycans, but only two of the glycosylation sites are fully occupied. 21 Given that the drug methotrexate binds to folate receptors, we synthesized a probe consisting of methotrexate and the fluorophore Oregon Green (Figure 2A. The synthesis is described in the Supplementary Information. Next, folate receptor expressing cells (cell line IGROV-1, receptor count: 0.14 +/- 0.03x106 per cell 21 ) were cultured in the presence of peracetyl N-azidoacetyl mannosamine. This monosaccharide readily penetrates the cell membrane, then gets deacetylated in the cytosol and incorporated as N-azidoacetyl neuraminic acid (Neu5Az) into cell-surface glycans (Fig. 1, step 1). As a possible alternative to Neu5Az labeling, N-azidoacetyl galactosamine can get incorporated into receptor glycans via conversion into N-azidoacetyl galactosamine, which is a building block of complex, hybrid and high-mannose N-linked glycans. However, we found that the labeling efficiency of N-glycans is usually higher with Neu5Az. 21 Once Neu5Az was installed, cells were incubated with the ADIBO-terbium chelate at a concentration of 5 µM for 1 h to equip cell-surface proteins with a TR-FRET donor. Finally, cells were aliquoted into 384-well HTS plates, treated with drug and incubated with TR-FRET probe. Energy transfer to Oregon Green was measured in the silent region between the first two terbium emission peaks (Fig. 1). The first terbium emission peak (centered between approximately 485 and 505 nm) overlaps with the maximum excitation peak of Oregon Green. Because it was important to measure energy transfer to Oregon Green without interference from terbium, a filter centered at 520 nm with a 25 nm bandpass was used. The emission of Oregon Green due to FRET was referenced to the emission of the first terbium peak, using a filter centered at 495 nm with a 10 nm bandpass (see Fig. 1 for the gating strategy). A saturation binding experiment showed a surprisingly low background signal when the receptor was blocked with methotrexate (Supplementary Figure S4). Furthermore, the saturation binding experiment indicated that a probe concentration of 100 nM resulted in the highest signal-to-background ratio. Competition experiments using folic acid, trimethoprim and methotrexate resulted in robust dose-response data and signal-to-background ratios of about 5. The measured affinities for the competitors were similar to those derived from other binding experiments reported in the literature (Figure 2B). 22 Thus, for the first time we could show that by using a lanthanide as a fluorescent donor, no background signal



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A

Page 4 of 9

B

Hill slope

IC50

IC50 (lit.)

-1.3

6.5 x 10-9

1.0 x 10-9

Trimethoprim

-0.8

5.9 x

10-6

5.3 x 10-5

Methotrexate

-1.9

1.1 x 10-7

5.3 x 10-6

Folic acid

Figure 2. GlycoFRET assay with the human folate receptor. The TR-FRET acceptor probe probe was based on methotrexate, which was linked to the fluorophore Oregon Green (A). IGROV-1 cells were cultured in the presence of N-azidoacetyl-D-mannosamine (ManNAz). This was followed by the attachment of a terbium chelate to the azidoglycans via click chemistry, treatment of cells with unlabeled receptor ligands (trimethoprim, folic acid or methotrexate, A), treatment with TR-FRET acceptor probe at 100 nM, and measurement of the TR-FRET signals. Dose-response curves are shown in (B). Literture data were obtained from Fujishima et al. 22

could be detected when every receptor was equipped with a lanthanide. The lanthanide’s favorable fluorescence emission spectrum with extremely narrow emission peaks allowed time- and wavelength gating, so that the interfering fluorescence signal from the donor and the matrix could be eliminated. Overall, this resulted in a method with very high sensitivity compared to a conventional FRET system. Because the method allows systemic labeling of the cell-surface proteome with a TR-FRET donor, it makes the technology extremely versatile and provides access to virtually every cell-surface receptor. Unlike previously described methods, 14 GlycoFRET does not require genetic engineering or antibodies. Importantly, no washing step was required after the cells had been treated with probe, making GlycoFRET a convenient mix-and-read technology where assays can be performed in very small volumes. The homogeneous assay format provides a straightforward binding assay particularly suitable in a high-throughput screening environment by providing a miniaturized format and thus an attractive alternative to radioactive assays. Given that receptors can be labeled through native glycans, the derivatization reaction is not expected to alter the receptors’ expression or functional behavior, while the distance dependence of the TRFRET signal ensures a high specificity of the assay for the receptor. Next, we tested the assay on the human histamine H3 receptor, which is one of the four human histamine receptor subtypes. It bears one asparagine-linked glycan and is a membrane-resident class A family G-protein-coupled receptor (GPCR) (Gi/o coupled) that is expressed in the central nervous system (CNS) and acts as an auto- as well as a het-

eroreceptor. 23 The receptor modulates the release of several neuronal neurotransmitters. Because of different central effects of several hH3R antagonists in preclinical and clinical trials, labeled hH3R ligands have been developed to investigate neurological disorders in the CNS based on receptor distribution, occupation, and regulation. 24 To investigate structure-activity relationships, we prepared a small library of probes. Our design was based on previous research where a novel series of fluorescent ligands for the histamine H3 receptor had been described. 24 One series of antagonists had produced a number of potent and selective ligands with a benzofuran core (e.g. ABT-239). The chemistry was well developed, so that probe 1 (Figure 3A) was designed based on this structure. 29 Another set of probes was based on a quinoline core, 30 where linkers were attached to the the methyl-pyrrolidine (probe 2) or one of the nitrogen atoms of the pyrazole (probes 3 and 4, Figure 3A). Synthetic details are provided in the Supplementary Information. We generated a stable HEK293 cell line that expressed full-length human histamine receptor 31 and metabolically introduced ManNAz, followed by a click reaction with ADIBO-terbium chelate, as described above for the folate receptor. With the probe library in hand, we investigated the correlation between probe specificity and assay window. Nonspecific binding was determined by the addition of 1 µM unlabeled ABT-239. Saturation binding experiments showed that all four probes were active in the GlycoFRET assay, but they had different levels of specificity, which directly impacted the assay window (Figure 3B and Supplementary Figure 5). For instance, probe 2 had a low receptor


Page 5 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

C

Histamine H3 receptor TR-FRET

Hill slope

IC50

IC50 (lit.) 1.6 x 10-9

Imetit

-0.7

4.9 x 10-9

Clobenpropit

-0.9

3.6 x 10-10 3.4 x 10-10

Thioperamide

-1.0

9.6 x 10-9

ABT-239

-1.1

3.3 x 10-10 4.5 x 10-10

1.4 x 10-8

B

Figure 3. Human histamine receptor H3 (HRH3) GlycoFRET assay. The TR-FRET acceptor probes were composed of Oregon Green, linked to HRH3 ligands shown in (A). HRH3-expressing HEK293 cells were cultured in the presence of N-azidoacetyl-D-mannosamine (ManNAz), which was followed by the attachment of a terbium chelate to the azidoglycans via click chemistry. Saturation-binding curves (B) were obtained by titrating the probes in the presence or absence of ABT-239 at 1 µM. Dose-response curves (C) were obtained by treatment of cells with unlabeled receptor ligands (imetit, clobenpropit, trimethoprim and ABT-239), treatment with TR-FRET acceptor probe 3 at 100 nM, and measurement of the TR-FRET signals. Literature data were obtained from Lim et al., 25 Wijtmans et al., 26 Zaragoza et al., 27 and Cowart et al. 28



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

specificity and hence a low signal-to-background ratio. Although this probe may be sufficient to obtain a qualitative binding assay, its utility for pharmacological studies would be limited. In contrast, probe 3 had the highest receptor specificity and was used in competition experiments with the drugs imetit, 25 clobenpropit, 26 thioperamide 27 and ABT239. 28 The probe yielded solid dose-response curves with signal-to-noise ratios of about 4 (Figure 3C), which was remarkable considering that the receptor only has one N-linked glycosylation site. The measured drug affinities were similar to those derived from other binding experiments reported in the literature. Taken together, GlycoFRET can yield excellent assay data, but the quality of the assay depends on the specificity of the probe. Since binding of biological macromolecules (e.g. proteins) to cell-surface receptors is key to both theoretical studies and drug development research 32 we next sought to assess the ability of GlycoFRET to quantify interactions between a protein ligand and a heterodimeric receptor. Here, we studied the IL-36 receptor (IL-36R), a member of interleukin-1 receptor superfamily, which is a cell-surface receptor that heterodimerizes with the IL-1-receptor accessory protein (IL-1RAcP) upon binding to the cytokine IL-36. 33,34 To develop the assay, we used recombinant IL-36 family members with Alexa Fluor 488 and cell lines that were expressing recombinant IL-36R and IL-1RAcP. For a proof of concenpt, we generated a stable transfected HEK293 cell line that expressed mouse IL-36R/IL-1RAcP. Cells were metabolically labeled with ManNAz and incubated with ADIBO-terbium chelate as described previously. In addition, we labeled mouse IL-36α using Alexa Fluor 488 N-hydroxysuccinimidyl ester. Successful labelling of the ligand was confirmed using mass spectrometry (Supplementary Figure S3). In order to test whether ligand labelling caused any changes in its ability to bind to the receptor dimer on cells we measured the release of known downstream effector of IL-36 signalling CXCL1. 35 The functional assay data demonstrates no difference in the ability of labeled cytokine to induce CXCL1 release compared to unlabelled cytokine (Supplementary Figure S6). Following assay optimization, clear dose-response inhibition was observed with three competitors:unlabeled mIL-36α ligand, mIL-36 receptor antagonist (mIL-36Ra) and an antagonistic antibody against mIL-36 receptor (anti-mIL-36R). Importantly, the functional and binding IC50 data are in accordance with unlabeled ligands. In human drug discovery, working with ligand/receptor systems from humans is more relevant than working with mouse proteins. Thus, we transiently transfected HEK293 cells with human IL-36R and IL-1RAcP. Shortly after transfection, cells were cultured in the presence of peracetyl N-azidoacetyl mannosamine and then stored as frozen aliquots. After thawing, cells were incubated with the ADIBO-terbium chelate, aliqoted into a multiwell plate and incubated with competitor together with Alexa Fluor 488 labeled IL-36γ. Similarly to the mouse data (above), the assay demonstrated dose-dependent response to competitors, with the functional assay data confirming the retention of biological activity of labelled IL-36γ (Figure 4). Again, functional and binding data demonstrated very similar characteristics further raising the confidence in the assay. Interestingly, the antagonistic antibody against human IL-36R did not fully compete with the probe in the binding assay suggesting that its ability to block IL-36R function may not completely rest in blocking the receptor/ligand interaction,

but perhaps working through a different mechanism such as blocking receptor dimerization induced by ligand binding. However, the binding constant was in full agreement with the compound’s IC50 value in functional assays. In addition to its versatility, GlycoFRET has been successfully applied to systems where other approaches have failed. For instance, we attempted to label membrane proteins with terbium chelate via lysine conjugation. Although the conjugation reaction was successful, which was reflected by a high donor fluorescence signal, a dose-dependent signal could not be obtained in any of the receptor systems under investigation. Despite the advantages of GlycoFRET over other technologies, its main limitation may be the reliance on probes with sufficient target specificity to achieve useful signal-to-background ratios. Our saturation-binding data indicated that probe specificity dictates the assay window (Figure 3B and Supplementary Figure S5). Although lowspecificity probes could still yield qualitative data, too low a signal window may impede pharmacological investigations where high-quality dose-response data are required. However, we believe that specific probes can be furnished for most receptor systems, although in certain cases this may require several iterations of structure optimization. CONCLUSIONS To summarize, we have provided a versatile procedure for a time-resolved fluorescence resonance energy transfer (TRFRET) assay to characterize ligands and membrane receptors in native, stable or transiently transfected cells. Endogenous receptor glycans were covalently tagged with TR-FRET reporters via metabolic cell-surface engineering with N-azidoacetyl neuraminic acid and a bio-orthogonal, catalyst-free [3+2] alkyne-azide cycloaddition. Excellent TR-FRET data were obtained for a folate-binding GPIanchored receptor, a histamine-binding GPCR, and a heterodimeric cytokine receptor and its fluorescently labeled cytoline ligand. We anticipate that the method can be tailored to other receptors and their labeled ligands with minimal modification and without the need to genetically engineer receptor constructs. Moreover, GlycoFRET assays can be run in 384-well format to enable high-throughput compound screening. Given that metabolic glycan engineering can be performed in living animals, 36,37 GlycoFRET may be a compelling method to assess target engagement in vivo or ex vivo. Taken together, compared to conventional ligandbinding assays, GlycoFRET can be carried out in a platebased format, is easy to perform, does not require washing steps and requires only a small number of cells. Although GlycoFRET can be conveniently performed manually, automation is a possible avenue to further increase utility and reduce variability. EXPERIMENTAL SECTION Chemical Compounds and Biological Materials Synthetic details are provided in the Supplementary Information, together with details on all biological materials and procedures. GlycoFRET Assay Cells expressing the receptor of interest (see Supplementary Information for details on cell culture) were cultured


Page 6 of 9

Page 7 of 9

A

Human IL-36 receptor TR-FRET

TR-FRET Ratio

0.5

hIL-36(unl.) hIL-36Ra anti-hIL-36R

0.4 0.3 0.2 0.1 0.0 -12

-10

-8

-6

-4

Concentration (log M) Hill slope

B

Human IL-36 receptor functional assay 100

hIL-36Ra anti-hIL-36R antibody

60 40 20 0 -12

C

IC50

hIL-36γ (unlabeled) -0.7

6.2 x 10-9

hIL-36Ra

-0.6

3.0 x 10-9

anti-hIL-36

-1.0

1.7 x 10-10

Human IL-36 receptor functional assay 100 80

% Activation

80

% Inhibition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 40 20

-10

-8

-6

-4

0 -12

-11

Concentration [log M]

-10

-9

-8

-7

hIL-36[log M]

hIL-36RaHill slope anti-hIL-36R antibody IC50 HillSlope hIL-36Ra0.8017 -0.8 1.0891.6 x 10-8 IC50 1.613e-008 3.042e-010 anti-hIL-36 -1.0 3.0 x 10-10

Hill slope EC50 hIL-36g

HillSlope hIL-36γ (unlabeled) -0.7 1.989 6.1 x 10-10 EC50

6.105e-010

Figure 4. GlycoFRET assay with the human IL-36 receptor. The TR-FRET acceptor probe was based on Alexa Fluor 488, linked to IL-36 via lysine labeling. HEK293 cells expressing human IL-36 receptor and IL-1-receptor accessory protein were cultured in the presence of N-azidoacetyl-D-mannosamine (ManNAz). terbium chelate was attached to the azidoglycans via click chemistry, followed by treatment of cells with unlabeled receptor ligands (hIL-36γ, hIL-36R antagonist and hIL-36 antibody), treatment with TR-FRET acceptor probe, and measurement of the TR-FRET signals. Dose-response curves are shown in (A). IC50 values for the antagonists (B) and the EC50 value for hIL-36γ (C) were determined with a CXCL1 release assay.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

for 48 h in media containing 50 µM Ac4 ManNAz. Next, the cells were washed with Dulbecco’s Phosphate-Buffered Saline (DPBS, Invitrogen), dissociated and suspended in labeling buffer (1 % Fetal Bovine Serum (FBS) in DPBS) at a density of 10x106 cells/mL. The cells were then incubated with ADIBO-Terbium chelate at a concentration of 5 µM for 1 h on ice. Following cell washing with labeling buffer, they were suspended in labeling buffer at a density of 10x106 cells/mL. Next, the cells were aliquoted into a 384-well TRFRET assay plate at 50,000 cells/well (5 µL). For competition experiments, the appropriate ligand in 5 µL of labeling buffer was added and the plate was incubated on ice for 10 min. Alternatively, 5 µL of labeling buffer was added. Next, the TR-FRET probe in labeling buffer (5 µL) was added and the plate was incubated for 10 min on ice. The GlycoFRET signal was measured with an EnVision plate reader (PerkinElmer) equipped with a TR-FRET module. The filter setup consisted of the following: excitation filter UV2 (TRF), 340 nm; emission filters 495 nm (donor) and 520 nm (acceptor); mirror module D400. The measurement parameters (height, delay and window) were optimized with the optimization wizard software. An optimized set of measurement parameters was as follows: 50-µs delay, 400-µs integration time, 6.5 mm measurement height and 20 flashes per well. To obtain the TR-FRET ratio, the emission signal at 520 nm due to FRET was divided by the emission of the first terbium peak, using a filter centered at 495 nm with a 10 nm bandpass (see Fig. 1 for the gating strategy). Dose-response curves were fitted to a four-parameter logistic equation (sigmoidal dose-response with variable slope): T op − Bottom 1 + 10(log(IC50)−X)∗HillSlope where ”X” is the logarithm of the competitor concentration and ”Y” reflects the amount of bound ligand at equilibrium obtained. ”Top” indicates the maximum amount of ligand binding without competitor. ”Bottom” expresses the amount of unspecific ligand binding in the presence of a very high concentration of competitor. ”HillSlope” reflects the slope factor, which indicates the steepness of the resulting inhibition curve. Y = Bottom +

Acknowledgement We thank Arlene Manelli for providing the human HRH3-expressing cell line as well as Noah Pefaur and Marla C. Harris for the human IL-36R antibody. We thank Jane Seagal, Sheeba Mathew and Amanda Horowitz for the mouse IL-36R antibody. DISCLOSURE All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. SUPPORTING INFORMATION AVAILABLE

(4) Handl, H. L.; Vagner, J.; Yamamura, H. I.; Hruby, V. J.; Gillies, R. J. Anal Biochem 2005, 343, 299–307. (5) Lea, W. A.; Simeonov, A. Expert Opin Drug Discov 2011, 6, 17–32. (6) de Jong, L. A.; Uges, D. R.; Franke, J. P.; Bischoff, R. J Chromatogr B Analyt Technol Biomed Life Sci 2005, 829, 1–25. (7) Degorce, F.; Card, A.; Soh, S.; Trinquet, E.; Knapik, G. P.; Xie, B. Curr Chem Genomics 2009, 3, 22–32. (8) Jia, Y.; Quinn, C. M.; Gagnon, A. I.; Talanian, R. Anal Biochem 2006, 356, 273–81. (9) Maurel, D.; Comps-Agrar, L.; Brock, C.; Rives, M. L.; Bourrier, E.; Ayoub, M. A.; Bazin, H.; Tinel, N.; Durroux, T.; Prezeau, L.; Trinquet, E.; Pin, J. P. Nat Methods 2008, 5, 561–7. (10) Hu, L. A.; Zhou, T.; Hamman, B. D.; Liu, Q. Assay Drug Dev Technol 2008, 6, 543–550. (11) Pietraszewska-Bogiel, A.; Gadella, T. J Microsc 2011, 241, 111–118. (12) Hoffmann, C.; Gaietta, G.; Bunemann, M.; Adams, S. R.; Oberdorff-Maass, S.; Behr, B.; Vilardaga, J. P.; Tsien, R. Y.; Ellisman, M. H.; Lohse, M. J. Nat Methods 2005, 2, 171–6. (13) Albizu, L.; Teppaz, G.; Seyer, R.; Bazin, H.; Ansanay, H.; Manning, M.; Mouillac, B.; Durroux, T. J Med Chem 2007, 50, 4976–85. (14) Zwier, J. M. et al. J Biomol Screen 2010, 15, 1248–59. (15) Bertozzi, C. R. Acc Chem Res 2011, 44, 651–3. (16) Lallana, E.; Riguera, R.; Fernandez-Megia, E. Angew Chem Int Ed Engl 2011, 50, 8794–804. (17) Selvin, P. R. Annu Rev Biophys Biomol Struct 2002, 31, 275– 302. (18) Oueslati, N.; Hounsou, C.; Belhocine, A.; Rodriguez, T.; Dupuis, E.; Zwier, J. M.; Trinquet, E.; Pin, J.-P.; Durroux, T. G Protein-Coupled Receptor Screening Assays: Methods and Protocols 2015, 23–36. (19) Assaraf, Y. G.; Leamon, C. P.; Reddy, J. A. Drug Resist Updat 2014, 17, 89–95. (20) Yang, J.; Vlashi, E.; Low, P. Subcell Biochem 2012, 56, 163–79. (21) Stockmann, H.; Marin, V. L.; Nimmer, P.; Balut, C. M.; Davidson, D. J.; Richardson, P. L.; Vasudevan, A. Chembiochem 2016, 17, 150–4. (22) Fujishima, S. H.; Yasui, R.; Miki, T.; Ojida, A.; Hamachi, I. J Am Chem Soc 2012, 134, 3961–4. (23) Walter, M.; Stark, H. Front Biosci (Schol Ed) 2012, 4, 461–88. (24) Cowart, M.; Gfesser, G. A.; Bhatia, K.; Esser, R.; Sun, M.; Miller, T. R.; Krueger, K.; Witte, D.; Esbenshade, T. A.; Hancock, A. A. Inflamm Res 2006, 55 Suppl 1, S47–8. (25) Lim, H. D.; Istyastono, E. P.; van de Stolpe, A.; Romeo, G.; Gobbi, S.; Schepers, M.; Lahaye, R.; Menge, W. M. B. P.; Zuiderveld, O. P.; Jongejan, A.; Smits, R. A.; Bakker, R. A.; Haaksma, E. E. J.; Leurs, R.; de Esch, I. J. P. Bioorg Med Chem 2009, 17, 3987–3994. (26) Wijtmans, M. et al. J Med Chem 2008, 51, 2944–2953. (27) Zaragoza, F.; Stephensen, H.; Peschke, B.; Rimvall, K. J Med Chem 2005, 48, 306–311. (28) Cowart, M.; Faghih, R.; Curtis, M. P.; Gfesser, G. A.; Bennani, Y. L.; Black, L. A.; Pan, L.; Marsh, K. C.; Sullivan, J. P.; Esbenshade, T. A.; Fox, G. B.; Hancock, A. A. J Med Chem 2005, 48, 38–55. (29) Esbenshade, T. A.; Fox, G. B.; Krueger, K. M.; Miller, T. R.; Kang, C. H.; Denny, L. I.; Witte, D. G.; Yao, B. B.; Pan, L.; Wetter, J.; Marsh, K.; Bennani, Y. L.; Cowart, M. D.; Sullivan, J. P.; Hancock, A. A. J Pharmacol Exp Ther 2005, 313, 165–75. (30) Liu, H.; Altenbach, R. J.; Diaz, G. J.; Manelli, A. M.; Martin, R. L.; Miller, T. R.; Esbenshade, T. A.; Brioni, J. D.; Cowart, M. D. Bioorg Med Chem Lett 2010, 20, 3295–300. (31) Yao, B. B.; Hutchins, C. W.; Carr, T. L.; Cassar, S.; Masters, J. N.; Bennani, Y. L.; Esbenshade, T. A.; Hancock, A. A. Neuropharmacology 2003, 44, 773–86. (32) Yakimchuk, K. Mater Methods 2011, 1, 199. (33) Gunther, S.; Sundberg, E. J. J Immunol 2014, 193, 921–30. (34) O’Neill, L. A. Immunol Rev 2008, 226, 10–8. (35) Foster, A. M.; Baliwag, J.; Chen, C. S.; Guzman, A. M.; Stoll, S. W.; Gudjonsson, J. E.; Ward, N. L.; Johnston, A. J Immunol 2014, 192, 6053–61. (36) Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Science 2008, 320, 664–667. (37) Neves, A. A.; Stockmann, H.; Wainman, Y. A.; Kuo, J. C.; Fawcett, S.; Leeper, F. J.; Brindle, K. M. Bioconjugate chemistry 2013, 24, 934–941.

Experimental procedures and characterization data for all new compounds available as Supporting Information at http://pubs.acs.org. References (1) Lagerstrom, M. C.; Schioth, H. B. Nat Rev Drug Discov 2008, 7, 339–57. (2) Lefkowitz, R. J. Trends Pharmacol Sci 2004, 25, 413–22. (3) Birdsall, N. J. Trends Pharmacol Sci 2010, 31, 499–508.


Page 8 of 9

Page 9 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical TOC Entry


Cell-Surface ReceptorâLigand Interaction Analysis with

Recommend Documents

Cell-Surface ReceptorâLigand Interaction Analysis with