Development of a NanoBRET-Based Sensitive Screening Method for

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Development of a NanoBRET-Based Sensitive Screening Method for CXCR4 Ligands Maxwell M. Sakyiamah,†,‡ Wataru Nomura,† Takuya Kobayakawa,† and Hirokazu Tamamura*,†,‡ †

Bioconjugate Chem. Downloaded from pubs.acs.org by UNIV OF SOUTHERN INDIANA on 04/12/19. For personal use only.

Department of Medicinal Chemistry, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 2-3-10 Kandasurugada, Chiyoda-ku, Tokyo 101-0062, Japan ‡ Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan S Supporting Information *

ABSTRACT: A critical part of the development of CXCR4 modulators is to have a simple and sensitive assay system to complement the search by screening and evaluating the binding affinity. Herein, a NanoBRET assay system was developed, and its feasibility as a high-throughput screening tool for potent CXCR4 ligands was ascertained. TAMRA-Ac-TZ14011, a fluorescentlabeled CXCR4 antagonist, was adopted as a fluorescent acceptor of bioluminescent energy from N-terminally fused NanoLucCXCR4 stably expressed in CHO cells. The ratio of fluorescence at 620 nm to the luminescence at 460 nm represents the interaction between test compounds and CXCR4. We have demonstrated in the present study that the NanoBRET assay system is applicable for the evaluation of CXCR4 ligands using the combination of TAMRA-Ac-TZ14011 as an acceptor and NanoLuc tagged to CXCR4 as a bioluminescent donor expressed in living cells. IC50 values of known CXCR4 ligands were determined and found to be compatible with the values obtained by other existing and sensitive methods, such as SDF-1:3.2 nM, Ac-TZ14011:15.3 nM, and FC131:4.5 nM, which confirmed the feasibility of our system (Z′ values ≥0.5). The introduction of an IL-6 secretory signaling peptide (secNluc-CXCR4) further enhanced the expression and trafficking of the tagged receptor, which, in turn, increased the dynamic range of the NanoBRET assay system. Thus, we have successfully developed a NanoBRET system in living cells, which is simple, homogeneous, and useful in multiwell plate screening of potent CXCR4 ligands.

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INTRODUCTION

requires skill in handling, and also leads to the generation of radioactive waste, making it undesirable.17 The present study therefore sought to improve upon existing methods by applying advanced screening systems to develop a high-throughput screening (HTS) technique devoid of any untoward effects. Recently, a few GPCR binding assays have been reported utilizing time-resolved resonance energy transfer (RET), which are suitable for high-throughput screening in terms of rapidity and safety.18 A NanoLuc (Nluc) bioluminescence resonance energy transfer (NanoBRET) assay system, an improved BRET which borders on the principle that a bioluminescent donor (NanoLuc luciferase) transfers energy, generated by enzymatic oxidation of furimazine, to a fluorescent acceptor in close proximity (∼10 nm) through dipole−dipole interaction, has been demonstrated to have prospects in future drug discovery and profiling applications.19,20 Presently, NanoBRET has become a preferred assay option due to its enhanced detection sensitivity and applied to real-time high-throughput screening of GPCR antagonists in living cells.21−23 However, its application to the studies of CXCR4 antagonists has not been reported. Previously, we

C-X-C chemokine receptor type 4 (CXCR4), a seven transmembrane (7TM) G-protein-coupled receptor (GPCR), together with its cognate ligand stromal cell-derived factor-1 (SDF-1: CXCL12), plays a key role in immune cell development and hematopoietic stem cell trafficking. However, CXCR4/SDF-1 signaling axis is also implicated in various pathological conditions including cancer progression, leukemia, cardiovascular diseases, and autoimmune diseases such as rheumatoid arthritis.1−7 Furthermore, CXCR4 also doubles as a co-receptor for T tropic strains of HIV-1 infection.8 Owing to its versatile role in various physiopathological conditions, the CXCR4/SDF-1 signaling axis represents a potential drug target, and as such, several antagonists (both peptides and nonpeptides as well as small compounds) against CXCR4 have been developed.9−16 Meanwhile, the continuous search for potent ligands to modulate the CXCR4/SDF-1 (CXCL12) signaling axis requires a complementary screening assay technique that is simple, homogeneous, and safe. Although there are many screening assays developed, most of these assays fall short in one or more of these enumerated criteria. A radioligand binding assay, for instance, a classical screening technique, makes use of radio-labeled ligands which is costly, © XXXX American Chemical Society

Received: March 10, 2019

A

DOI: 10.1021/acs.bioconjchem.9b00182 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 1. Model of a NanoBRET assay system to detect the ligand−CXCR4 interaction.

Figure 2. Binding characteristics of TAMRA-Ac-TZ14011 to CXCR4 using confocal laser scanning microscopy. (A) Expression of Nluc-CXCR4EGFP in transiently transfected HEK293 cells. (B) Characteristic binding of TAMRA-Ac-TZ14011 (0.1 μM) to HEK293 cells transiently expressing Nluc-CXCR4-EGFP incubated for 1 h at 25 °C. Cells were washed and observed using confocal laser scanning microscopy (scale bars: 20 μm scale). PC: Phase contrast.

characteristic binding of the fluorescent probe (TAMRA-AcTZ14011) to CXCR4, HEK293 cells transiently transfected with pcDNA3.1 coding for CXCR4 with Nluc tagged at the Nterminus and with EGFP tagged to the C-terminus (NlucCXCR4-EGFP) or without EGFP (Nluc-CXCR4) were incubated with 0.1 μM TAMRA-Ac-TZ14011 and observed using confocal laser scanning microscopy (Figure 2). Fluorescence from EGFP was observed to be localized mainly toward the cell membrane (Figure 2A), suggesting that the Nluc-CXCR4-EGFP fusion protein is expressed and trafficked to the surface of the cells. The observed trafficking and expression could be attributed to the presence of signal peptides which possibly predispose the receptor toward the secretory pathway, although the mechanism by which CXCR4 reaches the plasma membrane is not fully understood.29−31 When cells expressing Nluc-CXCR4-EGFP were incubated with TAMRA-Ac-TZ14011, colocalization of the EGFP and TAMRA-Ac-TZ14011 which merged at the cell membrane was observed (Figure 2B) due to the binding of TAMRA-AcTZ14011 to CXCR4, further confirming the expression and trafficking of the Nluc-CXCR4-EGFP fusion protein to the cell membrane. Similarly, binding of TAMRA-Ac-TZ14011 to CXCR4 was also found to be localized at the cell membrane of transfected cells expressing Nluc-CXCR4 (Supporting Information, Figure S1A) with little or no internalization of the fluorescent-labeled ligand after incubation for 1 h at 25 °C. CXCR4 used in this study has an alanine mutation at the series

synthesized the fluorescent-labeled T140 derivative with CXCR4 antagonistic activity, TAMRA-Ac-TZ14011, and demonstrated through a fluorescence-based assay the characteristic binding of ligands to CXCR4 and also evaluated the IC50 values of some CXCR4 ligands.24−27 The assay system, however, required intensive washing steps, which made it nonhomogenous. Herein, we have developed and proven the feasibility of a NanoBRET system in CXCR4-expressing mammalian cells, which is useful for high-throughput screening of CXCR4 ligands.

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RESULTS AND DISCUSSION The objective of the study was to develop a NanoBRET system as a sensitive and real-time high-throughput method for the screening of CXCR4 ligands. This has become necessary due to setbacks associated with existing techniques used in the screening of CXCR4 ligands. A model of the system is shown in Figure 1. Expression of Nluc-CXCR4 on the Surfaces of Mammalian Cells. A prerequisite for setting up the NanoBRET assay system was to express CXCR4 tagged with NanoLuc (Nluc) at its N-terminus (Nluc-CXCR4) so that the bioluminescent donor, Nluc, is displayed on the cell surface (Figure 1). Nluc, engineered from a naturally secreted protein, has been reported to suit N-terminus labeling of receptors as it enhances cell surface trafficking with the correct protein folding.28 To confirm the expression of Nluc-CXCR4 and the B

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Figure 3. NanoBRET assay system as a suitable tool for binding studies. (A) BRET signaling in transiently transfected HEK293 cells incubated with increasing concentrations of TAMRA-Ac-TZ14011 for 1 h at 25 °C. Values are mean ± SEM, n = 3 (Z′ values ≥0.3). (B,C) Competitive binding of TAMRA-Ac-TZ14011 in the presence of increasing concentrations of unlabeled Ac-TZ14011 using the NanoBRET assay system in (B) HEK293 cells and (C) HeLa cells transiently expressing Nluc-CXCR4. Values are mean ± SEM, n = 4. (D) Competitive binding of TAMRA-AcTZ14011 in the presence of increasing concentrations of FC131. Values are mean ± SEM, n = 3. Cells were incubated with increasing concentrations of a test compound in the presence of 0.1 μM TAMRA-Ac-TZ14011 for 1 h at 25 °C.

mer peptide was successfully synthesized by Fmoc-based solidphase peptide synthesis (Fmoc SPPS) using a Rink amide resin and purified by analytical HPLC. To curb any possible interference to the binding potency of Ac-TZ14011 by TAMRA due to proximity, we introduced a 6-aminohexanoic acid through an amide bond as a linker before conjugation with the TAMRA fluorescent dye, as conjugation of a 6-aminohexanoic acid with TAMRA was introduced through an amide bond in our previous study.37 TAMRA-Ac-TZ14011 was successfully synthesized and purified by HPLC in 37% yield (Supporting Information). Evaluation of the NanoBRET Assay System as a Tool for Binding Studies. The suitability of our NanoBRET assay system as a tool for binding studies was tested in HEK293 cells expressing Nluc-CXCR4. Prior to that, the functionality of the bioluminescent protein (NanoLuc) tagged to the N-terminus of CXCR4 was evaluated by luminescence (RLU; relative light unit), and concurrent increase in luminescence with increasing number of transfected cells was observed after the addition of the substrate furimazine (Supporting information, Figure S3A). This confirmed not only the expression of the Nluc-CXCR4 but also the functionality of NanoLuc. Incubation of HEK293 cells transiently expressing Nluc-CXCR4 with TAMRA-AcTZ14011 or vehicle (OptiMEM; background) also showed concurrent increase in BRET ratio (acceptor emission/donor

of C-terminal serines to reduce the tendency of ligandpromoted phosphorylation, which could lead to internalization of the receptor.32−34 To ascertain the specificity of the binding of TAMRA-AcTZ14011, transfected cells were incubated with labeled ligand in the presence of excess amounts of unlabeled Ac-TZ14011 (1.0 μM), and we observed a significant decrease in the fluorescent intensity of the bound TAMRA-Ac-TZ14011 (Supporting Information, Figures S1B and S2). The binding of TAMRA-Ac-TZ14011 was inhibited by Ac-TZ14011. This suggests the specificity of the binding of TAMRA-Ac-TZ14011 to Nluc-CXCR4. Ac-TZ14011, a derivative of T140, is a CXCR4 antagonist, which can bind to CXCR4 with high specificity.35 Therefore, the observed specificity in the binding of TAMRA-Ac-TZ14011 corroborates the results of our previous studies. Generally, labeled antagonists are preferred in fluorescent-based binding assays because agonists easily activate receptors and cause internalization, leading to possible reduction of receptor numbers.36 We adopted Ac-TZ14011 because of the existence of its free D-lysine residue at position 8, which is well-suited for conjugation to fluorescent probes through amide linkage, in addition to being a strong antagonist of CXCR4. It has been reported that conjugation of fluorophores to Ac-TZ14011 via its D-lysine residue does not interfere with the binding potency of Ac-TZ14011.37 The 14C

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Figure 4. Binding characteristics of TAMRA-Ac-TZ14011 to CXCR4 using confocal laser scanning microscopy. (A) Characteristic binding of TAMRA-Ac-TZ14011 (500 nM) to CHO cells stably expressing Nluc-CXCR4 incubated for 1 h at 25 °C. (B) Characteristic binding of TAMRAAc-TZ14011 (500 nM) to CHO cells stably expressing Nluc-CXCR4 in the presence of 5.0 μM Ac-TZ14011 incubated for 1 h at 25 °C. Cells were washed and observed using confocal laser scanning microscopy (scale bars: 20 μm scale). PC: Phase contrast.

Figure 5. Use of a NanoBRET assay system as a screening tool in CHO stable cells. (A) Saturation binding studies of TAMRA-Ac-TZ14011 for Nluc-CXCR4 in the presence (nonspecific (NS) binding) and absence (total binding) of 10 μM Ac-TZ14011 in CHO stable cells. (B) Competitive binding of TAMRA-Ac-TZ14011 in the presence of SDF-1α, FC131, unlabeled Ac-TZ14011, and maraviroc (negative control). Values are mean ± SEM, n = 3. Raw BRET ratio values are shown in Supporting Information, Figure S5.

expressing Nluc-CXCR4 (Figure 3D). The IC50 values correspond to those obtained by other existing and sensitive methods. For instance, the IC50 value of FC131 was evaluated to be 4 nM in a radioisotope-based assay using 125I-SDF-1α,38 and the IC50 value of Ac-TZ14011 was evaluated to be 14 nM using a fluorescent-based binding assay.25 Development of the NanoBRET Assay System in CHO Stable Cells. For purposes of reproducibility and time convenience, the NanoBRET assay system was developed with Chinese hamster ovary (CHO) cells stably expressing Nluc-CXCR4. The expression of Nluc-CXCR4 in stably transfected CHO cells was confirmed by observation with confocal laser scanning microscopy and check of the functionality of Nluc by luminescence as done with the transiently transfected cells. The binding of TAMRA-AcTZ14011 to Nluc-CXCR4 was found to be localized mainly at the cell membrane of stably transfected CHO cells with little or no internalization and was significantly inhibited in the presence of excess unlabeled Ac-TZ14011 (Figure 4). Furthermore, binding studies with nontransfected CHO host cells showed no significant binding of TAMRA-Ac-TZ14011 (Supporting Information, Figure S4). These observations suggest that the labeled ligand binds specifically to CXCR4 expressed and trafficked to the membrane of cells. The luminescence studies showed concurrent increase in luminescence with increasing number of CHO cells stably expressing

emission) with increasing concentrations of the labeled ligand (Figure 3A). This suggests that the labeled ligand, TAMRAAc-TZ14011, binds to the expressed Nluc-CXCR4, establishing a close proximity, a key factor for bioluminescent energy transfer where the Nluc serves as the bioluminescent donor and TAMRA as the fluorescent acceptor. In the BRET assay, an important underlying factor is to have the donor and acceptor in close proximity (∼10 nm) as well as an overlap between the donor emission spectra and acceptor excitation spectra to facilitate energy transfer. In addition to being a photostable fluorescent dye, the spectral characteristics of TAMRA dye (peak excitation 565 nm and emission 580 nm) present an appropriate spectral separation suitable for energy transfer from Nluc (with peak intensity of 460 nm).22 The small size, stability, and sustained highly intense luminescence of Nluc might also contribute to the feasibility and sensitivity of the NanoBRET assay system.21 Furthermore, the efficiency of the NanoBRET assay system as a potential screening tool was tested by a competitive binding assay. Transiently transfected HEK293 and HeLa cells, expressing Nluc-CXCR4, were incubated with 0.1 μM TAMRA-AcTZ1411 in the presence of increasing concentrations of Ac-TZ14011, and the IC50 values of Ac-TZ14011 were then evaluated to be 15.0 and 13.5 nM (Figure 3B,C), respectively. The IC50 value of FC131,38 a cyclo-pentapeptide derived from Ac-TZ14011, was evaluated to be 3.6 nM using the NanoBRET assay system in HEK293 cells transiently D

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secretory signal peptide from interleukin-6, IL-6 (sec), was introduced to the N-terminus of the Nluc-CXCR4 receptor, designated as secNluc-CXCR4/secNluc-CXCR4-EGFP. The receptors were expressed in HEK293 cells by transient transfection. The expression and characteristic binding of TAMRA-Ac-TZ14011 to the expressed receptors (secNlucCXCR4/secNluc-CXCR4-EGFP) were confirmed using confocal laser scanning microscopy. There was similar but enhanced expression of secNluc-CXCR4 and secNlucCXCR4-EGFP in the plasma membrane of the transfected cells to which the fluorescent probe (TAMRA-AcTZ14011) bound significantly with little or no internalization (Figure 7 and Supporting Information, Figure S6A) compared to cells expressing the receptor without the secretory peptide. Although GPCRs are generally predisposed to be expressed and trafficked to the plasma membrane,44−46 the introduction of the secretory signal peptide further enhanced the proper trafficking of the expressed receptor. The binding of the fluorescent probe to the receptor was significantly inhibited in the presence of excess unlabeled Ac-TZ14011 (Supporting Information, Figure S6B,C). Functionality of the NanoLuc of secNluc-CXCR4 was confirmed by evaluation of luciferase signals. The luciferase signals proportionally increased with increasing cell numbers, indicating that NanoLuc retains its functionality when the secretory peptide is fused to its Nterminus (Supporting Information, Figure S7). Therefore, the NanoBRET system as a tool for binding studies was evaluated in HEK293 cells transiently expressing secNluc-CXCR4. As this is similar to what was observed with cells expressing Nluc-CXCR4, the dose−response measurement of TAMRA-AcTZ14011 binding to HEK293 cells expressing secNluc-CXCR4 showed a concurrent increase in BRET ratio with increasing concentrations of the labeled ligand (Figure 8A), confirming the establishment of BRET. Evaluation of the binding potency of unlabeled Ac-TZ14011 using the NanoBRET system in transiently transfected cells expressing secNluc-CXCR4 showed a nanomolar degree IC50 value (that is, 23 nM) comparable to what was obtained using the NanoBRET system in cells expressing Nluc-CXCR4 (Figure 8B). However, the system with secNluc-CXCR4 showed higher dynamic range. This observation could be attributed to the enhanced expression and trafficking of the receptor due to the introduction of the IL-6 secretory signal peptide.47−49 Thus, in future studies, the NanoBRET system would be established in stable cell lines using secNluc-CXCR4. Taken together, the present NanoBRET assay system is simple and useful for rapid and real-time screening of a library of CXCR4 ligands, to evaluate the IC50 values as a measure of their binding affinity for CXCR4, and can also be designed for kinetic studies of ligands in the future. Furthermore, the NanoBRET assay system has the advantage of being homogeneous and less time-consuming over our previous fluorescence-based binding assay system because the NanoBRET assay system does not require a washing step. The current system is also less cumbersome as it can be performed at a temperature of 25 °C, which generally translates into room temperature, unlike most binding assays which may require incubation at 4 °C.

Nluc-CXCR4 (Supporting Information, Figure S3B), confirming the functionality of the NanoLuc. The NanoBRET assay system as a tool for saturation studies is shown in Figure 5A. The dissociation constant (Kd) of TAMRA-Ac-TZ14011, a measure of the affinity for specific binding to CXCR4, was estimated in the CHO cells stably expressing Nluc-CXCR4 to be 18.6 ± 0.5 nM. This implies that through time-resolved experiments, the NanoBRET system can be designed for CXCR4 ligand-binding kinetic studies in the future.39,40 Furthermore, through a competitive binding assay using CHO stable cells, the IC50 values of three known CXCR4 ligands, SDF-1α (CXCL12), Ac-TZ14011, and FC131, were estimated to be 3.2, 15.3, and 4.5 nM, respectively (Figure 5B), which are comparable to values obtained by other binding assay methods as well as in the transiently transfected HEK293 and HeLa cells in Figure 3B− D. As a control, the binding affinity of a known non-CXCR4 but CCR5 ligand, maraviroc (Pfizer Inc.),41 was also evaluated using the NanoBRET assay system and found to bind faintly to CXCR4 (Figure 5B). These results, therefore, confirmed the feasibility and efficiency of the developed NanoBRET assay system as a screening tool for CXCR4 ligands. To evaluate the suitability of the NanoBRET assay system for high-throughput screening, a Z′ factor assay was performed by incubating CHO cells stably expressing Nluc-CXCR4 with increasing concentrations of the fluorescent probe (test compounds) or vehicle (OptiMEM; negative control) for 1 h at 25 °C, and the Z′ values were derived from correlations between the mean and standard deviation of the test compounds and negative controls.42 Generally, there were positive Z′ values for all the concentrations evaluated, with concentrations between 1.0 and 1000 nM showing Z′ values of ≥0.5 (except 2.5 nM, Figure 6), an indication of a feasible and efficient high-throughput assay system.21,42,43 The signal-to-noise ratio, especially at lower concentrations, was, however, observed to be weak in the assay system despite the high sensitivity. One possible cause could be due to low expression and trafficking of the receptor, Nluc-CXCR4. To enhance the expression and trafficking of the receptor, a

Figure 6. Z′ factor values of TAMRA-Ac-TZ14011 binding to CXCR4 using the NanoBRET assay system. CHO cells stably expressing Nluc-CXCR4 (5 × 104/well) were incubated with increasing concentrations of TAMRA-Ac-TZ14011 (test compound) or vehicle (OptiMEM; negative control) for 1 h at 25 °C. Z′ values are calculated based on the correlations between the mean and standard deviation of test compounds and negative controls as described in Materials and Methods (red horizontal line shows Z′ factor of 0.5 as an indication of high assay performance). Values are mean ± SEM, n = 3.

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CONCLUSION Existing binding assays, although saddled with limitations, can be improved by applying advanced screening systems. We have successfully developed a NanoBRET assay system that is useful E

DOI: 10.1021/acs.bioconjchem.9b00182 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 7. Binding characteristics of TAMRA-Ac-TZ14011 to CXCR4 using confocal laser scanning characteristic binding of TAMRA-Ac-TZ14011 (0.1 μM) to HEK293 cells transiently expressing secNluc-CXCR4-EGFP incubated for 1 h at 25 °C. Cells were washed and observed using confocal laser scanning microscopy (scale bars: 20 μm scale). PC: Phase contrast.

Figure 8. NanoBRET assay system in HEK293 cells expressing secNluc-CXCR4. (A) Dose−response measurement in transiently transfected HEK293 cells incubated with increasing concentrations of TAMRA-Ac-TZ14011 for 1 h at 25 °C. Values are mean ± SEM, n = 3 (Z′ values ≥0.5). (B) Competitive binding of 0.1 μM TAMRA-Ac-TZ14011 in the presence of increasing concentrations of unlabeled Ac-TZ14011 using the NanoBRET assay system in HEK293 cells transiently expressing secNluc-CXCR4. Values are mean ± SEM, n = 3.

Expression Plasmids. To the gene of CXCR4 were introduced alanine mutations using site-directed mutagenesis at S338, S339, S341, S344, S346, S347, and S348. The sitedirected mutagenesis was done on pEGFP-N-CXCR4, and the obtained plasmid was designated as pEGFP-N-CXCR4SAm. CXCR4SAm and CXCR4SAm-EGFP genes were amplified by PCR using pEGFP-N-CXCR4SAm as a template. The amplified genes were introduced to NanoBRET positive control vector (Promega, Madison, WI, USA) as XhoI/XbaI fragments, resulting in pNLucCXCR4SAm and pNLucCXCR4SAm-EGFP. To add the secretory signal peptide sequence of IL-6, the NanoLuc gene was amplified by long PCR primers coding the signal peptide sequence. The amplified secNanoLuc fusion gene was introduced to NanoLuc-CXCR4 and NanoLuc-CXCR4-EGFP expression plasmids as the NheI/XhoI fragment. Amino acid sequences for constructed fusion proteins are described in the Supporting Information. Construction of Expression Plasmids for Establishment of Stable Cell Lines. The genes of NanoLuc-

for real-time high-throughput screening of CXCR4 potent ligands in living cells. The method will be utilized for multiwell plate-based screening of potent CXCR4 ligands and, furthermore, for functional studies of CXCR4 ligands.

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MATERIALS AND METHODS Materials. All reagents were obtained from Sigma-Aldrich unless stated otherwise. Cell Lines and Cell Culture. Human embryonic kidney (HEK293) cells and human epithelial (HeLa) cells were obtained from the American Type Culture Collection (ATCC, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Wako Pure Chemical Corp., Japan) supplemented with 10% fetal bovine serum (FBS) purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Chinese hamster ovary cells were also obtained from the ATCC and maintained in Ham F-12 medium (Wako Pure Chemical Corp., Japan) supplemented with 10% FBS. Construction of NanoLuc-CXCR4/secNanoLuc-CXCR4 and NanoLuc-CXCR4-EGFP/secNanoLuc-CXCR4-EGFP F

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incubated for 24 h at 37 °C under 5% CO2 prior to experiments. The medium was removed from each well and replaced with 100 μL of OptiMEM (reduced serum) after being washed with PBS. The luminescence was read immediately after adding 25 μL of the Nluc substrate, furimazine, using a Tecan Genios Pro plate reader. NanoBRET Binding Assay. Post-transfected cells expressing Nluc-CXCR4/secNluc-CXCR4 (5 × 104) were seeded in a white Thermo Scientific Matrix 96-well microplate and incubated for 24 h at 37 °C under 5% CO2 prior to experiments. The medium in each well was removed and replaced with OptiMEM (reduced serum). Competitive binding assay in transiently transfected cells (HEK293 and HeLa cells) was performed by incubation with 0.1 μM TAMRA-Ac-TZ14011 in the presence of increasing concentrations of test compounds for 1 h at 25 °C. Competitive binding assays in the CHO stable cells were performed by incubating seeded cells with 10 nM TAMRA-Ac-TZ14011 in the presence of increasing concentrations of the test compounds. Saturation studies in CHO stable cells were done by incubating seeded cells with serially diluted TAMRAAc-TZ14011 without unlabeled Ac-TZ14011 (total binding) and in the presence of excess unlabeled 10 μM Ac-TZ14011 (nonspecific binding) to deduce the specific binding of TAMRA-Ac-TZ14011. The substrate furimazine, reconstituted in the buffer (30 mM Tris·HCl (pH 7.6), 10 mM EDTA), was then added to each well (25 μL) to a final concentration of 10 μM, according to manufacturer’s protocol. BRET was read immediately using a Wallac 1420 ARVO MX plate reader (fitted with 100 nm filter) at room temperature. The donor emission of Nluc was measured at 460 nm (80 nm band-pass), and the acceptor emission of TAMRA was measured at >610 nm (long-pass). Raw BRET ratio was given as

CXCR4SAm and NanoLuc-CXCR4SAm-EGFP were cut out by NheI/NotI digestion from pNLuc plasmids. The fragments were introduced to pcDNA5/FRT by ligation, resulting pcDNA5NLucCXCR4SAm and pcDNA5NLucCXCR4SAmEGFP. Establishment of Stably Expressing Cell Lines. Flp-in CHO cells were maintained in Ham F-12 medium supplemented with 10% FBS, penicillin/streptomycin, and 1 μg/mL hygromycin (Nacalai Tesque Inc., Japan) at 37 °C in 5% CO2. Transfection and antibiotic selection were performed following the manufacturers’ instruction. Briefly, pcDNA5NLucCXCR4SAm or pcDNA5NLucCXCR4SAm-EGFP (0.25 μg) was co-transfected with pOG44 (2.25 μg) using Lipofectamine 3000 (Invitrogen) in 6-well plates. Medium was changed to hygromycin B (500 μg/mL, Wako Chemicals) containing medium. After 3−4 weeks of selection, survived cells were split into a 96-well plate to obtain single-cell clones. Cell culture was continued for 1 week after transfection. The expression of NLuc-CXCR4 or NLuc-CXCR4SAm-EGFP was checked by fluorescent microscopy directly for EGFP or after being stained with TAMRA-Ac-TZ14011. TAMRA-Ac-TZ14011. TAMRA-Ac-TZ14011 was synthesized according to the method by Nomura et al.25 with modifications (Supporting Information). Confocal Laser Scanning Microscopy. The expression of Nluc-CXCR4/secNluc-CXCR4 and Nluc-CXCR4-EGFP/ secNluc-CXCR4-EGFP, and subsequently the characteristic binding of TAMRA-Ac-TZ14011 to CXCR4 in mammalian cells, was observed using a confocal laser scanning microscope, Fluoview FV10i (Olympus Corp., Tokyo, Japan). HEK293 cells (1 × 106) were transiently transfected with 5 μg of pcDNA3 coding Nluc-CXCR4/secNluc-CXCR4 or NlucCXCR4-EGFP/secNluc-CXCR4-EGFP using Lipofectamine 3000 and incubated for 24 h in a tissue culture incubator at 37 °C under 5% CO2. Transfected cells were then seeded in a 35 mm optically clear plate (Greiner Bio-one Co., Ltd.) at a density of 2 × 105 cells per plate in 2 mL of DMEM supplemented with 10% FBS and incubated for 24 h at 37 °C under 5% CO2. The growth medium was then replaced with 1 mL of OptiMEM (reduced serum) after the cells were washed with PBS(−) and incubated with the CXCR4 ligands (TAMRA-Ac-TZ14011 and Ac-TZ14011 at final concentrations of 0.1 and 1.0 μM, respectively) for 1 h at 25 °C. The cells were then washed twice with PBS(+) [PBS(−) containing Ca2+ and Mg2+], and the medium was replaced with 1 mL of OptiMEM (reduced serum). CHO cells stably expressing Nluc-CXCR4 were seeded in a 35 mm optically clear plate at a density of 2 × 105 cells per plate in 2 mL of Ham F-12 medium supplemented with 10% FBS and incubated for 48 h at 37 °C under 5% CO2 prior to experiments. Cells were washed and incubated with TAMRAAc-TZ14011 and Ac-TZ14011 at final concentrations of 0.5 and 5.0 μM, respectively, in OptiMEM for 1 h at 25 °C. The medium was replaced with 1 mL of OptiMEM (reduced serum) after the cells were washed three times with PBS(+). All images were acquired using the software Olympus Fluoview version 4.2a. Measurement of Luminescence Emitted by Nluc. The luminescence emitted by Nluc tagged to CXCR4 was measured as an indicator of functionality of the bioluminescent protein in transiently transfected HEK293 cells and CHO stable cells. Increasing cell density of post-transfected cells expressing Nluc-CXCR4 was seeded in a 96-well plate and

raw BRET ratio =

acceptor emission donor emission

Z′ Factor Assay. Post-transfected CHO cells stably expressing Nluc-CXCR4 (5 × 104) were seeded in a white Thermo Scientific Matrix 96-well microplate and incubated for 24 h at 37 °C under 5% CO2 prior to experiments. The medium in each well was removed and replaced with OptiMEM (reduced serum). The cells were incubated with serially diluted TAMRA-Ac-TZ14011 or the vehicle (OptiMEM; negative control) for 1 h at 25 °C. BRET was detected immediately after adding 25 μL of furimazine using the Wallac 1420 ARVO MX plate reader (fitted with 100 nm filter) at room temperature. The Z′ factor for each concentration point was calculated using the formula below according to Zhang et al.42 1 − (3δt + 3δb)/|μt − μb|

where δt and δb are the standard deviations of a test compound and a negative control, respectively, and μt and μb are the mean BRET ratios of the test compound and the negative control, respectively. Data Presentation and Statistical Analysis. The data were presented and analyzed using GraphPad Prism software. The IC50 values were generated from nonlinear regression analysis. G

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.9b00182. Experimental procedure for the synthesis of TAMRAAc-TZ14011, data of binding characteristics of TAMRAAc-TZ14011 to CXCR4 using confocal laser scanning microscopy, characteristic binding of TAMRA-AcTZ14011 to Nluc-CXCR4-EGFP/secNluc-CXCR4EGFP in the presence of 1.0 μM Ac-TZ14011, expression and functionality of Nluc-CXCR4/secNlucCXCR4 in HEK293 cells and CHO stable cells, and binding characteristics of TAMRA-Ac-TZ14011 to CHO cells without Nluc-CXCR4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Wataru Nomura: 0000-0001-8348-7544 Hirokazu Tamamura: 0000-0003-2788-2579 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Japan Society for the Promotion of Science (JSPS) for providing financial support (JSPS KAKENHI Grant No. JP15H04652) to H.T., the Platform for Drug Discovery, Informatics, and Structural Life Science of MEXT, Japan, and the Cooperative Research Project of Research Center for Biomedical Engineering. M.S. was also supported by MEXT through the award of a scholarship. We are also thankful to Prof. Takamitsu Hosoya and Assoc. Prof. Suguru Yoshida, TMDU, who donated furimazine, and to Dr. Shigeyoshi Harada, National Institute of Infectious Diseases, who donated maraviroc. Our profound gratitude also goes to Prof. Shoji Yamaoka for his immense support.

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REFERENCES

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DOI: 10.1021/acs.bioconjchem.9b00182 Bioconjugate Chem. XXXX, XXX, XXX−XXX