Affinity-based fluorescence polarization assay for screening molecules acting on insect ryanodine receptors

Abstract

Insect ryanodine receptors constitute calcium ion channels, and are the target of novel diamide insecticides containing two types of phthalic and anthranilic diamides. Ryanodine receptors in Periplaneta americana leg muscle were extracted through homogenization and centrifugation. Based on the potential usefulness of a fluorescent probe identified in a previous study, a fluorescence polarization assay was developed with extracted Periplaneta ryanodine receptors, and this technique was used to test the affinity binding of two types of active diamide compounds. Results showed that anthranilic diamides could replace the binding of the fluorescent probe on the ryanodine receptor, but phthalic analogs, including flubendiamide, did not affect the binding of fluorescent probe. Therefore, the developed fluorescence polarization (FP) assay is an easier and more efficient technique to study the affinity binding of small molecules on fluorescent probe binding sites on ryanodine receptors than the [³H] labeled-based assay.

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1. Introduction

Ryanodine receptors (RyRs) were derived from natural ryanodine (Ry), which possesses insecticidal activity, in the 1940s.¹ Since then, great effort has been focused on discovering new insecticidal molecules,^2–5 and some phthalic diamide compounds have been found to have toxic symptoms similar to those of Ry by Nihon Nohyaku, Japan, who optimized a pyrazinedicarboxamide herbicide.⁶ From 1998 to 2012, two kinds of diamide compounds, a phthalic diamide, flubendiamide (Flu), by Nihon Nohyaku and Bayer; and anthranilic diamides, chlorantraniliprole (Chlo) and cyantraniliprole (Cyan) by DuPont, have been developed as novel commercial insecticides.^7–11 Presently, they are applied extensively against Lepidopteran pests on crops, vegetables, and fruits. Meanwhile, much research has been carried out on the configuration of RyRs and its binding with active compounds in the past few decades. Genes encoding RyRs have been characterized from various species, including Heliothis virescens, Myzus persicae, Aphis gossypii, Peregrinus maidis, and Drosophila melanogaster,^12–16 and more recently Toxoptera citricida and Spodoptera exigua.^17,18 Photoaffinity labeling revealed that Flu interacts in the insect transmembrane domain and that the N-terminus plays an important role in sensitivity.¹⁹ Unfortunately, no clear information about the binding sites of active compounds has been reported so far because insect RyRs are large tetrametric transmembrane protein that are made up of four monomers, each 565 kDa in size.²⁰

RyRs constitute Ca²⁺ channels, and Ca²⁺ is an important messenger in neural signal transmission.²¹ To identify small molecules that can regulate Ca²⁺ channels, many activity-based assays have been developed. Electrophysiological techniques were first employed to study the intracellular Ca²⁺ flow change induced by active compounds acting on RyRs like the natural alkaloid Ry.^22–24 This technique studied the active compounds regulating Ca²⁺, and did not differentiate the binding difference among different classes of compounds, such as Ry, Flu, and Chlo. The binding assays based on [³H]-labeled Chlo, Flu, and Ry revealed that phthalic and anthranilic diamides and Ry act at different allosterically-coupled sites on the RyRs of Musca domestica.^25,26 Although it is sensitive and reliable, it is not a convenient and easy technique owing to many limitations, such as the use of expensive radiochemicals, related facilities to handle and dispose of such compounds, and personnel training to prevent the exposure of researchers to radiation, etc.

Fluorescence Polarization (FP) has a number of key advantages as an assay technique. It is carried out in solution phase, is nonradioactive, does not require any separation of bound and free ligands, and is readily adaptable to low volumes (in the order of 10 μL). These features can be exploited to design FP probes for competitive binding assays.^27,28

In our previous study,²⁹ fluorescent ligands (F-Chlo) were designed and synthesized through modifying N-methyl in Chlo as N-allyl following by coupling with 3-azido-7-hydroxy-coumarin via a click reaction, and F-Chlo showed good characteristics as a potent fluorescent probe, with affinity binding nearly as high as that of Chlo, inhibition of [³H]Chlo-binding in house fly thoracic muscle RyR, and moderate activity in insect larvicidal activity assays, as well as suitable chemical properties. Although FP binding assays have usually employed purified receptors or enzymes of interest, several reports have indicated that this method can be used with membranes isolated from cells.³⁰

The aim of the present study was to develop an easy and convenient FP assay to study active molecules acting on RyRs based on our previous prepared fluorescent probe, F-Chlo. Several tests were conducted to optimize the interaction conditions between F-Chlo and the membrane extract, such as temperature, interaction equilibrium time, the concentrations of membrane extract, F-Chlo, and organic solvent. Finally, an FP assay for a specific binding site on RyRs in P. americana muscle was developed, and can be used as an alternative to radioligand binding and Ca²⁺ flux measurements to determine the affinity of new compounds to insect RyRs. Unlike radioligand binding and Ca²⁺ flux measurements, FP does not require the separation of bound and free species and has a simple read-out.

2. Results and discussion

The characterization of RyR's configuration for studying the mode of action of active compounds is necessary to establish practical and fast in vitro assays to discover new molecules. Techniques, including isolation, purification, and gene-encoded cloning and expression, were used in an attempt to obtain purified RyRs with functional Ca²⁺ channels; unfortunately, none of these techniques were successful. However, RyR membrane extract containing functional Ca²⁺ channels was obtained from some insect pests, and met the requirements of the FP assay. Thus, the RyR membranes in P. americana legs were extracted in accordance with the reported process,^31,32 and the presence of the RyRs in the membranes was confirmed by ELISA in the present study.

The interaction of receptor proteins, membranes, and enzymes with small molecules were conducted in a buffer solution, and the buffer solution influenced affinity binding, the stability of protein or membrane extract, fluorescent probes, etc. To minimize its impact on the FP assay, the fluorescence characteristics of three buffer solutions Tris–HCl (50 nM, pH 7.4), HEPES (25 nM, pH 7.4), and PBS (pH 7.4) were tested, and the stability of the fluorescent probe, F-Chlo, and the membranes was also studied in three buffer solutions, respectively. The results showed that Tris–HCl was the most suitable buffer solution as an affinity-binding medium because F-Chlo at 1–50 nM displayed stable fluorescence density and low fluorescence polarization. In addition, F-Chlo was derived from Chlo, and the K_d value of the binding of Chlo to RyR was 15 nM.³³ Thus, an initial concentration of 15 nM was selected as suitable for the fluorescent probe (Fig. 1).

Fig. 1 Chemical structures of the fluorescent probe (F-Chlo) and anthranilic diamide compound Prop-Cl.

In an initial experiment, membranes isolated from P. americana leg muscle were titrated at a constant F-Chlo concentration of 15 nM. A significant increase in FP signal was observed with increasing membrane concentrations (12.5, 25, 50, 100, 200, and 400 μg), and 15 nM of the fluorescent probe bound with half the binding sites on 25 μg of membrane extract (Fig. 2).

Fig. 2 Binding of the fluorescent probe F-Chlo (15 nM) with RyR membranes from Periplaneta americana at different concentrations. Each datapoint represents the average of triplicate determination. Vertical error bars represent ±standard deviations (SD) from the average adsorbance.

Next, membrane extracts from P. americana were titrated at several fixed concentrations of the probe to optimize probe and membrane concentrations to provide the greatest dynamic range (ΔmP[millipolarization units] = total binding − nonspecific binding) and sensitivity. We determined that the use of a 15 nM tracer and 25 μg of membranes per reaction provided a dynamic range of 65 mP under conditions where an estimated 50% of the tracer was bound to the receptor. Under these conditions, not all the specific binding sites on the receptors were occupied by probe molecules, and the assay would be the most sensitive to competition with compounds of interest (Fig. 3).

Fig. 3 Binding of the fluorescent probe F-Chlo (3.75, 7.5, 15, and 30 nM) with RyR membranes from Periplaneta americana at different concentrations. Each datapoint is the average of triplicate determination. Error bars represent ±SD about the mean.

Active compounds bind quickly to receptors in some cases,^34,35 and more slowly in others.^30,36 Time course experiments to determine the interaction between fluorescent probes and membrane extracts indicated that FP values increased gradually within 1 h; however, they changed very little, and decreased after 3 h. This showed that the equilibrium of action between F-Chlo and RyRs was reached by 1 to 2 h, and we chose 2 h as a final time point for the assay (Fig. 4).

Fig. 4 Binding of the fluorescent probe F-Chlo (15 nM) with RyR membranes from Periplaneta americana (25 μg) at 4 or 25 °C. Each datapoint is mean ± SD of data from three independent triplicate experiments.

Tested compounds and fluorescent probes are organic compounds, and are difficult to dissolve in aqueous buffer solution, but can be dissolved in the organic solvent dimethyl sulfoxide (DMSO). To dissolve tested compounds and fluorescent ligands in buffer solution, tested compounds and fluorescent probes were initially dissolved in DMSO. Thus, the F-Chlo-based FP polarization assay should tolerate the presence of low volumes of DMSO. Tests of the impact of DMSO on FP assays determined that the effect of up to 5% DMSO on the competition experiments was less than 10%. In subsequent binding and competition assays, a maximum 1.0% DMSO in each well (v/v) was included (Fig. 5).

Fig. 5 Binding of fluorescent ligand F-Chlo (15 nM) with RyR membranes from Periplaneta americana (25 μg) at 4 °C with different concentrations of DMSO. All measurements were carried out in triplicate, and each datapoint is representative of repeat experiments. Vertical bars indicate ±SD about the mean.

Z′ is a measure when the assay quality integrates variability and the signal window as determined by the difference in the signal between free (in the presence of 200-fold unlabeled competitor) and bound fluorescent probe controls across a number of assay wells.³⁷ An ideal assay has a Z′ of 1, whereas the requirement for a reliable assay is 0.5 to 1. The calculated Z′ factor for this FP assay was 0.78, reflecting its suitability for studying the rapid interaction of small molecules with insect RyR by the FP assay.

Under the optimized conditions, we performed a series of homologous competition tests with 96-well plates using F-Chlo and unlabeled Chlo for masking non-specific binding, and software OriginPro 8.0 was used to treated obtained data to get inhibition curve and the corresponding IC₅₀s (Fig. 6). The results showed that Chlo, Cyan, and Prop-Cl could competitively replace the binding of F-Chlo on membrane extracts containing RyRs from P. americana with IC₅₀s of 102, 151, and 182 nM, respectively, but Fluo and Fluo analogs ZS-IV and IPP (Fig. 7) did not influence the affinity binding of F-Chlo. The results illustrated again that the two types of biological active compounds, phthalic and anthranilic insecticides, acting on insect RyRs could bind at the different binding sites, and the developed FP assay could study the affinity-binding action of small molecules on Chlo-binding sites.

Fig. 6 Inhibitory activity of known biological molecules on binding of the fluorescent ligand F-Chlo (15 nM) with RyR membranes from Periplaneta americana (25 μg) at 4 °C. Each datapoint reflect mean ± SD of data from three independent experiments.

Fig. 7 Chemical structures of Fluo analogs ZS-IV and IPP.

3. Experimental

3.1 General methods and materials

Commercial reagents were obtained from Sigma-Aldrich (St. Louis., MO, US), Alfa Aesar (Ward Hill, MA, US), and J&K (Beijing, China). Technical grade Chlo (95% purity) was obtained from DuPont Agricultural Chemicals Ltd. (Shanghai, China). Sources for Cyan and other relevant chemicals were reported earlier.^15,16 Hepes was produced in-house. FP was measured with a Cary Eclipse Fluorescence Spectrophotometer (Agilent, Santa Clara, US) and a SpectraMax M5 (Molecular Device, US).

The preparation of Prop-Cl and F-Chlo has been described in a previously published paper,²⁹ and IPP and ZS-IV were obtained from the East China University of Science and Technology and Nankai University.

3.2 Preparation of RyR

Periplaneta adults were purchased from the Tianyu Medicinal Insects Breeding Base (Bozhou, China); their leg muscles were used to extract RyR membranes as described by Schmitt et al.³¹ All operation steps were carried out on ice,¹¹ and the following two buffers were used: (C) 50 mM Tris–HCl, pH 7.4, containing 1 μg mL⁻¹ each of aprotinin, pepstatin, and leupeptin; (D) 1.5 M KCl, 300 mM sucrose, 0.5 mM CaCl₂, 20 mM Tris–HCl, pH 8.0, containing 1 μg mL⁻¹ each of aprotinin, pepstatin, and leupeptin. Excised mid and hind legs from 8 wk-old P. americana were collected and immediately placed in liquid nitrogen, and stored at −80 °C for several months. Generally, legs (1 g wet weight) were homogenized in 9 mL of ice-cold buffer C. The homogenate was centrifuged at 10 000g for 10 min at 4 °C. The recovered supernatant was centrifuged for 1 h at 100 000g again and the resulting pellet was re-suspended in a minimum volume (3–5 mL) of buffer D to obtain membrane extracts that were stored at −80 °C. The protein concentration was determined by a Bradford assay using bovine serum albumin (BSA) as a standard.³¹

3.3 Assay conditions

To determine optimal assay conditions, the stability of the fluorescent probe was tested in three buffer solutions to select a suitable buffer solution, and then the fluorescence density and polarization value of the fluorescent probe at different concentrations (10⁻³ to 10³ nM) in a suitable buffer solution was tested to determine the optimal concentration of the fluorescent probe. The extracted membranes were diluted in 50 nM Tris–HCl buffer to different concentrations, and titrated with optimal concentrations of the fluorescent probe to determine the optimal dilution factor. Once the optimal quantity of fluorescent probe and extracted membranes were determined, all affinity-binding assays were conducted in a total volume of 120 μL per well of a 96-well plate. Reagents were added as follows: 80 μL membranes extract dilution in Tris–HCl buffer, 10 μL fluorescent probe (15 nM) in Tris–HCl buffer, and 20 μL Tris–HCl buffer. Following a 30 min incubation with agitation at 4 °C in the dark, 20 μL tested compounds in Tris–HCl buffer were added. The plate was then incubated for 2 h with agitation at 4 °C in the dark. FP was measured using SpectroMax M5. All measurements were carried out in triplicate. The Z′ value for a 96-well plate format was calculated as follows:

Z′ factor = 1 − 3 × (SD_max + SD_min)/(mP_max − mP_min)

where SD_max is the standard deviation of the bound signal in the absence of competitor, SD_min is the standard deviation of the free signal in the presence of a saturating concentration of competitor, mP_max is the mean bound signal, and mP_min is the mean free signal.

4. Conclusions

Based on the fluorescent probe F-Chlo reported in 2014, a novel homogeneous competition FP binding assay was established with several advantages over other assays, such as simpler ligand synthesis and purification, a faster mix-and-read assay, a lack of requirement for radioactive materials, and improved reagent stability over time. The developed FP binding assay for insect RyRs could supplement or replace radiolabeled technology and monitoring Ca²⁺ flux to study the interaction of active compounds and RyRs, and is very useful to screen new candidate insecticides in vitro, especially through high throughput screening.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (NNSFC) (No. 21172256), the National Basic Research Program of China (No. 2010CB126104).

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