Fluorescent Probes for Insect Ryanodine Receptors: Candidate Anthranilic Diamides

Diamide insecticides with high efficacy against pests and good environmental safety are broadly applied in crop protection. They act at a poorly-defined site in the very complex ryanodine (Ry) receptor (RyR) potentially accessible to a fluorescent probe. Two N-propynyl analogs of the major anthranilic diamide insecticides chlorantraniliprole (Chlo) and cyantraniliprole (Cyan) were accordingly synthesized and converted into two fluorescent ligands by click reaction coupling with 3-azido-7-hydroxy-2H-chromen-2-one. The new diamide analogs and fluorescent ligands were shown to be nearly as potent as Chlo and Cyan in inhibition of [3H]Chlo binding and stimulation of [3H]Ry binding in house fly thoracic muscle RyR. Although the newly synthesized compounds had only moderate activity in insect larvicidal activity assays, their high in vitro potency in a validated insect RyR binding assay encourages further development of fluorescent probes for insect RyRs.


Introduction
Natural ryanodine (Ry) from Ryania speciosa was used as a botanical insecticide in the 1940s [1]. By the late 1990s, two types of diamides were found to possess excellent insecticidal activity, producing poisoning signs similar to those of Ry. Radioligand binding and Ca 2+ flux studies at Nihon Nohyaku and Bayer [2][3][4][5][6][7][8][9] and at DuPont [10][11][12][13] defined the mode of action of the diamides as Ry receptor (RyR) modulators keeping Ca 2+ channels open and depleting Ca 2+ stores, leading to gradual muscle contraction and paralysis. RyRs form intracellular Ca 2+ channels located mostly on the sarcoplasmic reticulum of muscle. With commercialization of the anthranilic diamides chlorantraniliprole (Chlo) [11] and cyantraniliprole (Cyan) [14] (Figure 1) and phthalic diamide flubendiamide (Flu) [2], the insect RyR became one of the most important insecticide targets. Photoaffinity labeling revealed that Flu interacts in the insect transmembrane domain and that the N-terminus plays an important role in sensitivity [8]. Radioligand binding assays with [ 3 H]Chlo and [ 3 H]Ry established that anthranilic diamides and Ry act at the Musca domestica RyR [15,16]. The genes encoding the RyRs have been characterized for various species (Heliothis virescens, Myzus persicae, Aphis gossypii, Peregrinus maidis and Drosophila melanogaster) [10], and more recently for Plutella xylostella [17]. However, up to now, very little is known about the structure of the insect tetrameric transmembrane RyRs and particularly their active binding site. This study takes the first steps in developing a fluorescent probe for the insect RyR. Chlo and Cyan were selected as prototypes because they are exceptionally potent and readily recognizable insecticides. 7-Hydroxycoumarin was used as the fluorescent substituent to be introduced. Retention of the required biological properties was established by high potency in a validated insect (Musca) RyR radioligand binding assay and moderate larvicidal activity on an important pest (Spodoptera).

Fluorescent Ligands Design and Synthesis
The key step in preparing the effective fluorescent ligands was solved by coupling of Chlo and Cyan as the pharmacophore skeletons and hydroxycoumarin as the fluorescent chromophore with a click reaction (Scheme 1). Both fluorescent ligands obtained are very stable and possess good fluorescent properties, for instance, both their maximum excitation wavelength 396 nm and the maximum emission wavelength 475 nm and their large stokes shift are suitable for fluorescence assay. Scheme 1. Synthesis routes for two N-propynyl intermediates and two fluorescent ligands.

Fluorescent Ligands Bind Potently to Specific Site in Musca RyR
The  Figure 2A) consistent with the reported value of 3-15 nM for Chlo and Cyan [15]. In addition, Fluor-Cl and Fluor-CN inhibited [ 3 H]Chlo binding with similar IC 50 's of 34-39 nM ( Figure 2B), compared to 6-14 nM for Chlo and Cyan [15], which indicated that they all work at the same binding site on Musca RyRs.

Insecticidal Activity of Fluorescent Ligands
The results of assays with Plutella larvae are shown in Table 1. Although the two N-propynyl compounds were less potent than Chlo, Cyan and Flu, they all showed good insecticidal activity with LC 50 s for Prop-Cl and Prop-CN of 0.08 ppm and 0.34 ppm, respectively. After incorporating the hydroxycoumarin fluorophore, the ligands were 22-32 fold less potent than the propynyl compounds, with LC 50 s of 2.6-7.6 ppm. Thus the new compounds retain good insecticidal activity despite the propynyl and large polar fluorescent substituents were introduced. Most importantly the fluorescent probes are very potent in vitro in binding assays, although less toxic in vivo with penetration barriers.

Insecticidal Activity
The diamondback moth, (Plutella xylostella) was reared in the laboratory for over ten years using vermiculite cultured radish (Raphanus sativusL. var. cuiqing) seedlings. Bioassays were conducted using a leaf-dip method slightly adapted from the methods of Liang et al. [20] and He et al. [21]. Cabbage (Brassica oleracea variant L.) leaves measuring 6 × 6 cm were immersed for 10 s in various concentrations of test compound prepared with distilled water containing 1 g L -1 Triton X-100. Each leaf was left to air dry for 1.5 h and then placed into a Petri dish lined with filter paper. A total of 15 first day fourth instar larvae was introduced into each dish, and three replicates were prepared. Five to seven concentrations of test compound and one control (distilled water with 1 g/L Triton X-100) were examined in each bioassay. Mortality was assessed after 96 h of exposure as individuals that did not move when pushed gently with a brush. The LC 50 value was calculated using PoLoPlus 2.0 software (LeOra Software, Petaluma, CA, USA) with data corrected for control mortality by Abbott's formula [22]. Following incubation for 2 h at 37 °C, the mixtures were filtered through GF/B filters (Whatman, presoaked in ice-cold washing buffer) immediately after dilution with 5 mL of ice-cold washing buffer (150 mM KCl, 10 mM Hepes, pH 7.4). Each assay tube was further rinsed twice with 5 mL of ice-cold washing buffer, and the rinses were passed through the same filter. The filters were then transferred to scintillation vials containing 10 mL of Safety-Solve (Research Products International Corporation, Mount Prospect, IL, USA) and held overnight in the dark before scintillation counting. Binding data were analyzed and plotted by GraphPad Prism 5.0. All data reported are mean ± standard error for two independent experiments with triplicate samples.

Conclusions
The goal to prepare a highly potent and specific fluorescent probe for the insect RyR has been achieved with an anthranilic diamide analog of Chlo containing a hydroxycoumarin substituent. In a validated Musca RyR assay Fluor-Cl is similar to Chlo in potency for stimulating [ 3 H]Ry binding and inhibiting [ 3 H]Chlo binding all in the range of 5-39 nM. The fluorescent probes are less active than Chlo and Cyan as insecticides as expected for compounds with large polar substituents affecting transport and stability. Large species differences are evident in RyR ligand binding with house fly and honeybee quite sensitive [15,16] and therefore possibly preferred RyR sources. Replacing radioligands with fluorescent probes will allow broader use of binding assays without restrictions associated with radioactive materials.