Ryanodine insect

Ryania consists of the powdered stem of the tropical shrub, Ryania speciosa. The extract contains ryanodine and related compounds, and has a low toxicity to mammals. The powder is used as a stomach poison on vegetables and fruit. Ryanodine induces paralysis in insects by direct action on the muscles, resulting in sustained contraction and paralysis. 

New agrochemicals introduced in the past five years include new chemistries with known modes of action, such as the protoporphyrinogen inhibitor bencarba-zone, the phytoene desaturase picolinafen and beflutamid, and sodium channel pyrethroids new chemistries with possibly new modes of action, such as flonic-amid and pyridalyl and new chemistries with established new modes of actions, such as flubendiamide, which activates ryanodine-sensitive intracellular calcium release channels, ryanodine receptors RyR, in insects. 

Flubendiamide, a benzenedicarboxamide insecticide, also affects calcium channels. This insecticide induces unique symptomology in poisoned insects, showing a gradual contraction of the insect body. It is believed that flubendiamide induces intracellular Ca2+ release mediated by a calcium channel such as the ryanodine receptor resulting in the contraction of insect muscle (Tohnishi et al., 2005 Ebbinghaus-Kintscher et al., 2006 Nauen, 2006). 

Ebbinghaus-Kintscher, U., Luemmen, P, Lobitz, N., Schsulte, T., Funke, C., Fischer, R., Masaki, T., Yasokawa, N., and Tohnishi, M., Phthalic acid diamides activate ryanodine-sensitive Ca2+ release channels in insects, Cell Calcium, 39, 21, 2006. 

The insecticidal effect of Ryana surpasses that of the other insecticides of natural origin, as well as of a major part of chlorinated hydrocarbon insecticides (Clark and Laudani, 1953). Ryanodin entering the insect organism in 2-5 ppm quantities substantially reduces oxygen uptake, which results in paralysis. 

Ryanodine-type compounds act primarily at the Ca2+ release channel in both mammals and insects [12], Ryanodol-type compounds, however, are more selective toxicants for insects than they are for mammals, suggesting a different mode of action for these compounds [13, 14]. 

This hypothesis was further supported using molecular biology and cellular tools where the insect ryanodine receptor gene was heterologously expressed in appropriate cells. In untransfected CHO cells, application of flubendiamide sulfoxide (a better soluble analogue) did not cause an [Ca ] increase (Figure 8). In CHO cells transfected with the RyR from Drosophila (CHO-RyR), flubendiamide sulfoxide induced Ca responses with similar kinetic responses to those found in Heliothis neurons (Figure 8). 

Therefore it was concluded that flubendiamide acts as a selective activator of the insect ryanodine receptor, inducing ryanodine-sensitive cytosolic Ca transients. Furthermore, radioligand binding studies using microsomal membranes from Heliothis flight muscles demonstrated that flubendiamide allosterically increased the ryanodine affinity. Flubendiamide was found to bind to Heliothis microsomal membranes with an apparent of 4.7 nM. Known ryanodine receptor ligands such as cyclic ADP-ribose, caffeine, ryanodine, and dantrolene did not interfere... 

Last but not least, it was also shown that fiubendiamide and its sulfoxide are specihc to insect ryanodine receptors and do not affect mammalian ryanodine receptors. Even high concentrations of fiubendiamide sulfoxide applied on differentiated mouse muscle C2C12 cells which express the muscle Subtype 1 and Subtype 111 did not either elicit Ca signals nor did they prevent the Ca transients elicited by caffeine (Figure 9). Therefore, we conclude that fiubendiamide and related compounds do not affect mammalian RyR Type I and III. These observations provide a good explanation for the excellent toxicological profile observed in the case of fiubendiamide. 

Figure 1. Rynaxypyr 3-bromo-N-[4-chloro-2-methyl-6-[(methylamino)carbonyl -phenyl -l-(3-chloro-2-pyridinyl)-l H-pyrazole-5-carboxamide. A new anthranilic diamide insecticide acting at the insect ryanodine receptor.

To assess activity at the insect ryanodine receptor, pyridyl pyrazoles of Table II were tested in a calcium mobilization assay, using neurons from the American cockroach, Periplaneta americana. These studies have confirmed the mode of action to be RyR activation. Compounds D11-D17 showed exceptional potency in this assay with activity in the range of0.03-0.30 pM. The data shows the ability of anthranilic diamides to release internal calcium stores while failing to activate voltage-gated calcium channels. Furthermore, calcium mobilization induced by anthranilic diamides is blocked following treatment with 1 pM ryanodine, consistent with action at the ryanodine receptor. 

In summary, a novel class of chemistry has been discovered with exceptional insecticidal activity against a broad spectrum of lepidoptera. These compounds have been found to exhibit their action through release of intracellular Ca stores mediated by the ryanodine receptor. The first commercial member of this class, Rynaxypyr, demonstrates outstanding lab and field activity on all major species of lepidoptera with lab rates in the range of 0.01-0.06 ppm. This level of activity is significantly better than current commercial standards and shows remarkable consistency across a broad insect spectrum. Rynaxypyr thus offers exceptional promise as a new product for crop protection based on this combination of a new mode of action with outstanding insecticidal properties. 

Mammals possess three isoforms of the ryanodine receptor RyRl and RyR2, distributed primarily in skeletal and cardiac muscle, respectively, and RyR3 distributed more heterogeneously. Insects, however, express a single form of the receptor, sharing only 47% sequence homology [9]. Comparative studies were conducted to determine Rynaxypyr s ability to activate mammalian RyRs. 

Though ryanodine and caffeine show similar potency against insect and mammalian receptors (not shown), differential selectivity is observed for Rynaxypyr . 

Flubendiamide is most effective on larvae followed by adults, but it has no ovicidal activity. In the course of extensive research on the mode of action of flubendiamide, it was determined that flubendiamide was a ryanodine receptor modulator. Flubendiamide Axes the Ca-channel of insect ryanodine receptors (RyR) in the open state, and subsequently induces calcium release from the membrane vesicle... 

Flubendiamide is a novel insecticide possessing potent and selective activity against lepidopterous insects [1], This insecticidal activity has been clarified to be mediated by a ryanodine-sensitive calcium release channel (RyR) [2-3], The stabilization of RyR to open state by the compound induces robust calcium release from intracellular calcium store (Figure 1). This implies a significant impact on components involved in intracellular calcium homeostasis such as the pump, a pivotal component which reuptakes released Ca into SR. In this study, we examined effects on the Ca pump, as a consequence of the calcium mobilization induced by flubendiamide. 

Chlorantraniliprole. Diamide insecticides are another class of recently introduced crop protection agents, which behave as activators of ryanodine receptors in the insect. This leads to uncontrolled calcium release in muscles. Chlorantraniliprole is a member of this family and is in commercial use for protection from various pests. 

A novel mode of action was first suggested by the characteristic symptoms induced by flubendiamide, such as gradual contractions of insect body, thickening and shortening without convulsions (Fig. 31.2). Though the symptoms were obviously different from those of existing insecticides, similar symptoms were also seen in insects treated with the plant alkaloid ryanodine, a modulator of a... 

This ranking of halogen atoms can be exemplified by the recently described SAR of halogenated phthalic acid diamides (F < Cl < Br < I) in the development product fiubendiamide (54 ISO-proposed, Hal = I Nihon Nohyaku Co., Ltd./ Bayer Crop Science) [24], which activates selective ryanodine-sensitive intracellular Ca + release channels in insects as novel mode of action [25]. Introduction of a bulky and moderate lipophilic halogen such as iodine into 3-position of the phthalic acid aryl moiety increased the insecticidal activity considerably (Fig. 35.4) (for more details see Chapter 34). 

Flubendiamide (54 ISO-proposed Nihon Nohyaku Co., Ltd./Bayer CropSdence) [125, 126,127] (Fig. 35.13) with a heptafluoro-isopropyl moiety in the anilide part of the molecule (Chapter 34) induces ryanodine-sensitive cytosoUc Ca + transients that were independent of extracellular Ca concentration in isolated neurons from the pest insect Heliothis virescens as well as in transfected CHO cells expressing the RyR from Drosophila mdanogaster. Binding studies on microsomal membranes from H. virescens flight muscle revealed that 54 interacts with a site distinct from the ryanodine binding site and disrupted the Ca + regulation of ryanodine binding by an allosteric mechanism. 

Here we have presented a new class of highly potent insecticides, the anthranilamides, which control pest insects via a novel mode of action. This chemistry exhibits broad-spectrum insect control with exceptional activity against lepidopteran larvae. RyR activation has clearly been demonstrated as the mode of action for the anthranilamides. Unregulated receptor activation results in depletion of internal calcium stores, muscle paralysis and ultimately insect death. In addition to this chemistry representing one of the first examples of a potent, synthetic RyR-active molecule, anthranilamides exploit a unique binding site on the receptor, distinct from that of ryanodine or caffeine. 

Phthalic Acid Diamides Activate Insect Ryanodine... 

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