Ryanodine receptors insects

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). 

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. 

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... 

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. 

Ryanodine receptors (RyR) mediate the release of calcium from intracellular stores of the sarcoplasmic/endoplasmic reticulum (SR/ER) following a small calcium influx via voltage-gated calcium channels of the cytoplasmic membrane after depolarization. The process, referred to as calcium-induced calcium release, triggers muscle contraction after electrical excitation in mammalian cardiac as well as in insect striated muscles (I). Three RyR isoforms exist in mammals Type 1, which is predominantly expressed in skeletal muscles Type 2, which is expressed in cardiac muscle and the more ubiquitously expressed Type 3 2 . 

Equilibrium binding of [ H]ryanodine to Heliothis flight muscle microsomes clearly revealed cooperativity between the binding sites at 100 pM [Ca ]. Based on the assumption that RyRs are arranged in arrays of calcium release units in insect as well as in mammalian sarcpplasmic reticulum membranes (22), a plausible explanation would be that ryanodine receptors opened by calcium may have activated adjacent RyR channels by intermolecular interactions. Phthalic acid diamides completely converted the receptors to the high-affinity conformation as revealed by the hyperbolic binding iso erm. 

Flubendiamide Selectively Activated Insect ryanodine Receptors... 

Calcium channels represent an attractive biological target for insect control because of their role in multiple cell functions such as muscle contraction. One new insecticide [45] that causes release of intracellular Ca is RynaxypyrTM. it is in group 28, ryanodine receptor modulators. The ryanodine receptors contract the muscles the contraction requires a release of calcium ions. Depletion of the calcium ion results in insect death. 

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

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. 

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