Divergent Nine-Step Syntheses of Perhydrohistrionicotoxin Analogs and Their Inhibition Activity Toward Chicken α4ß2-Neuronal Nicotinic Acetylcholine Receptors.

Histrionicotoxin (HTX) alkaloids, which are isolated from Colombian poison dart frogs, are analgesic neurotoxins that modulate nicotinic acetylcholine receptors (nAChRs) as antagonists. Perhydrohistrionicotoxin (pHTX) is the potent synthetic analogue of HTX and possesses a 1-azaspiro[5.5]undecane skeleton common to the HTX family. Here, we show for the first time the divergent nine-step synthesis of pHTX and its three stereoisomers from the known aldehyde through a one-step construction of the 1-azaspiro[5.5]undecane framework from a linear amino ynone substrate. Surprisingly, some pHTX diastereomers exhibited antagonistic activities on the chicken α4ß2-neuronal nAChRs that were more potent than pHTX.

Investigation Of The Biosynthesis Pathway That Generates cis-Jasmone.

Researchers have established that (+)-7-iso-jasmonic acid ((+)-7-iso-JA) is an intermediate in the production of cis-jasmone (CJ); however, the biosynthetic pathway of CJ has not been fully described. Previous reports stated that CJ, a substructure of pyrethrin II produced by pyrethrum (Tanacetum cinerariifolium), is not biosynthesized through this biosynthetic pathway. To clarify the ambiguity, stable isotope-labelled jasmonates were synthesized, and compounds were applied to apple mint (Mentha suaveolens) via air propagation. The results showed that cis-jasmone is not generated from intermediate (+)-7-iso-JA, and (+)-7-iso-JA is not produced from 3,7-dideydro-JA (3,7-ddh-JA); however, 3,7-didehydro-JA and 4,5-didehydro-7-iso-JA were converted into CJ and JA, respectively.

cis-Decalin-containing tetramic acids as inhibitors of insect steroidogenic glutathione S-transferase Noppera-bo.

Decalin-containing tetramic acid is a bioactive scaffold primarily produced by filamentous fungi. The structural diversity of this group of compounds is generated by characteristic enzymes of fungal biosynthetic pathways, including polyketide synthase/nonribosomal peptide synthetase hybrid enzymes and decalin synthase, which are responsible for the construction of a linear polyenoyl tetramic acid structure and stereoselective decalin formation via the intramolecular Diels-Alder reaction, respectively. Compounds that differed only in the decalin configuration were collected from genetically engineered mutants derived from decalin-containing tetramic acid-producing fungi and used for a structure-activity relationship study. Our evaluation of biological activities, such as cytotoxicity against several cancer cell lines and antibacterial, antifungal, antimalarial, and mitochondrial inhibitory activities, demonstrated that the activity for each assay varies depending on the decalin configurations. In addition to these known biological activities, we revealed that the compounds showed inhibitory activity against the insect steroidogenic glutathione S-transferase Noppera-bo. Engineering the decalin configurations would be useful not only to find derivatives with better biological activities but also to discover overlooked biological activities.

Creating Pyrethrin Mimetic Phosphonates as Chemical Genetics Tools Targeting the GDSL Esterase/Lipase TcGLIP to Investigate Pyrethrin Biosynthesis.

Pyrethrins from Tanacetum cinerariifolium are natural pesticides that exhibit high knockdown and killing activities against flying insects such as disease-spreading mosquitoes. Despite the increasing demand for pyrethrins, the mechanism of pyrethrin biosynthesis remains elusive. To elucidate it, we for the first time created pyrethrin mimetic phosphonates targeting the GDSL esterase/lipase (GELP or TcGLIP) underpinning pyrethrin biosynthesis. The compounds were synthesized by reacting mono-alkyl or mono-benzyl-substituted phosphonic dichloride with pyrethrolone, the alcohol moiety of pyrethrin I and II, and then p-nitrophenol. n-Pentyl (C5) and n-octyl (C8)-substituted compounds were the most potent of the (S)p,(S)c, and (R)p,(S)c diastereomers, respectively. The (S)-pyrethrolonyl group is more effective than the (R)-pyrethrolonyl group in blocking TcGLIP, consistent with the features predicted by TcGLIP models complexed with the (S)p,(S)c-C5 and (R)p,(S)c-C8 probes. The (S)p,(S)c-C5 compound suppressed pyrethrin production in T. cinerariifolium, demonstrating potential as a chemical tool for unravelling pyrethrin biosynthesis.

Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dß1, and Dß2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dß3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dß3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

Effects of cofactors RIC-3, TMX3 and UNC-50, together with distinct subunit ratios on the agonist actions of imidacloprid on Drosophila melanogaster Dα1/Dß1 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

Insect nicotinic acetylcholine receptors (nAChRs) require cofactors for functional heterologous expression. A previous study revealed that TMX3 was crucial for the functional expression of Drosophila melanogaster Dα1/Dß1 nAChRs in Xenopus laevis oocytes, while UNC-50 and RIC-3 enhanced the acetylcholine (ACh)-induced responses of the nAChRs. However, it is unclear whether the coexpression of UNC-50 and RIC-3 with TMX3 and the subunit stoichiometry affect pharmacology of Dα1/Dß1 nAChRs when expressed in X. laevis oocytes. We have investigated the effects of coexpressing UNC-50 and RIC-3 with TMX3 as well as changing the subunit stoichiometry on the agonist activity of ACh and imidacloprid on the Dα1/Dß1 nAChRs. UNC-50 and RIC-3 hardly affected the agonist affinity of ACh and imidacloprid for the Dα1/Dß1 nAChRs formed by injecting into X. laevis oocytes with an equal amount mixture of the subunit cRNAs, but enhanced current amplitude of the ACh-induced response. Imidacloprid showed higher affinity for the Dß1 subunit-excess Dα1/Dß1 (Dα1/Dß1 = 1/5) nAChRs than the Dα1 subunit-excess Dα1/Dß1 (Dα1/Dß1 = 5/1) nAChRs, suggesting that imidacloprid prefers the Dα1-Dß1 orthosteric site over the Dα1-Dα1 orthosteric site.

TcGLIP GDSL Lipase Substrate Specificity Co-determines the Pyrethrin Composition in Tanacetum cinerariifolium.

Natural pesticides pyrethrins biosynthesized by Tanacetum cinrerariifolium are biodegradable and safer insecticides for pest insect control. TcGLIP, a GDSL lipase underpinning the ester bond formation in pyrethrins, exhibits high stereo-specificity for acyl-CoA and alcohol substrates. However, it is unknown how the enzyme recognizes the other structural features of the substrates and whether such specificity affects the product amount and composition in T. cinrerariifolium. We report here that the cysteamine moiety in (1R,3R)-chrysanthemoyl CoA and the conjugated diene moiety in (S)-pyrethrolone play key roles in the interactions with TcGLIP. CoA released from chrysanthemoyl CoA in the pyrethrin-forming reaction reduces the substrate affinity for TcGLIP by feedback inhibition. (S)-Pyrethrolone shows the highest catalytic efficiency for TcGLIP, followed by (S)-cinerolone and (S)-jasmololone, contributing, at least in part, to determine the pyrethrin compositions in T. cinerariifolium.

Competitive chrodrimanin B interactions with rat brain GABAA receptors revealed by radioligand binding assays.

Meroterpenoid compounds chrodrimanins produced by Talaromyces sp. YO-2 have been shown to act as competitive antagonists of silkworm larval GABAA receptors using electrophysiology, yet no further evidence has been provided to support such an action. We have investigated the actions of chrodrimanin B on rat brain GABAA receptors by binding assays with non-competitive ligand of GABAA receptors [3H]EBOB and competitive ligands [3H]gabazine and [3H]muscimol. Chrodrimanin B did not significantly affect the binding of [3H]EBOB while reducing the binding of [3H]gabazine and [3H]muscimol to the rat membrane preparations. Chrodrimanin B increased the dissociation constant Kd of [3H]gabazine and [3H]muscimol without significantly affecting the maximum binding, pointing to competitive interactions of chrodrimanin B with rat GABAA receptors in support of our previous observation that the compound acts as a competitive antagonist on the silkworm larval GABA receptor.

Chemical and biological studies of natural and synthetic products for the highly selective control of pest insect species.

Tanacetum cinerariifolium was known to produce pyrethrins, but the mechanism of pyrethrin biosynthesis was largely unclear. The author showed that the nonmevalonate and oxylipin pathways underlie biosynthesis of the acid and alcohol moieties, respectively, and a GDSL lipase joins the products of these pathways. A blend of the green leaf volatiles and (E)-ß-farnesene mediates the induction of wounding responses to neighboring intact conspecies by enhancing pyrethrin biosynthesis. Plants fight against herbivores underground as well as aboveground, and, in soy pulps, some fungi produce compounds selectively modulating ion channels in insect nervous system. The author proposed that indirect defense of plants occurs where microorganisms produce defense substances in the rhizosphere. Broad-spectrum pesticides, including neonicotinoids, may affect nontarget organisms. The author discovered cofactors enabling functional expression of insect nicotinic acetylcholine receptors (nAChRs). This led to understanding the mechanism of insect nAChR-neonicotinoid interactions, thus paving new avenues for controlling crop pests and disease vectors.

Identification of multiple odorant receptors essential for pyrethrum repellency in Drosophila melanogaster.

Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-ß-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.

A dual-target molecular mechanism of pyrethrum repellency against mosquitoes.

Pyrethrum extracts from flower heads of Chrysanthemum spp. have been used worldwide in insecticides and repellents. While the molecular mechanisms of its insecticidal action are known, the molecular basis of pyrethrum repellency remains a mystery. In this study, we find that the principal components of pyrethrum, pyrethrins, and a minor component, (E)-ß-farnesene (EBF), each activate a specific type of olfactory receptor neurons in Aedes aegypti mosquitoes. We identify Ae. aegypti odorant receptor 31 (AaOr31) as a cognate Or for EBF and find that Or31-mediated repellency is significantly synergized by pyrethrin-induced activation of voltage-gated sodium channels. Thus, pyrethrum exerts spatial repellency through a novel, dual-target mechanism. Elucidation of this two-target mechanism may have potential implications in the design and development of a new generation of synthetic repellents against major mosquito vectors of infectious diseases.

Robust functional expression of insect nicotinic acetylcholine receptors provides new insights into neonicotinoid actions and new opportunities for pest and vector control.

Neonicotinoids are selective modulators of insect nicotinic acetylcholine receptors (nAChRs). These widely deployed insecticides interact with the orthosteric sites of nAChRs, not only to activate nAChRs on their own, but also to block the desensitizing component of nAChR responses. To date recombinant vertebrate or insect/vertebrate hybrid nAChRs have been deployed to understand the mechanism of selectivity and diversity of neonicotinoid actions as well as to show that both α/α and α/non-α interfaces are involved in the interactions with neonicotinoids. However, many of the fine details of insecticide interactions with sites on nAChRs remain to be resolved. The breakthrough of functional expression of insect nAChRs allows such questions to be addressed, not only for neonicotinoids but for other insecticides targeting insect nAChRs. © 2020 Society of Chemical Industry.

A unique primary structure of RDL (resistant to dieldrin) confers resistance to GABA-gated chloride channel blockers in the two-spotted spider mite Tetranychus urticae Koch.

The primary structure of the second transmembrane (M2) segment of resistant to dieldrin (RDL), an ionotropic γ-aminobutyric acid receptor (GABAR) subunit, and the structure-function relationships in RDL are well conserved among insect species. An amino acid substitution at the 2' position in the M2 segment (Ala to Ser or Gly) confers resistance to non-competitive antagonists (NCAs) of GABARs. Here, a cDNA encoding RDL was cloned from the two-spotted spider mite Tetranychus urticae Koch. Unlike insect homologs, native TuRDL has His at the 2' position (H305) and Ile at 6' (I309) in the M2 segment and is insensitive to NCAs. Single and multiple mutations were introduced in the M2 segment of TuRDL, and the mutant proteins were expressed in Xenopus oocytes and examined for the restoration of sensitivity to NCAs. The sensitivity of a double mutant (H305A and I309T in the M2 segment) was greatly increased but was still considerably lower than that of insect RDLs. We therefore constructed chimeric RDLs consisting of TuRDL and Drosophila melanogaster RDL and examined their sensitivities to NCAs. The results show that the N-terminal region containing the Cys-loop as well as the M2 segment confers functional specificity; thus, our current understanding of the mechanism underlying NCA binding to GABARs requires reappraisal.

Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions.

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.

Modulation by neonicotinoids of honeybee α1/chicken ß2 hybrid nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

Neonicotinoids targeting insect nicotinic acetylcholine (ACh) receptors (insect nAChRs) are used for crop protection, but there is a concern about adverse effects on pollinators such as honeybees (Apis mellifera). Thus, we investigated the agonist actions of neonicotinoids (imidacloprid, thiacloprid and clothianidin) on A. mellifera α1 (Amα1)/chicken ß2 hybrid nAChRs in Xenopus laevis oocytes according to the subunit stoichiometry of (Amα1)3(ß2)2 and (Amα1)2(ß2)3 using voltage-clamp electrophysiology. ACh activated (Amα1)3(ß2)2 and (Amα1)2(ß2)3 nAChRs with similar current amplitude. We investigated the agonist activity of imidacloprid, thiacloprid and clothianidin for the two hybrid nAChRs and found that: 1) imidacloprid showed higher affinity than clothianidin, whereas clothianidin showed higher efficacy than imidacloprid for the nAChRs; 2) Thiacloprid showed the highest agonist affinity and the lowest efficacy for the nAChRs. The Amα1/ß2 subunit ratio influenced the efficacy of imidacloprid and thiacloprid, but hardly affected that of clothianidin. Hydrogen bond formation by the NH group in clothianidin with the main chain carbonyl of the loop B may account, at least in part, for the unique agonist actions of clothianidin on the hybrid nAChRs tested.

The mechanism of loop C-neonicotinoid interactions at insect nicotinic acetylcholine receptor α1 subunit predicts resistance emergence in pests.

Neonicotinoids selectively modulate insect nicotinic acetylcholine receptors (insect nAChRs). Studies have shown that serine with ability to form a hydrogen bond in loop C of some insect nAChR α subunits and glutamate with a negative charge at the corresponding position in vertebrate nAChRs may contribute to enhancing and reducing the neonicotinoid actions, respectively. However, there is no clear evidence what loop C properties underpin the target site actions of neonicotinoids. Thus, we have investigated the effects of S221A and S221Q mutations in loop C of the Drosophila melanogaster Dα1 subunit on the agonist activity of imidacloprid and thiacloprid for Dα1/chicken ß2 nAChRs expressed in Xenopus laevis oocytes. The S221A mutation hardly affected either the affinity or efficacy for ACh and imidacloprid, whereas it only slightly reduced the efficacy for thiacloprid on the nAChRs with a higher composition ratio of ß2 to Dα1 subunits. The S221Q mutation markedly reduced the efficacy of the neonicotinoids for the nAChRs with a higher composition of the ß2 subunit lacking basic residues critical for binding neonicotinoids. Hence, we predict the possibility of enhanced neonicotinoid resistance in pest insect species by a mutation of the serine when it occurs in the R81T resistant populations lacking the basic residue in loop D of the ß1 subunit.

Jasmonic acid is not a biosynthetic intermediate to produce the pyrethrolone moiety in pyrethrin II.

Pyrethrum (Tanacetum cinerariifolium) produces insecticidal compounds known as pyrethrins. Pyrethrins are esters; the acid moiety is either trans-chrysanthemic acid or pyrethric acid and the alcohol moiety of pyrethrins is either pyrethrolone, cinerolone, or jasmolone. It was generally accepted that cis-jasmone was biosynthetic intermediate to produce the alcohol moieties of pyrethrin, and the biosynthetic origin of the cis-jasmone was postulated to be jasmonic acid. However, there was no direct evidence to prove this hypothesis. In order to uncover the origin of pyrethrolone moiety in pyrethrin II, feeding experiments were performed employing deuterium- and 13C-labeled compounds as substrates, and the expected labeled compounds were analyzed using UPLC MS/MS system. It was found that the pyrethrolone moiety in pyrethrin II was derived from 12-oxo-phytodienoic acid (OPDA), iso-OPDA and cis-jasmone but not from methyl jasmonate and 3-oxo-2-(2'-[Z]-pentenyl)-cyclopentane-1-hexanoic acid. The results supported that the biosynthesis of the pyrethrolone moiety in pyrethrin II partially used part of the jasmonic acid biosynthetic pathway, but not whole.

Metabolome Analysis Identified Okaramines in the Soybean Rhizosphere as a Legacy of Hairy Vetch.

Inter-organismal communications below ground, such as plant-microbe interactions in the rhizosphere, affect plant growth. Metabolites are shown to play important roles in biological communication, but there still remain a large number of metabolites in soil to be uncovered. Metabolomics, a technique for the comprehensive analysis of metabolites in samples, may uncover the molecules that intermediate these interactions. We conducted a multivariate analysis using liquid chromatography (LC)-mass spectrometry (MS)-based untargeted metabolomics in several soil samples and also targeted metabolome analysis for the identification of the candidate compounds in soil. We identified okaramine A, B, and C in the rhizosphere soil of hairy vetch. Okaramines are indole alkaloids first identified in soybean pulp (okara) inoculated with Penicillium simplicissimum AK-40 and are insecticidal. Okaramine B was detected in the rhizosphere from an open field growing hairy vetch. Okaramine B was also detected in both bulk and rhizosphere soils of soybean grown following hairy vetch, but not detected in soils of soybean without hairy vetch growth. These results suggested that okaramines might be involved in indirect defense of plants against insects. To our knowledge, this is the first report of okaramines in the natural environment. Untargeted and targeted metabolomics would be useful to uncover the chemistry of the rhizosphere.

Neonicotinoid Insecticides: Molecular Targets, Resistance, and Toxicity.

Neonicotinoids have been used to protect crops and animals from insect pests since the 1990s, but there are concerns regarding their adverse effects on nontarget organisms, notably on bees. Enhanced resistance to neonicotinoids in pests is becoming well documented. We address the current understanding of neonicotinoid target site interactions, selectivity, and metabolism not only in pests but also in beneficial insects such as bees. The findings are relevant to the management of both neonicotinoids and the new generation of pesticides targeting insect nicotinic acetylcholine receptors.