Shisa reduces the sensitivity of homomeric RDL channel to GABA in the two-spotted spider mite, Tetranychus urticae Koch.

The γ-aminobutyric acid receptors (GABARs) mediate fast inhibitory transmission in central nervous system of insects and are important targets of insecticides. An auxiliary subunit, Shisa7, was identified in mammals as a single-passing transmembrane protein. However, the homology gene(s) of Shisa in invertebrates has not been reported to date. In the present study, a homolog Shisa gene was identified from the two-spotted spider mite, Tetranychus urticae Koch. Its open reading frame had 927 base pairs and encoded 308 amino acid residues, which has a typical Shisa domain at 13th-181st amino acid residues. According to the phylogenetic tree, the invertebrate Shisa was categorized apart with those of vertebrate, and TuShisa showed closest relationship with the Shisa9 of velvet mite, Dinothrombium tinctorium (L.). In the electrophysiological assay with two-electrode voltage clamp, the GABA-activated TuRDL channel was functionally formed in the Africa clawed frog Xenopus laevis (Daudin) oocytes (EC50 = 53.34 µM). No GABA-activated current could be observed in TuShisa-expressed oocytes, whereas TuShisa could reduce the sensitivity of TuRDL/TuShisa (mass ratio of 1: 4) channel to GABA. The homology structural models of TuRDL and TuShisa were built by the SWISS-MODEL server, their interaction was predicted using Z-DOCK and three predicted hydrogen bonds and interface residues were analysed by PyMOL. Meanwhile, the key interface residues of TuShisa affected the stability of complex were calculated by Discovery Studio 2019. In conclusion, the TuShisa, as the first reported invertebrate Shisa, was explored and functionally examined as the GABARs auxiliary subunit. Our findings provide a basis for research of invertebrate Shisa.

The Haemonchus contortus LGC-39 subunit is a novel subtype of an acetylcholine-gated chloride channel.

The nematode genome exhibits a vast array of Cys-loop receptors that are activated by a diverse set of neurotransmitters and anthelmintic drugs such as ivermectin and levamisole. While many Cys-loop receptors have been functionally and pharmacologically characterized, there remains a large subset of orphan receptors where the agonist remains unknown. We have identified an orphan Cys-loop receptor, LGC-39, from the parasitic nematode Haemonchus contortus that is a novel type of cholinergic-sensitive ligand-gated chloride channel. This receptor groups outside of the acetylcholine-gated chloride channel family, in the previously named GGR-1 (GABA/Glycine Receptor-1) group of Cys-loop receptors. We found that LGC-39 forms a functional homomeric receptor when expressed in Xenopus laevis oocytes and is activated by several cholinergic ligands including acetylcholine, methacholine and surprisingly, atropine with an EC50 for atropine on the low µM range. A homology model was generated which revealed some key features of the LGC-39 ligand-binding pocket that may explain some of the elements important for atropine recognition of the LGC-39 receptor. Overall these results suggest that the GGR-1 family (now called LGC-57) of Cys-loop receptors includes novel acetylcholine-gated chloride channel subtypes and may represent important future drug targets.

Solid-state NMR study of structural heterogeneity of the apo WT mouse TSPO reconstituted in liposomes.

In the last decades, ligand binding to human TSPO has been largely used in clinical neuroimaging, but little is known about the interaction mechanism. Protein conformational mobility plays a key role in the ligand recognition and both, ligand-free and ligand-bound structures, are mandatory for characterizing the molecular binding mechanism. In the absence of crystals for mammalian TSPO, we have exploited solid-state nuclear magnetic resonance (ssNMR) spectroscopy under magic-angle spinning (MAS) to study the apo form of recombinant mouse TSPO (mTSPO) reconstituted in lipids. This environment has been previously described to permit binding of its high-affinity drug ligand PK11195 and appears therefore favourable for the study of molecular dynamics. We have optimized the physical conditions to get the best resolution for MAS ssNMR spectra of the ligand-free mTSPO. We have compared and combined various ssNMR spectra to get dynamical information either for the lipids or for the mTSPO. Partial assignment of residue types suggests few agreements with the published solution NMR assignment of the PK11195-bound mTSPO in DPC detergent. Moreover, we were able to observe some lateral chains of aromatic residues that were not assigned in solution. 13C double-quantum NMR spectroscopy shows remarkable dynamics for ligand-free mTSPO in lipids which may have significant implications on the recognition of the ligand and/or other protein partners.

Effect of amphiphilic environment on the solution structure of mouse TSPO translocator protein.

The translocator protein (TSPO) is a ubiquitous transmembrane protein of great pharmacological interest thanks to its high affinity to many drug ligands. The only high-resolution 3D-structure known for mammalian TSPO was obtained by NMR for the mouse mTSPO in DPC detergent only in presence of the high-affinity PK 11195 ligand. An atomic structure of free-ligand mTSPO is still missing to better understand the interaction of ligands with mTSPO and their effects on the protein conformation. Here, we decipher the solution structures of the recombinant mTSPO without ligand both in (i) SDS, the detergent used to extract and purify the protein from E. coli inclusion bodies, and (ii) DPC, the detergent used to solve the PK 11195-binding mTSPO NMR structure. We report partially refolded and less flexible mTSPO helices in DPC compared to SDS. Besides, DPC stabilizes the tertiary structure of mTSPO, as shown by a higher intrinsic Trp fluorescence and changes in indole environment. We evaluate by SEC-MALLS that ∼135 SDS and ∼100 DPC molecules are bound to mTSPO. SEC-small-angle X-ray (SAXS) and neutron (SANS) scattering confirm a larger mTSPO-detergent complex in SDS than in DPC. Using the contrast-matching technique in SEC-SANS, we demonstrate that mTSPO conformation is more compact and less flexible in DPC than in SDS. Combining ab initio modeling with SANS, we confirm that mTSPO conformation is less elongated in DPC than in SDS. However, the free-ligand mTSPO envelope in DPC is not as compact as the PK 11195-binding protein NMR structure, the ligand stiffening the protein.

Characterization of adjacent charged residues near the agonist binding site of the nematode UNC-49 GABA receptor.

The UNC-49 receptor is a Cys-loop GABA receptor that is unique to the nematode phylum. The receptor differs from mammalian GABA receptors both in amino acid sequence and pharmacology which highlights its potential as a novel anthelmintic target. Sequence differences within and near the various ligand-binding loops of the nematode receptor suggest that there could be structural differences compared to mammalian receptors that result in different pharmacological and functional features. Here we investigated three residues in the UNC-49 receptor from the parasitic nematode Haemonchus contortus: K181, E183, and T230. Analysis of these residues was conducted via site-directed mutagenesis, electrophysiology, MD simulations, and mutant cycling analysis. In the UNC-49 receptor, E183 lies in close proximity to K181 where together they appear to play a role in GABA sensitivity and pharmacology, possibly interacting via an ionic bond. While the introduction of single alanine residues at each position separately had a negative impact on GABA EC50, the double alanine mutant (K181A/E183A) exhibited wildtype-level GABA EC50 and some differences in pharmacology. Overall, this study has revealed a potentially novel role for these two residues in nematode UNC-49 GABA receptors that could aid in understanding their function.

Identification of the probable structure of the sAPPα-GABABR1a complex and theoretical solutions for such cases.

Amyloid precursor protein (APP) is the core of the pathogenesis of Alzheimer's disease (AD). Existing studies have shown that the soluble secreted APP (sAPPα) fragment obtained from the hydrolysis of APP by α-secretase has a synaptic function. Thereinto, a nine-residue fragment (APP9mer) of the extension domain region of sAPPα can bind directly and selectively to the N-terminal sushi1 domain (SD1) of the γ-aminobutyric acid type B receptor subunit 1a (GABABR1a) protein, which can influence synaptic transmission and plasticity by changing the GABABR1a conformation. APP9mer is a highly flexible, disordered region, and as such it is difficult to experimentally determine the optimal APPmer-SD1 binding complex. In this study we constructed two types of APP9mer-SD1 complexes through molecular docking and molecular dynamics simulation, aiming to explore the recognition function and mechanism of the specific binding of APP9mer with SD1, from which the most probable APPmer-SD1 model conformation is predicted. All the data from the analyses of RMSD, RMSF, PCA, DCCM and MM/PBSA binding energy as well as comparison with the experimental dissociation constant Kd suggest that 2NC is the most likely conformation to restore the crystal structure of the experimental APP9mer-SD1 complex. Of note, the key recognition residues of APP9mer are D24, D25, D27, W29 and W30, which mainly act on the 9-45 residue domain of SD1 (consisting of two loops and three short ß-chains at the N-terminus of SD1). The mini-model with key residues identified establishes the molecular basis with deep insight into the interaction between APP and GABABR1a and provides a target for the development of therapeutic strategies for modulating GABABR1a-specific signaling in neurological and psychiatric disorders. More importantly, the study offers a theoretical solution for how to determine a biomolecular structure with a highly flexible, disordered fragment embedded within. The flexible fragment involved in a protein structure has to be deserted usually during the structural determination with experimental methods (e.g. X-ray crystallography, etc.).

The Repertoire of Small-Molecule PET Probes for Neuroinflammation Imaging: Challenges and Opportunities beyond TSPO.

Neuroinflammation is an adaptive response of the central nervous system to diverse potentially injurious stimuli, which is closely associated with neurodegeneration and typically characterized by activation of microglia and astrocytes. As a noninvasive and translational molecular imaging tool, positron emission tomography (PET) could provide a better understanding of neuroinflammation and its role in neurodegenerative diseases. Ligands to translator protein (TSPO), a putative marker of neuroinflammation, have been the most commonly studied in this context, but they suffer from serious limitations. Herein we present a repertoire of different structural chemotypes and novel PET ligand design for classical and emerging neuroinflammatory targets beyond TSPO. We believe that this Perspective will support multidisciplinary collaborations in academic and industrial institutions working on neuroinflammation and facilitate the progress of neuroinflammation PET probe development for clinical use.

Muscimol Directly Activates the TREK-2 Channel Expressed in GABAergic Neurons through Its N-Terminus.

The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons.

Neuroactive Type-A γ-Aminobutyric Acid Receptor Allosteric Modulator Steroids from the Hypobranchial Gland of Marine Mollusk, Conus geographus.

In a program to identify pain treatments with low addiction potential, we isolated five steroids, conosteroids A-E (1-5), from the hypobranchial gland of the mollusk Conus geographus. Compounds 1-5 were active in a mouse dorsal root ganglion (DRG) assay that suggested that they might be analgesic. A synthetic analogue 6 was used for a detailed pharmacological study. Compound 6 significantly increased the pain threshold in mice in the hot-plate test at 2 and 50 mg/kg. Compound 6 at 500 nM antagonizes type-A γ-aminobutyric acid receptors (GABAARs). In a patch-clamp experiment, out of the six subunit combinations tested, 6 exhibited subtype selectivity, most strongly antagonizing α1ß1γ2 and α4ß3γ2 receptors (IC50 1.5 and 1.0 µM, respectively). Although the structures of 1-6 differ from those of known neuroactive steroids, they are cell-type-selective modulators of GABAARs, expanding the known chemical space of neuroactive steroids.

Mechanistic evaluation of a novel cyclohexenone derivative's functionality against nociception and inflammation: An in-vitro, in-vivo and in-silico approach.

The synthesis of a novel cyclohexanone derivative (CHD; Ethyl 6-(4-metohxyphenyl)-2-oxo-4-phenylcyclohexe-3-enecarboxylate) was described and the subsequent aim was to perform an in vitro, in vivo and in silico pharmacological evaluation as a putative anti-nociceptive and anti-inflammatory agent in mice. Initial in vitro studies revealed that CHD inhibited both cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes and it also reduced mRNA expression of COX-2 and the pro-inflammatory cytokines TNF-α and IL-1ß. It was then shown that CHD dose dependently inhibited chemically induced tonic nociception in the abdominal constriction assay and also phasic thermal nociception (i.e. anti-nociception) in the hot plate and tail immersion tests in comparison with aspirin and tramadol respectively. The thermal test outcomes indicated a possible moderate centrally mediated anti-nociception which, in the case of the hot plate test, was pentylenetetrazole (PTZ) and naloxone reversible, implicating GABAergic and opioidergic mechanisms. CHD was also effective against both the neurogenic and inflammatory mediator phases induced in the formalin test and it also disclosed anti-inflammatory activity against the phlogistic agents, carrageenan, serotonin, histamine and xylene compared with standard drugs in edema volume tests. In silico studies indicated that CHD possessed preferential affinity for GABAA, opioid and COX-2 target sites and this was supported by molecular dynamic simulations where computation of free energy of binding also favored the formation of stable complexes with these sites. These findings suggest that CHD has prospective anti-nociceptive and anti-inflammatory properties, probably mediated through GABAergic and opioidergic interactions supplemented by COX-2 and 5-LOX enzyme inhibition in addition to reducing pro-inflammatory cytokine expression. CHD may therefore possess potentially beneficial therapeutic effectiveness in the management of inflammation and pain.

5-O-methylcneorumchromone K Exerts Antinociceptive Effects in Mice via Interaction with GABAA Receptors.

The proper pharmacological control of pain is a continuous challenge for patients and health care providers. Even the most widely used medications for pain treatment are still ineffective or unsafe for some patients, especially for those who suffer from chronic pain. Substances containing the chromone scaffold have shown a variety of biological activities, including analgesic effects. This work presents for the first time the centrally mediated antinociceptive activity of 5-O-methylcneorumchromone K (5-CK). Cold plate and tail flick tests in mice showed that the 5-CK-induced antinociception was dose-dependent, longer-lasting, and more efficacious than that induced by morphine. The 5-CK-induced antinociception was not reversed by the opioid antagonist naloxone. Topological descriptors (fingerprints) were employed to narrow the antagonist selection to further investigate 5-CK's mechanism of action. Next, based on the results of fingerprints analysis, functional antagonist assays were conducted on nociceptive tests. The effect of 5-CK was completely reversed in both cold plate and tail-flick tests by GABAA receptor antagonist bicuculline, but not by atropine or glibenclamide. Molecular docking studies suggest that 5-CK binds to the orthosteric binding site, with a similar binding profile to that observed for bicuculline and GABA. These results evidence that 5-CK has a centrally mediated antinociceptive effect, probably involving the activation of GABAergic pathways.

Direct and specific binding of cholesterol to the mitochondrial translocator protein (TSPO) using PhotoClick cholesterol analogue.

The translocator protein (TSPO) is a five-helix transmembrane protein localized to the outer mitochondria membrane. Radioligand binding assays and chemical crosslinking showed TSPO to be a high affinity cholesterol-binding protein. In this report, we show that TSPO in mitochondrial fractions from MA-10 mouse tumour Leydig cells can interact directly and competitively with the clickable photoreactive cholesterol analogue. PhotoClick cholesterol showed saturable photoaffinity labelling of TSPO that could be specifically immunoprecipitated with anti-TSPO antibody, following the click reaction with the fluorescent-azide probe, tetramethylrhodamine (TAMRA)-azide. Moreover, excess cholesterol reduced the photolabelling of both total mitochondrial proteins and TSPO. Together, the results of this study demonstrated direct binding of PhotoClick cholesterol to TSPO and that this interaction occurs at physiologically relevant site(s).

The Interplay of Cholesterol and Ligand Binding in hTSPO from Classical Molecular Dynamics Simulations.

The translocator protein (TSPO) is a 18kDa transmembrane protein, ubiquitously present in human mitochondria. It is overexpressed in tumor cells and at the sites of neuroinflammation, thus representing an important biomarker, as well as a promising drug target. In mammalian TSPO, there are cholesterol-binding motifs, as well as a binding cavity able to accommodate different chemical compounds. Given the lack of structural information for the human protein, we built a model of human (h) TSPO in the apo state and in complex with PK11195, a molecule routinely used in positron emission tomography (PET) for imaging of neuroinflammatory sites. To better understand the interactions of PK11195 and cholesterol with this pharmacologically relevant protein, we ran molecular dynamics simulations of the apo and holo proteins embedded in a model membrane. We found that: (i) PK11195 stabilizes hTSPO structural fold; (ii) PK11195 might enter in the binding site through transmembrane helices I and II of hTSPO; (iii) PK11195 reduces the frequency of cholesterol binding to the lower, N-terminal part of hTSPO in the inner membrane leaflet, while this impact is less pronounced for the upper, C-terminal part in the outer membrane leaflet, where the ligand binding site is located; (iv) very interestingly, cholesterol most frequently binds simultaneously to the so-called CRAC and CARC regions in TM V in the free form (residues L150-X-Y152-X(3)-R156 and R135-X(2)-Y138-X(2)-L141, respectively). However, when the protein is in complex with PK11195, cholesterol binds equally frequently to the CRAC-resembling motif that we observed in TM I (residues L17-X(2)-F20-X(3)-R24) and to CRAC in TM V. We expect that the CRAC-like motif in TM I will be of interest in future experimental investigations. Thus, our MD simulations provide insight into the structural features of hTSPO and the previously unknown interplay between PK11195 and cholesterol interactions with this pharmacologically relevant protein.

Chiral Pd-Catalyzed Enantioselective Syntheses of Various N-C Axially Chiral Compounds and Their Synthetic Applications.

Biaryl atropisomers are key structural components in chiral ligands, chiral functional materials, natural products, and bioactive compounds, and their asymmetric syntheses have been reported by many groups. In contrast, although the scientific community has long been aware of atropisomers due to rotational restriction around N-C bonds, they have attracted scant attention and have remained an unexplored research area. In particular, their catalytic asymmetric synthesis and the synthetic applications were unknown until recently. This Account describes studies conducted by our group on the catalytic enantioselective syntheses of N-C axially chiral compounds and their applications in asymmetric reactions.In the presence of a chiral Pd catalyst, the reactions of achiral secondary ortho-tert-butylanilides with 4-iodonitrobenzene proceeded in a highly enantioselective manner (up to 96% ee), affording N-C axially chiral N-arylated ortho-tert-butylanilides in good yields. The application of the present chiral Pd-catalyzed N-arylation reaction to an intramolecular version gave N-C axially chiral lactams with high optical purity (up to 98% ee). These reactions were the first highly enantioselective syntheses of N-C axially chiral compounds with a chiral catalyst. Since the publication of these reactions, N-C axially chiral compounds have been widely accepted as new target molecules for catalytic asymmetric reactions. Furthermore, chiral-Pd-catalyzed intramolecular N-arylations were applied to the enantioselective syntheses of N-C axially chiral quinoline-4-one and phenanthridin-6-one derivatives. We also succeeded in the enantioselective syntheses of various N-C axially chiral compounds using other chiral Pd-catalyzed reactions. That is, optically active N-C axially chiral N-(2-tert-butylphenyl)indoles, 3-(2-bromophenyl)quinazolin-4-ones, and N-(2-tert-butylphenyl)sulfonamides were obtained through chiral Pd-catalyzed 5-endo-hydroaminocyclization, monohydrodebromination (reductive asymmetric desymmetrization), and Tsuji-Trost N-allylation, respectively. The study of the catalytic asymmetric synthesis of axially chiral indoles has contributed to the development of not only N-C axially chiral chemistry but also the chemistry of axially chiral indoles. Subsequently, the catalytic asymmetric syntheses of various indole derivatives bearing a C-C chiral axis as well as an N-C chiral axis have been reported by many groups. Moreover, axially chiral quinazlolin-4-one derivatives, which were obtained through chiral Pd-catalyzed asymmetric desymmetrization, are pharmaceutically attractive compounds; for example, 2-methyl-3-(2-bromophenyl)quinazolin-4-one product is a mebroqualone possessing GABA agonist activity.Most of the N-C axially chiral products have satisfactory rotational stability for synthetic applications, and their synthetic utility was also demonstrated through application to chiral enolate chemistry. That is, the reaction of various alkyl halides with the enolate prepared from the optically active anilide, lactam, and quinazolinone products proceeded with high diastereoselectivity by asymmetric induction due to the N-C axial chirality.At the present time, N-C axially chiral chemistry has become a popular research area, especially in synthetic organic chemistry, and original papers on the catalytic asymmetric syntheses of various N-C axially chiral compounds and their synthetic applications have been published.

Rational approaches for the design of various GABA modulators and their clinical progression.

GABA (γ-amino butyric acid) is an important inhibitory neurotransmitter in the central nervous system. Attenuation of GABAergic neurotransmission plays an important role in the etiology of several neurological disorders including epilepsy, Alzheimer's disease, Huntington's chorea, migraine, Parkinson's disease, neuropathic pain, and depression. Increase in the GABAergic activity may be achieved through direct agonism at the GABAA receptors, inhibition of enzymatic breakdown of GABA, or by inhibition of the GABA transport proteins (GATs). These functionalities make GABA receptor modulators and GATs attractive drug targets in brain disorders associated with decreased GABA activity. There have been several reports of development of GABA modulators (GABA receptors, GABA transporters, and GABAergic enzyme inhibitors) in the past decade. Therefore, the focus of the present review is to provide an overview on various design strategies and synthetic approaches toward developing GABA modulators. Furthermore, mechanistic insights, structure-activity relationships, and molecular modeling inputs for the biologically active derivatives have also been discussed. Summary of the advances made over the past few years in the clinical translation and development of GABA receptor modulators is also provided. This compilation will be of great interest to the researchers working in the field of neuroscience. From the light of detailed literature, it can be concluded that numerous molecules have displayed significant results and their promising potential, clearly placing them ahead as potential future drug candidates.

Membrane-embedded TSPO: an NMR view.

Translocator Protein (18 kDa) (TSPO) is a mitochondrial transmembrane protein commonly used as a biomarker for neuroinflammation and is also a potential therapeutic target in neurodegenerative diseases. Despite intensive research efforts, the function of TSPO is still largely enigmatic. Deciphering TSPO structure in the native lipid environment is essential to gain insight into its cellular activities and to design improved diagnostic and therapeutic ligands. Here, we discuss the influence of lipid composition on the structure of mammalian TSPO embedded into lipid bilayers on the basis of solid-state NMR experiments. We further highlight that cholesterol can influence both the tertiary and quaternary TSPO structure and also influence TSPO localization in mitochondria-associated endoplasmic reticulum membranes.

Synthesis and Properties of Pentafluorosulfanyl Group (SF5)-Containing Meta-Diamide Insecticides.

Herein, we describe novel pentafluorosulfanyl (SF5) group-containing meta-diamide insecticides. For the facile preparation of the SF5-based compounds 4a-d, practical synthetic methods were applied. Among newly synthesized compounds, 3-benzamido-N-(2,6-dimethyl-4-(pentafluoro-λ6-sulfanyl)phenyl)-2-fluorobenzamide 4d showed (i) a high insecticidal activity, (ii) an excellent selectivity to insects, and (iii) good levels of water solubility and log P values. In this study, we demonstrated that the pentafluorosulfanyl moiety could serve as an attractive functionality for the discovery of a new scope of crop-protecting agents.

Design, Synthesis, and Insecticidal Activity of 5,5-Disubstituted 4,5-Dihydropyrazolo[1,5-a]quinazolines as Novel Antagonists of GABA Receptors.

To control the development of resistance to conventional insecticides acting as γ-aminobutyric acid (GABA) receptor antagonists (e.g., fipronil), new GABAergic 5,5-disubstituted 4,5-dihydropyrazolo[1,5-a]quinazolines were designed via a scaffold-hopping strategy and synthesized with a facile method. Among the 50 target compounds obtained, compounds 5a, 5b, 7a, and 7g showed excellent insecticidal activities against a susceptible strain of Plutella xylostella (LC50 values ranging from 1.03 to 1.44 µg/mL), which were superior to that of fipronil (LC50 = 3.02 µg/mL). Remarkably, the insecticidal activity of compound 5a was 64-fold better than that of fipronil against the field population of fipronil-resistant P. xylostella. Electrophysiological studies against the housefly GABA receptor heterologously expressed in Xenopus oocytes indicated that compound 5a could act as a potent GABA receptor antagonist, and IC50 was calculated to be 32.5 nM. Molecular docking showed that the binding poses of compound 5a with the housefly GABA receptor can be different compared to fipronil, which explains the effectiveness of compound 5a against fipronil-resistant insects. These findings have suggested compound 5a as a lead compound for a novel GABA receptor antagonist controlling field-resistant insects and provided a basis for further design, structural modification, and development of 4,5-dihydropyrazolo[1,5-a]quinazoline motifs as new insecticidal GABA receptor antagonists.

Exploring the Interaction Mechanism of Desmethyl-broflanilide in Insect GABA Receptors and Screening Potential Antagonists by In Silico Simulations.

Broflanilide, a novel insecticide, is classified as a negative allosteric modulator (NAM) of insect γ-aminobutyric acid (GABA) receptors (GABARs) as desmethyl-broflanilide (DMBF) allosterically inhibits the GABA-induced responses. The G277M mutation of the Drosophila melanogaster GABAR subunit has been reported to abolish the inhibitory activity of DMBF. The binding mode of DMBF in insect GABARs needs to be clarified to understand the underlying mechanism of this mutation and to develop novel, efficient NAMs of insect GABARs. Here, we found that a hydrogen bond formed between DMBF and G277 of the D. melanogaster GABAR model might be the key interaction for the antagonism of DMBF by in silico simulations. The volume increase induced by the G277M mutation blocks the entrance of the binding pocket, making it difficult for DMBF to enter the binding pocket and thereby decreasing its activity. The following virtual screening and bioassay results identified a novel NAM candidate of insect GABARs. Overall, we reported a possible binding mode of DMBF in insect GABARs and proposed the insensitivity mechanism of the G277M mutant GABAR to DMBF using molecular simulations. The identified NAM candidates might provide more alternatives or potentials for the design of GABAR-targeting insecticides.