Identification of highly active compounds from insecticidal plant Oroxylum indicum L. (Vent.) and the induction of apoptosis by lapachol on Sf9 cells.

The extraction of biopesticides from plants has become a promising field for agricultural development. To explore a high-efficiency and viable method for the screening of plant compounds with insecticidal activity, we screened for active ingredients in the insecticidal plant, Oroxylum indicum L. Vent, using Sf9 cells. A CCK-8 cytotoxicity assay kit was used for high-throughput screening of 34 compounds contained in O. indicum. The apoptosis-inducing effect of the highly cytotoxic compound on Sf9 cells was investigated by morphological characterization using inverted microscopy, caspase-3 activity assay, and DNA gel electrophoresis. Finally, the biological activity of compounds against aphids was evaluated using the leaf-pest dipping methods and leaf dipping methods. Results showed that among the main compounds identified, lapachol, chrysin, and baicalein had good proliferation inhibitory effects on Sf9 cells, with their recorded IC50 being 11.53 mg/L, 38.39 mg/L, and 42.10 mg/L, respectively. Moreover, the IC50 value of lapachol was lower than the control insecticides rotenone (18.03 mg/L) and fipronil (21.04 mg/L). Apoptosis assay further showed that lapachol promoted the production of caspase-3 and led to DNA fragmentation in Sf9 cells. Lapachol showed high biological activity against Aphis gossypii, Sitobion avenae, and Semiaphis heraclei, with its recorded LC50 being 104.40, 101.80, and 110.29 mg/L, respectively, which were comparable to the activity of the control insecticide rotenone. High-throughput screening of active ingredients in the insecticidal plant O. indicum using Sf9 cells is feasible, and the identification of lapachol as the main aphidicidal active substance is valuable for further study.

Does Differential Receptor Distribution Underlie Variable Responses to a Neuropeptide in the Lobster Cardiac System?

Central pattern generators produce rhythmic behaviors independently of sensory input; however, their outputs can be modulated by neuropeptides, thereby allowing for functional flexibility. We investigated the effects of C-type allatostatins (AST-C) on the cardiac ganglion (CG), which is the central pattern generator that controls the heart of the American lobster, Homarus americanus, to identify the biological mechanism underlying the significant variability in individual responses to AST-C. We proposed that the presence of multiple receptors, and thus differential receptor distribution, was at least partly responsible for this observed variability. Using transcriptome mining and PCR-based cloning, we identified four AST-C receptors (ASTCRs) in the CG; we then characterized their cellular localization, binding potential, and functional activation. Only two of the four receptors, ASTCR1 and ASTCR2, were fully functional GPCRs that targeted to the cell surface and were activated by AST-C peptides in our insect cell expression system. All four, however, were amplified from CG cDNAs. Following the confirmation of ASTCR expression, we used physiological and bioinformatic techniques to correlate receptor expression with cardiac responses to AST-C across individuals. Expression of ASTCR1 in the CG showed a negative correlation with increasing contraction amplitude in response to AST-C perfusion through the lobster heart, suggesting that the differential expression of ASTCRs within the CG is partly responsible for the specific physiological response to AST-C exhibited by a given individual lobster.

A Commercial Extract of Cyanotis arachnoidea Roots as a Source of Unusual Ecdysteroid Derivatives with Insect Hormone Receptor Binding Activity.

Ecdysteroids act as molting hormones in insects and as nonhormonal anabolic agents and adaptogens in mammals. A wide range of ecdysteroid-containing herbal extracts are available worldwide as food supplements. The aim of this work was to study such an extract as a possible industrial source of new bioactive ecdysteroids. A large-scale chromatographic isolation was performed from an extract of Cyanotis arachnoidea roots. Ten ecdysteroids (1-10) including eight new compounds were isolated and characterized by extensive nuclear magnetic resonance studies. Highly unusual structures were identified, including a H-14ß (1, 2, 4, and 10) moiety, among which a 14ß(H)17ß(H) phytosteroid (1) is reported for the first time. Compounds with an intact side chain (4-10) and 11 other natural or semisynthetic ecdysteroids (11-21) were tested for insect ecdysteroid receptor (EcR) binding activity. Two new compounds, i.e., 14-deoxydacryhainansterone (5) and 22-oxodacryhainansterone (6), showed strong EcR binding activity (IC50 = 41.7 and 380 nM, respectively). Six compounds were identified as EcR agonists and another two as antagonists using a transgenic ecdysteroid reporter gene assay. The present results demonstrate that commercial C. arachnoidea extracts are rich in new, unusual bioactive ecdysteroids. Because of the lack of an authentic plant material, the truly biosynthetic or artifactual nature of these compounds cannot be confirmed.

Diacylglycerol acyltransferase inhibitory new meroterpenes from the seeds of Psoralea corylifolia, and their structure-activity relationship study.

Five new meroterpenes, 12α-Psoracorylifol F (1), 7ß,8α-hydroxy-12ß-Psoracorylifol F (2), 8-ketone-Cyclobakuchiol C (3), 7α,8ß-hydroxy-12ß-Cyclobakuchiol C (4) and 8α-hydroxy-Cyclobakuchiol C (5) together with six known compounds (6-11) were isolated from seeds of Psoralea corylifolia, and their structures were elucidated on the basis of spectroscopic and physicochemical analyses. All the isolates were evaluated for in vitro inhibitory activity against DGAT1/2. Among them, compounds 1-6 were found to exhibit selective inhibitory activity on DGAT1 with IC50 values ranging from 61.5 ± 1.1 to 89.1 ± 1.2 µM.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation.

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.

The inducible amphisome isolates viral hemagglutinin and defends against influenza A virus infection.

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection.

Characterization of Terpene Synthase from Tea Green Leafhopper Being Involved in Formation of Geraniol in Tea (Camellia sinensis) Leaves and Potential Effect of Geraniol on Insect-Derived Endobacteria.

When insects attack plants, insect-derived elicitors and mechanical damage induce the formation and emission of plant volatiles that have important ecological functions and flavor properties. These events have mainly been studied in model plants, rather than crop plants. Our study showed that tea green leafhopper (Empoasca (Matsumurasca) onukii Matsuda), a major pest infesting tea attack significantly induced the emission of geraniol from tea leaves, but did not affect the crude enzyme activity of geraniol synthase in tea leaves. An enzyme extract of E. (M.) onukii specifically produced geraniol from geraniol diphosphate. Furthermore, a terpene synthase (EoTPS) was isolated from E. (M.) onukii. This terpene synthase was able to convert geraniol diphosphate to geraniol in vitro. In addition, geraniol had in vitro ability to inhibit the growth of Acinetobacter johnsonii that is endobacterial isolated from E. (M.) onukii. This information illustrates that elicitors from piercing-sucking insects can induce the formation of volatiles from crop plants and advances our understanding of the roles of plant volatiles in the interaction among crops-insects-microorganisms.

Neochamaejasmin A extracted from Stellera chamaejasme L. induces apoptosis involving mitochondrial dysfunction and oxidative stress in Sf9 cells.

To explore the toxicity mechanisms of neochamaejasmin A (NCA), extracted from Stellera chamaejasme L., we first evaluated its cytotoxicity on the Spodoptera frugiperda (Sf9) cell line. The results confirmed that NCA inhibited Sf9 cell survival in both a dose- and time-dependent manner. Then, intracellular biochemical assays showed that NCA induced apoptosis in Sf9 cells. Evidence of apoptosis was confirmed by morphological changes and the activation of caspases-3/9. We also observed that NCA induced apoptosis via mitochondrial-dependent intrinsic apoptotic pathway by upregulating cytochrome c and proapoptotic protein (Bax) and downregulating the mitochondrial membrane potential (MMP) and antiapoptotic protein (Bcl-2). Moreover, we found a dose-dependent increase in reactive oxygen species (ROS), accumulation of lipid peroxidation product and an inactivation of the antioxidant enzymes in treated cells. Additionally, the cleavage of PARP and G2/M arrest were also detected in Sf9 cells exposed to NCA. These findings provide critical information that NCA effectively induced apoptosis in Sf9 cells through mitochondrial pathways.

PEGylated cyanine dye nanoparticles as photothermal agents for mosquito and cancer cell control.

Conversion of light energy to heat via photothermal conversion agents (PTCAs) is of great interest and has potential applications. Here, we described a heptamethine cyanine (Cy7) dye nanoparticles (Cy7-PEG NPs) prepared from heptamethine cyanine and poly(ethylene glycol) (PEG400) via a simple solvothermal process as novel PTCA. Cy7-PEG NPs have absorption maximum at about 808 nm and good photothermal conversion ability. Upon irradiation, Cy7-PEG NPs can effectively kill living mosquito larva (Aedes albopictus) through heat generation. Furthermore, Cy7-PEG NPs have excellent phototoxic activity to Sf9, HeLa and MCF-7 cells. Our results indicated that Cy7-PEG NPs can be used as controlling agent for mosquito larvae and cancer cells.

Acetyl-CoA carboxylase inhibition regulates microtubule dynamics and intracellular transport in cystic fibrosis epithelial cells.

The use of high-dose ibuprofen as an anti-inflammatory therapy in cystic fibrosis (CF) has been shown to be an effective intervention although use is limited due to potential adverse events. Identifying the mechanism of ibuprofen efficacy would aid in the development of new therapies that avoid these adverse events. Previous findings demonstrated that ibuprofen treatment restores the regulation of microtubule dynamics in CF epithelial cells through a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism. The goal of this study is to define the AMPK pathway that leads to microtubule regulation. Here, it is identified that inhibition of acetyl-CoA carboxylase (ACC) is the key step in mediating the AMPK effect. ACC inhibition with 5-(tetradecyloxy)-2-furoic acid (TOFA) increases microtubule reformation rates in cultured and primary CF epithelial cells to wild-type (WT) rates. TOFA treatment also restores microtubule-dependent distribution of cholesterol and Rab7-positive organelles, as well as reduces expression of the proinflammatory signaling molecule RhoA to WT levels. ACC activation with citrate replicates these CF phenotypes in WT cells further supporting the role of AMPK signaling through ACC as a key mediator in CF cell signaling. It is concluded that ACC inhibition is the key step in the efficacy of AMPK activation at the cellular level and could represent a novel site of therapeutic intervention to address inflammation in CF.

Astratides: Insulin-Modulating, Insecticidal, and Antifungal Cysteine-Rich Peptides from Astragalus membranaceus.

Astragalus membranaceus root, Huang Qi in Chinese, is a popular medicinal herb traditionally used to regulate blood glucose. Herein, the identification and characterization of two families of cysteine-rich peptides (CRPs), designated α- and ß-astratides, from A. membranaceus roots are reported. Proteomic analysis showed that α-astratide aM1 and ß-astratide bM1 belong to two distinct CRP families. The six-cysteine-containing and proline-rich α-astratide aM1 displayed high sequence identity to Pea Albumin 1 Subunit b (PA1b), while the eight-cysteine-containing ß-astratide bM1 showed sequence similarity to plant defensins. An antifungal assay revealed that bM1 possessed potent antifungal activity. In contrast, aM1 showed a cytotoxic effect against insect Sf9 cells. More importantly, aM1 decreased insulin secretion in mouse pancreatic ß cells, suggesting it could interfere in glucose homeostasis, which accounts for the adaptogenic property of A. membranaceus. Phylogenetic clustering analysis suggested that the proline-rich aM1 is a putative prolyl oligopeptidase inhibitor and belongs to a novel subfamily of PA1b-like peptides, while bM1 belongs to a new subfamily of plant defensins. Together, the study reveals that astratides are multifunctional CRPs in plants, which expand the existing library of PA1b-like peptides and plant defensins and further our understanding of their roles in host-defense system and leads as peptidyl therapeutics.

Synthesis, characterization and in vitro biological evaluation of two matrine derivatives.

Matrine is a traditional Chinese medicine and botanical pesticide with broad biological activities, including pharmacological and agricultural activities. In present work, two matrine derivatives have been successfully synthesized via introducing indole and cyclohexylamino to 13 position of matrine, respectively, with sophocarpine as starting material, and structurally characterized via infrared spectroscopy(IR), MS, 1 H NMR, 13 C NMR and X-ray crystal diffraction. The results of the in vitro biological activity tests showed that these two matrine derivatives exhibited even better activities against human cancer cells Hela229 and insect cell line Sf9 from Spodoptera frugiperda (J. E. Smith) than that of parent matrine, suggesting that the heterocyclic or cyclic group can dramatically increase the biological activity of matrine. It is worth to mention that 13-indole-matrine could possibly inhibit the growth of insect cells or human cancer cells by inducing cell apoptosis. The results of the present study provide useful information for further structural modifications of these compounds and for exploring new, potent anti-cancer agents and environment friendly pesticides.

Cholesterol Unbound RORγt Protein Enables a Sensitive Inverse Agonist Screening.

The retinoic acid-related orphan receptor gamma T (RORγt) plays an important role in Th17 cell proliferation and functionality. Thus, RORγt inverse agonists are thought to be potent therapeutic agents for Th17-mediated autoimmune diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, and psoriasis. Although RORγt has constitutive activity, it is recognized that the receptor is physiologically regulated by various cholesterol derivatives. In this study, we sought to identify RORγt inverse agonists through a high-throughput screening campaign. To this end, we compared an apo-RORγt protein from Escherichia coli and a cholesterol-bound RORγt protein from insect cells. The IC50 of the known RORγt inverse agonist TO901317 was significantly lower for the apoprotein than for the cholesterol-bound RORγt. Through high-throughput screening using a fluorescence-based cholesterol binding assay with the apoprotein, we identified compound 1 as a novel cholesterol-competitive RORγt inverse agonist. Compound 1 inhibited the RORγt-TopFluor cholesterol interaction, coactivator recruitment, and transcriptional activity of RORγt. Cell-based reporter gene assay demonstrated that compound 1 showed higher potency by lipid depletion treatment. Collectively, our findings indicate that eliminating cholesterol from the RORγt protein is suitable for sensitive high-throughput screening to identify RORγt inverse agonists.

Pyrethrum-extract induced autophagy in insect cells: A new target?

Pyrethrum extract (PY) is a natural insecticide that is extensively used across the world, and its insecticidal activity is attributed to the presence of six active esters known as pyrethrins. PY targets the nervous systems of insects by delaying the closure of voltage-gated sodium ion channels in the nerve cells. However, limited information is available regarding the toxicity and detailed mechanisms of PY activity. This study is aimed at understanding the toxicity effect and the underlying mechanisms of PY in cellular level, which have not yet been investigated on the non-nervous system of insects. Results of the MTT assay showed that the viability of Sf9 cells was inhibited by PY in a time- and concentration-dependent manner, and observation under a microscope revealed accumulation of intracellular vacuoles. Monodansylcadaverine staining analysis and transmission electron microscope images revealed typical autophagic morphological changes in PY-treated Sf9 cells. Autophagy-related proteins such as LC3, p62, and beclin-1 were detected using by Western blotting. Protein expression levels of LC3-II and beclin-1 were upregulated while that of p62 was markedly downregulated in a dose-dependent manner upon the PY treatment in Sf9 cells. In conclusion, these results indicate that PY could induce autophagy in the non-nervous system of insects which may contribute to its insecticidal mechanism.

Inhibition of Breast Cancer Resistance Protein and Multidrug Resistance Associated Protein 2 by Natural Compounds and Their Derivatives.

The food and dietary supplements we consume contain a wide variety of plant secondary metabolites and other compounds, which, like drugs, can be absorbed, metabolized, distributed, and excreted from the body. In the intestine, these compounds can interact with transport proteins such as the multidrug resistance associated protein 2 (MRP2, ABCC2) and the breast cancer resistance protein (BCRP, ABCG2) that regulate the absorption of drugs and other compounds. Inhibition of these transporters by dietary components could lead to increased exposure and adverse effects of concomitantly administered drugs. Therefore, we screened a library of 124 natural compounds and their derivatives using the vesicular transport assay to evaluate their inhibitory potential on MRP2 and BCRP. Of the library compounds, 36% were identified as BCRP inhibitors, whereas the number was only 3.2% for MRP2. BCRP inhibitors are described by higher molecular weight, number of rings, aromaticity, and LogD7.4 than noninhibitors. IC50 values were measured for six dual inhibitors, among which three novel inhibitors, gossypin, nordihydroguaiaretic acid, and octyl gallate, were identified. Our results confirm that flavonoids are avid inhibitors of BCRP, and flavones and flavonols appear to be important subclasses of flavonoids for this inhibition. The strong inhibition of BCRP transport by some compounds suggests that their presence at high levels in the diet could cause food-drug interactions, but this seems to be a minor cause of concern for MRP2.

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Using Bicistronic Baculovirus Expression Vector System to Screen the Compounds That Interfere with the Infection of Chikungunya Virus.

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever and has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. A bicistronic baculovirus expression system was utilized to co-express CHIKV structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium is mediated by the CHIKV E1 allowing it to identify chemicals that can prevent syncytium formation. The compounds characterized by this method could be anti-CHIKV drugs.

A sub-milligram-synthesis protocol for in vitro screening of HDAC11 inhibitors.

This work demonstrated the high efficiency of a sub-milligram-synthesis based medicinal chemistry method. Totally 72 compounds, consisting a tri-substituted pyrrolidine core, were prepared. Around 0.1mg of each compound was solid-phase synthesized. Based on the additive property of UV absorptions of unconjugated chromophores of a molecule, these compounds were quantified by UV measurement. A hit, whose IC50 value was 1.2µM in HDAC11 inhibition assays, highlights the applicability of the approach reported here in future optimization works.

ROLE OF SERUM AND ION CHANNEL BLOCK ON GROWTH AND HORMONALLY-INDUCED DIFFERENTIATION OF Spodoptera frugiperda (Sf21) INSECT CELLS.

A neuronal morphological phenotype can be induced in cultured Spodoptera frugiperda insect cells (Sf21) by supplementing serum-containing media with 20-hydroxyecdysone (20-HE) and/or insulin. In this study, the primary objectives were to determine any role of ion channels in mediating the morphological change in cells treated with 20-HE and insulin, and whether serum was required to observe this effect. Results showed serum-free media also induced growth of processes in Sf21 cells, but at a lower percentage than that found previously in cells bathed in serum-containing media. Veratridine, a sodium channel activator, increased cell survival when applied in combination with 20-HE to Sf21 cells, and the effect was blocked by tetrodotoxin (1 µM) a known sodium channel blocker. Cobalt, a calcium channel blocker, showed significant inhibition of cell process growth when applied in combination with both 20-HE and 20-HE plus veratridine. Cobalt also showed significant inhibition of cell process growth when applied in combination with insulin. Thus, some type of sodium channel, as well as a mechanism for transmembrane calcium ion movement, are apparently expressed in Sf21 cells and are involved in the differentiation process. These cell lines may be used in a wide variety of endeavors, including the screening of insecticides, as well as foster basic studies of neurodevelopment and ecdysone action.