ZIKV Inhibitors Based on Pyrazolo[3,4-d]pyridazine-7-one Core: Rational Design, In Vitro Evaluation, and Theoretical Studies.

The Zika virus (ZIKV) is believed to cause birth defects, and no anti-ZIKV drugs have been approved by medical organizations to date. Starting from antimicrobial lead compounds with a pyrazolo[3,4-d]pyridazine-7-one scaffold, we synthesized 16 derivatives and screened their ability to interfere with ZIKV infection utilizing a cell-based phenotypic assay. Of these, five compounds showed significant inhibition of ZIKV with a selective index value greater than 4.6. In particular, compound 9b showed the best anti-ZIKV activity with a selectivity index of 22.4 (half-maximal effective concentration = 25.6 µM and 50% cytotoxic concentration = 572.4 µM). Through the brine shrimp lethality bioassay, 9b, 10b, 12, 17a, and 19a showed median lethal dose values in a range of 87.2-100.3 µg/mL. Compound 9b was also targeted to the NS2B-NS3 protease of ZIKV using molecular docking protocols, in which it acted as a noncompetitive inhibitor and strongly bound to five key amino acids (His51, Asp75, Ser135, Ala132, Tyr161). Utilizing the pharmacophore model of 9b, the top 20 hits were identified as prospective inhibitors of NS2B-NS3 protease, and six of them were confirmed for their stability with the protease via redocking and molecular dynamics simulations.

The insecticidal capacity of ethanol extract from Cascabela peruviana (L.) Lippold against fruit fly.

Cascabela peruviana (L.) Lippold (C. peruviana) has been extensively used for its antifungal and antibacterial properties. However, its role in anti-insect is still under investigation. To investigate the ability of the ethanol extract of C. peruviana against insects, we used the fruit fly (Drosophila melanogaster) as a model to gain more insight into the toxic effects of this extract. We found that the ethanol extract from the stem and leaves of C. peruviana was effective against insects and contained polyphenol and flavonoid compounds. C. peruviana could induce mortality of 2nd-instar larvae and reduce growth and reproduction of fruit flies. Interestingly, the toxicity of C. peruviana extract has been remained to affect the development of the next generation of fruit flies. The locomotor activity and feeding ability of the F1 generation of this insect were significantly reduced by C. peruviana. In addition, flavonoids and polyphenols, as well as saponins and tannins were detected in the ethanol extract of C. peruviana. We assume that the ability of the extract of C. peruviana to control insects may be related to the presence of high levels of these compounds. The findings highlighted that the extract from the leaves of Cascabela peruviana has the potential to be used as an insecticide.

Dysfunction of lipid storage droplet-2 suppresses endoreplication and induces JNK pathway-mediated apoptotic cell death in Drosophila salivary glands.

The lipid storage droplet-2 (LSD-2) protein of Drosophila is a homolog of mammalian perilipin 2, which is essential for promoting lipid accumulation and lipid droplet formation. The function of LSD-2 as a regulator of lipolysis has also been demonstrated. However, other LSD-2 functions remain unclear. To investigate the role of LSD-2, we performed tissue-specific depletion in the salivary glands of Drosophila using a combination of the Gal4-upstream activating sequence system and RNA interference. LSD-2 depletion inhibited the entry of salivary gland cells into the endoreplication cycle and delayed this process by enhancing CycE expression, disrupting the development of this organ. The deficiency of LSD-2 expression enhanced reactive oxygen species production in the salivary gland and promoted JNK-dependent apoptosis by suppressing dMyc expression. This phenomenon did not result from lipolysis. Therefore, LSD-2 is vital for endoreplication cell cycle and cell death programs.

Effects of Launaea sarmentosa Extract on Lipopolysaccharide-Induced Inflammation via Suppression of NF-κB/MAPK Signaling and Nrf2 Activation.

Launaea sarmentosa has been extensively used as a nutrient herb in traditional Vietnamese remedies for the treatment of various diseases, especially inflammatory diseases. However, no detailed research has been conducted examining the molecular mechanisms involved in the suppression of inflammatory response. Here, we studied the effects of L. sarmentosa methanol extract on lipopolysaccharide (LPS)-induced inflammation using RAW 264.7 macrophages. The extract demonstrated potent antioxidant activity owing to the presence of polyphenolic and flavonoid components. Pretreatment with the extract inhibited LPS-mediated secretion of nitric oxide, reactive oxygen species, and tumor necrosis factor-α as well as the expression of inflammatory cytokines. Furthermore, the activation of the nuclear factor-kappa B pathway and phosphoinositide-3-kinase/protein kinase B pathways was blocked by the extract by inhibiting Akt phosphorylation. Additionally, the mitogen-activated protein kinase pathway was suppressed, and endoplasmic reticulum stress was attenuated. Furthermore, the extract promoted the activity of nuclear factor erythroid-2-related factor 2 resulting in the up-regulation of heme oxygenase-1 pathway, leading to the suppression of oxidative stress and inflammatory response. Taken together, the results indicate that L. sarmentosa exhibits anti-inflammatory effects, and hence, can be further developed as a novel drug for the treatment of diseases associated with excessive inflammation.

Role of Serotonin Transporter in Eye Development of Drosophila melanogaster.

Serotonin transporter (SerT) in the brain is an important neurotransmitter transporter involved in mental health. However, its role in peripheral organs is poorly understood. In this study, we investigated the function of SerT in the development of the compound eye in Drosophila melanogaster. We found that SerT knockdown led to excessive cell death and an increased number of cells in S-phase in the posterior eye imaginal disc. Furthermore, the knockdown of SerT in the eye disc suppressed the activation of Akt, and the introduction of PI3K effectively rescued this phenotype. These results suggested that SerT plays a role in the healthy eye development of D. melanogaster by controlling cell death through the regulation of the PI3K/Akt pathway.

Dysfunction of LSD-1 induces JNK signaling pathway-dependent abnormal development of thorax and apoptosis cell death in Drosophila melanogaster.

Perilipins are evolutionarily conserved from insects to mammals. Lipid storage droplet-1 (LSD-1) is a member of the lipid droplet's surface-binding protein family and counterpart to mammalian perilipin 1. The role of LSD-1 has already been reported in lipid metabolism of Drosophila. However, the function of this gene during specific tissue development is still under investigation. Here, we found that LSD-1 is expressed in the notum of the wing imaginal disc, and notum-specific knockdown of Lsd-1 by pannir-GAL4 driver leads to split thorax phenotype in adults, suggesting an essential role of LSD-1 in development of Drosophila thorax. As overexpression of JNK homolog, bsk (basket) suppresses Lsd-1 knockdown phenotype, the role of LSD-1 in thorax development was proved to be dependent on the activity of the Drosophila c-Jun N-terminal kinase (JNK). The puckered (puc) expression led to significant decrease in the JNK activity in wing discs of Lsd-1 knockdown flies. In addition, we also detected that depletion of Lsd-1 enhances apoptotic cell death in the wing notum area. Taken together, these data demonstrated that LSD-1 functions in Drosophila thorax development by regulating JNK pathway.

LSD-2 dysfunction induces dFoxO-dependent cell death in the wing of Drosophila melanogaster.

Lipid storage droplet-2 (LSD-2) of Drosophila melanogaster is a member of the lipid storage droplet membrane surface-binding protein family. LSD-2 is detected in many specific tissues: germline precursor cells, fat body, and is associated with lipid metabolism, lipid storage, and regulation of lipid droplet transport. However, the roles of this gene in development remain unclear. To investigate these functions, we performed tissue-specific knockdown of Lsd-2 in Drosophila using the combination of GAL4/UAS system and RNAi. Here we report that the knockdown of Lsd-2 in the wing led to abnormal wing phenotype and cell death in the wing pouch of 3rd-instar larvae, suggesting an essential role of Lsd-2 in development of the Drosophila wing. This function of Lsd-2 is dependent on the transcription factor dFoxO, as dFoxO depletion suppresses cell death and the abnormal wing pattern formation induced by Lsd-2-knockdown. Furthermore, Lsd-2-knockdown up-regulated the expression of the dFoxO transcription target reaper, which constitutes a pro-apoptosis gene. This study provides the first evidence that Lsd-2-knockdown causes cell death mediated by dfoxO.

Function of Lipid Storage Droplet 1 (Lsd1) in Wing Development of Drosophila melanogaster.

Perilipins are evolutionarily conserved from Drosophila to humans, the lipid storage droplet 1 (Lsd1) is a Drosophila homolog of human perilipin 1. The function of Lsd1 as a regulator of lipolysis in Drosophila has been demonstrated, as the Lsd1 mutant causes an increase of lipid droplet size. However, the functions of this gene during development are still under investigation. In order to determine the function of Lsd1 during development, Lsd1 was knocked down in Drosophila using the GAL4-UAS system. Selective knockdown of Lsd1 in the dorsal wing disc caused an atrophied wing phenotype. The generation of reactive oxygen species in the wing pouch compartment of the Lsd1-knockdown flies was significantly higher than in the control. Immunostaining with caspase-3 antibody revealed a greater number of apoptotic cells in Lsd1-knockdown wing discs than in the control. Cell death by autophagy was also induced in the knockdown flies. Moreover, cells deprived of Lsd1 showed mitochondrial expansion and decreased ATP levels. These results strongly suggest that knockdown of Lsd1 induces mitochondrial stress and the production of reactive oxygen species that result in cell death, via apoptosis and the autophagy pathway. These results highlight the roles of Drosophila Lsd1 during wing development.