Antifungal Activity and Multi-Target Mechanism of Action of Methylaervine on Candida albicans.

The discovery of a lead compound against Candida albicans is urgently needed because of the lack of clinically available antifungal drugs and the increase in drug resistance. Herein, a ß-carboline alkaloid methylaervine (MET) exhibited potential activity against C. albicans (MIC = 16-128 µg/mL), no hemolytic toxicity, and a low tendency to induce drug resistance. An antifungal mechanism study indicated that MET effectively inhibited the biofilm formation and disrupted the mature biofilm. Moreover, filamentation formation and spore germination were also weakened. The electron microscopy analysis revealed that MET could damage the cell structure, including the cell wall, membrane, and cytoplasm. In particular, the permeability and integrity of the cell membrane were destroyed. When it entered the fungi cell, it interfered with the redox homeostasis and DNA function. Overall, MET can inhibit the growth of C. albicans from multiple channels, such as biofilm, filamentation, cell structure, and intracellular targets, which are difficult to mutate at the same time to generate drug resistance. This work provides a promising lead compound for the creation of new antifungal agents against C. albicans.

Quaternized antimicrobial peptide mimics based on harmane as potent anti-MRSA agents by multi-target mechanism covering cell wall, cell membrane and intracellular targets.

Infectious disease caused by methicillin-resistant Staphylococcus aureus (MRSA) seriously threatens public health. The design of antimicrobial peptide mimics (AMPMs) based on natural products (NPs) is a new strategy to kill MRSA and slow the development of drug resistance recently. Here, we reported the design and synthesis of novel AMPMs based on harmane skeleton. Notably, compound 9b exhibited comparable or even better anti-MRSA activity in vitro and in vivo with minimum inhibitory concentration (MIC) of 0.5-2 µg/mL than the positive drug vancomycin. The highly active compound 9b not only showed low cytotoxicity, no obvious hemolysis and good plasma stability, but also presented low tendency of developing resistance. Anti-MRSA mechanism revealed that compound 9b could destroy cell wall structure by interacting with lipoteichoic acid and peptidoglycan, cause membrane damage by depolarization, increased permeability and destructed integrity, reduce cell metabolic activity by binding to lactate dehydrogenase (LDH), interfere cellular redox homeostasis, and bind to DNA. Overall, compound 9b killed the MRSA by multi-target mechanism, which provide a promising light for combating the growing MRSA resistance.

Canthin-6-one analogs block Newcastle disease virus proliferation via suppressing the Akt and ERK pathways.

Newcastle disease virus, a member of the Paramyxoviridae family, causes significant economic losses in poultry worldwide. To identify novel antiviral agents against NDV, 36 canthin-6-one analogs were evaluated in this study. Our data showed that 8 compounds exhibited excellent inhibitory effects on NDV replication with IC50 values in the range of 5.26 to 11.76 µM. Besides, these analogs inhibited multiple NDV strains with IC50 values within 12 µM and exerted antiviral activity against peste des petits ruminants virus (PPRV) and canine distemper virus (CDV). Among these analogs, 16 presented the strongest anti-NDV activity (IC50 = 5.26 µM) and minimum cytotoxicity (CC50 > 200 µM) in DF-1 cells. Furthermore, 16 displayed antiviral activity in different cell lines. Our results showed that 16 did not affect the viral adsorption while it can inhibit the entry of NDV by suppressing the Akt pathway. Further study found that 16-treatment inhibited the NDV-activated ERK pathway, thereby promoting the expression of interferon-related genes. Our findings reveal an antiviral mechanism of canthin-6-one analogs through inhibition of the Akt and ERK signaling pathways. These results point to the potential value of canthin-6-one analogs to serve as candidate antiviral agents for NDV.

An inulin-based glycovesicle for pathogen-targeted drug delivery to ameliorate salmonellosis.

The gut microbiota plays a significant role in the pathogenesis and remission of inflammatory bowel disease. However, conventional antibiotic therapies may alter microbial ecology and lead to dysbiosis of the gut microbiome, which greatly limits therapeutic efficacy. To address this challenge, novel nanomicelles that couple inulin with levofloxacin via disulfide bonds for the treatment of salmonellosis were developed in this study. Owing to their H2S-responsiveness, the nanomicelles can target the inflamed colon and rapidly release levofloxacin to selectively fight against enteric pathogens. Moreover, the embedded inulin can serve as prebiotic fiber to increase the amount of Bifidobacteria and Lactobacilli in mice with salmonellosis, thus maintaining the intestinal mechanical barrier and regulating the balance of the intestinal flora. Therefore, multifunctional nanomicelles had a better curative effect than pure levofloxacin on ameliorating inflammation in vivo. The pathogen-targeted glycovesicle represents a promising drug delivery platform to maximize the efficacy of antibacterial drugs for the treatment of inflammatory bowel disease.

Synthesis of 3-formyl-eudistomin U with anti-proliferation, anti-migration and apoptosis-promoting activities on melanoma cells.

The discovery of new lead skeleton against melanoma are urgently needed due to its highly malignant and mortality. Herein, a new molecular entity (EU-5) derived from eudistomin U was synthesized with total yield of 46%, which displayed potent activity against malignant melanoma A375 cells (IC50 = 4.4 µM), no hemolytic toxicity and good physicochemical properties in silico. Colony formation and cell cycle arrest assays revealed that EU-5 suppressed cell proliferation by causing cell cycle arrest at G0/G1 phase. Wound healing and transwell assays suggested that EU-5 could effectively inhibit migration of A375 cells in a dose-dependent manner. Calcein-AM/PI staining, Annexin V-FITC/PI apoptosis detection, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), transcriptomics, quantitative real­time polymerase chain reaction (qRT­PCR), spectrometric titration and molecular docking assays indicated that EU-5 could activate p53 signaling pathway and trigger mitochondria-mediated cell apoptosis. Taken together, this study provided a promising lead structure for the design of a new generation of anti-melanoma drugs.

Discovery of new quaternized norharmane dimers as potential anti-MRSA agents.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA)-caused infections greatly threaten public health. The discovery of natural-product-based anti-MRSA agents for treating infectious diseases has become one of the current research focuses. OBJECTIVES: This study aims to identify promising anti-MRSA agents with a clear mechanism based on natural norharmane modified by quaternization or dimerization. METHODS: A total of 32 norharmane analogues were prepared and characterized. Their antibacterial activities and resistance development propensity were tested by the broth double-dilution method. Cell counting kit-8 and hemolysis experiments were used to assess their biosafety. The plasma stability, bactericidal mode, and biofilm disruption effects were examined by colony counting and crystal violet staining assays. Fluorescence microscopy, metabolomic analysis, docking simulation and spectra titration revealed its anti-MRSA mechanisms. The mouse skin infection model was used to investigate the in vivo efficacy. RESULTS: Compound 5a was selected as a potential anti-MRSA agent, which exhibited potent anti-MRSA activity in vitro and in vivo, low cytotoxicity and hemolysis under an effective dose. Moreover, compound 5a showed good stability in 50% plasma, a low tendency of resistance development and capabilities to disrupt bacterial biofilms. The mechanism studies revealed that compound 5a could inhibit the biosynthesis of bacteria cell walls, damage the membrane, disturb energy metabolism and amino acid metabolism pathways, and interfere with protein synthesis and nucleic acid function. CONCLUSIONS: These results suggested that compound 5a is a promising candidate for combating MRSA infections, providing valuable information for further exploiting a new generation of therapeutic antibiotics.

Click chemistry-based enrichment strategy for tracing cellular fatty acid metabolism by LC-MS/MS.

Fatty acids (FAs), which were initially recognized as energy sources and essential building blocks of biomembranes, serve as the precursors of important signaling molecules. Tracing FA metabolism is essential to understanding the biochemical activity and role of FAs in physiological and pathological events. Inspired by the advances in click chemistry for protein enrichment, we herein established a click chemistry-based enrichment (CCBE) strategy for tracing the cellular metabolism of eicosapentaenoic acid (EPA, 20:5 n-3) in neural cells. Terminal alkyne-labeled EPA (EPAA) used as a surrogate was incubated with N2a, mouse neuroblastoma cells, and alkyne-labeled metabolites (ALMs) were selectively captured by an azide-modified resin via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction for enrichment. After removing unlabeled metabolites, ALMs containing a triazole moiety were cleaved from solid-phase resins and subjected to liquid chromatography mass spectrometry (LC-MS) analysis. The proposed CCBE strategy is highly selective for capturing and enriching alkyne-labeled metabolites from the complicated matrices. In addition, this method can overcome current detection limits by enhancing MS sensitivity of targets, improving the chromatographic separation of sn-position glycerophospholipid regioisomers, facilitating structural characterization of ALMs by a specific MS/MS fragmentation signature, and providing versatile fluorescence detection of ALMs for cellular distribution. This CCBE strategy might be expanded to trace the metabolism of other FAs, small molecules, or drugs.

Recent Developments of Gramine: Chemistry and Biological Activity.

The natural alkaloid gramine has attracted significant attention in both academic and industrial circles because of its potential and diverse biological activities, including antiviral, antibacterial, antifungal, anti-inflammatory and antitumor activities; application in therapy for Alzheimer's disease; serotonin-receptor-related activity; insecticidal activity; and application as an algicide. In this review, we focus on the research advances that have been made for gramine-based molecules since their discovery, providing key information on their extraction and separation, chemical synthesis and diverse biological activities. Data regarding their mechanisms of action are also presented. This comprehensive and critical review will serve as a guide for developing more drug candidates based on gramine skeletons.

9-Butyl-Harmol Exerts Antiviral Activity against Newcastle Disease Virus through Targeting GSK-3ß and HSP90ß.

The paramyxoviruses represent a large family of human and animal pathogens that cause significant health and economic burdens worldwide. However, there are no available drugs against the virus. ß-carboline alkaloids are a family of naturally occurring and synthetic products with outstanding antiviral activities. Here, we examined the antiviral effect of a series of ß-carboline derivatives against several paramyxoviruses, including Newcastle disease virus (NDV), peste des petits ruminants virus (PPRV), and canine distemper virus (CDV). Among these derivatives, 9-butyl-harmol was identified as an effective antiviral agent against these paramyxoviruses. Further, a genome-wide transcriptome analysis in combination with target validation strategies reveals a unique antiviral mechanism of 9-butyl-harmol through the targeting of GSK-3ß and HSP90ß. On one hand, NDV infection blocks the Wnt/ß-catenin pathway to suppress the host immune response. 9-butyl-harmol targeting GSK-3ß dramatically activates the Wnt/ß-catenin pathway, which results in the boosting of a robust immune response. On the other hand, NDV proliferation depends on the activity of HSP90. The L protein, but not the NP protein or the P protein, is proven to be a client protein of HSP90ß, rather than HSP90α. 9-butyl-harmol targeting HSP90ß decreases the stability of the NDV L protein. Our findings identify 9-butyl-harmol as a potential antiviral agent, provide mechanistic insights into the antiviral mechanism of 9-butyl-harmol, and illustrate the role of ß-catenin and HSP90 during NDV infection. IMPORTANCE Paramyxoviruses cause devastating impacts on health and the economy worldwide. However, there are no suitable drugs with which to counteract the viruses. We determined that 9-butyl-harmol could serve as a potential antiviral agent against paramyxoviruses. Until now, the antiviral mechanism of ß-carboline derivatives against RNA viruses has rarely been studied. Here, we found that 9-butyl-harmol exerts dual mechanisms of antiviral action, with its antiviral activities being mediated by two targets: GSK-3ß and HSP90ß. Correspondingly, the interaction between NDV infection and the Wnt/ß-catenin pathway or HSP90 is demonstrated in this study. Taken together, our findings shed light on the development of antiviral agents against paramyxoviruses, based on the ß-carboline scaffold. These results present mechanistic insights into the polypharmacology of 9-butyl-harmol. Understanding this mechanism also deepens the host-virus interaction and reveals new drug targets for anti-paramyxoviruses.

Novel N1 or N9 modified α-carboline analogues as potential ligands in Alzheimer's disease therapy: Synthesis and neurobiological activity evaluation.

A series of new α-carboline analogues modified at N1 or N9 positions by alkyl, benzyl and phenyl were synthesized and characterized as potential ligands for AD therapy. These compounds exhibited multifunctional neurobiological activities including anti-neuroinflammatory, neuroprotective and cholinesterase inhibition. Among them, compound 5d with good drug-like properties and no cytotoxicity, showed potent inhibitory activity against NO production (IC50 = 1.45 µM), which could suppress the expression levels of iNOS and COX-2 in a dose-dependent manner. Further mechanism exploration indicated that compound 5d could regulate the NF-κB signaling pathway by decreasing the phosphorylation of IκB-α and p65. Notably, compound 5d could effectively decrease the LPS-induced aberrations in zebrafish. Compounds 3b, 4f, 5c, 5g, 5m and 6i exhibited potential neuroprotective activity (cell viability > 70 %) in the H2O2-induced PC-12 neuronal death model and rescued the SOD activity. In particular, compounds 3b, 4f, and 5g activated the Nrf2 signaling pathway, and improved the expressions of antioxidant proteins NQO-1 and HO-1, which alleviated the head cell apoptosis in zebrafish. Additionally, compound 6i exhibited potential inhibitory activity against BuChE with IC50 of 0.77 µM. Overall, this work provided some lead compounds based on α-carboline used for AD therapy.

ß-Carboline dimers inhibit the tumor proliferation by the cell cycle arrest of sarcoma through intercalating to Cyclin-A2.

ß-Carbolines are potentially strong alkaloids with a wide range of bioactivities, and their dimers exhibit stronger antitumor activity other than the monomers. However, the detailed mechanisms of the ß-carboline dimers in inhibiting sarcoma (SARC) remain unclear. The results showed that ß-carboline-3-carboxylic acid dimers Comp1 and Comp2, which were synthesized in our lab and modified at the N9 position and linked at the C3 position, exhibited effective inhibition activity on MG-63 proliferation (IC50 = 4.6µM). Meanwhile, the large scale transcriptome profiles of SARC from The Cancer Genome Atlas (TCGA) were analyzed, and found that abnormal expression of genes relevant to apoptosis, cell cycle, and signaling pathways of Hedgehog, HIF, Ras involved in the SARC pathogenesis. Interestingly, both dimers could promote the apoptosis and arrest the cell cycle in S phase to inhibit proliferation of MG-63. Moreover, Comp1 and Comp2 inhibited the expression CDK2, CCNA2, DBF4, and PLK1 associated with various immune cells and cell cycle in MG-63. Remarkably, drug-target interaction network analysis showed that numerous proteins involved in cell cycle were the potential targets of Comp1 and Comp2, especially CCNA2. Further molecular docking, isothermal titration calorimetry (ITC) and Cellular Thermal Shift Assay (CETSA) confirmed that both dimers could directly interact with CCNA2, which is significantly correlated with CD4+ T cells, by strong hydrophobic interactions (Kd=5.821 ×106 N). Meanwhile, the levels of CCNA2 and CDK2 were inhibited to decrease in MG-63 by both dimer treatments at transcription and protein levels, implying that Comp1 and Comp2 blocked the interaction between CCNA2 and CDK2 through competitive binding with CCNA2 to arrest the cell cycle of MG-63 cells in the S phase. Additionally, the transcriptome profiles of ß-carboline-treated mice from Gene Expression Omnibus (GEO) were obtained, and found that similar antitumor mechanism was shared among ß-carboline derivatives. Overall, our results elucidated the antitumor mechanisms of Comp1 and Comp2 through dual-suppressing the function of CCNA2 to profoundly arrest cell cycle of MG-63, then effectively inhibited cell proliferation of MG-63. These results provide new insights into the antitumor mechanism of ß-carboline dimers and new routes of various novel cancer-related drug targets for future possible cancer therapy.

Antibacterial quaternary ammonium agents: Chemical diversity and biological mechanism.

Bacterial infections have seriously threatened public health especially with the increasing resistance and the cliff-like decline of the number of newly approved antibacterial agents. Quaternary ammonium compounds (QACs) possess potent medicinal properties with 95 successfully marketed drugs, which also have a long history as antibacterial agents. In this review, we summarize the chemical diversity of antibacterial QACs, divided into chain-like and aromatic ring, reported over the past decade (2012 to mid-2022). Additionally, the structure-activity relationships, mainly covering hydrophobicity, charges and skeleton features, are discussed. In the cases where sufficient information is available, antibacterial mechanisms including biofilm, cell membrane, and intracellular targets are presented. It is hoped that this review will provide sufficient information for medicinal chemists to discover the new generation of antibacterial agents based on QACs.

Methylaervine as Potential Lead Compound Against Cervical Carcinoma: Pharmacologic Mechanism Prediction based on Network Pharmacology.

BACKGROUND: The discovery of therapeutic anticancer agents based on natural products is one of the current research focuses. Network pharmacology will broaden our understanding of drug actions by bioinformatics analysis. OBJECTIVE: To explore the potential and provide scientific evidence for methylaervine as a lead compound against cervical carcinoma. METHODS: Methylaervine was synthesized, and its activity against four cancer cell lines was evaluated by MTT assay. Pharmacokinetic properties were obtained by in silico approaches, and the pharmacologic mechanism was predicted by network pharmacology. Then we validated and investigated our predictions of candidate targets using a molecular docking study. RESULTS: Methylaervine was synthesized with a total yield of 54.9%, which displayed activity against HeLa (IC50 = 14.8 µM) with good predicted pharmacokinetic properties, thus it was considered a potential lead compound. The network pharmacology study indicated that methylaervine could act against cervical carcinoma by regulating the function of multiple pivotal targets, such as CTNNB1, PTPRJ, RPA1, and TJP1, mainly covering cell growth, cell motility, and cell proliferation. Moreover, docking analysis showed that hydrogen bonds and hydrophobic interactions were the main forms of interactions. CONCLUSION: This work would provide new insight into the design of anti-cervical carcinoma drugs based on methylaervine.

Natural and synthetic ß-carboline as a privileged antifungal scaffolds.

The discovery of antifungal agents with novel structure, broad-spectrum, low toxicity, and high efficiency has been the focus of medicinal chemists. Over the past decades, ß-carboline scaffold has attracted extensive attention in the scientific community due to its potent and diverse biological activities with nine successfully marketed ß-carboline-based drugs. In this review, we summarized the current states and advances in the antifungal activity of natural and synthetic ß-carbolines. Additionally, the structure-activity relationships and their antifungal mechanisms targeting biofilm, cell wall, cell membrane, and fungal intracellular targets were also systematically discussed. In summary, ß-carbolines have the great potential to develop new efficient scaffolds to combat fungal infections.

1-Formyl-ß-carboline Derivatives Block Newcastle Disease Virus Proliferation through Suppressing Viral Adsorption and Entry Processes.

Newcastle disease virus (NDV) is one of the highly contagious pathogens causing devastating economic effects on the global poultry industry. In the present study, three 1-formyl-ß-carboline derivatives (compounds 6, 7, and 9) were found to be potent inhibitors of different genotypes of NDV with IC50 values within 10 µM, which are similar to ribavirin. The virus titers were decreased by the presence of 1-formyl-ß-carboline derivatives in a dose-dependent manner, and the inhibition rate was found to exceed 90% at the concentration of 20 µM. These compounds mainly suppressed the adsorption and entry processes of NDV lifecycle. Through DARTS, CETSA, and RBC binding assay, these compounds were identified as novel HN inhibitors, which could directly interact with the NDV HN protein to affect the adsorption of NDV. Furthermore, they could inhibit the entry of NDV through suppressing the PI3K/Akt pathway rather than the ERK pathway. The PI3K/Akt pathway was proved to be involved in NDV entry. Our findings reveal a unique mechanism through which 1-formyl-ß-carboline derivatives restrain NDV infection. Moreover, these compounds represent suitable scaffolds for designing novel HN inhibitors.

Cellular and non-target metabolomics approaches to understand the antifungal activity of methylaervine against Fusarium solani.

Botanical fungicides are promising replacements for pure chemical synthetic pesticides in agriculture and organic food production. Methylaervine with good physicochemical properties exhibited effective activity against F. solani (EC50 = 10.56 µM) better than the positive control thiophanate-methyl (EC50 = 27.94 µM). The activity changes of malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) showed that methylaervine could significantly induce lipid peroxidation and activate the antioxidant enzymes. According to the metabolomics analysis, fifty-one differential metabolites and two major antifungal-related pathways covering tricarboxylic acid (TCA) cycle and steroid biosynthesis were identified. Moreover, the disturbance for TCA cycle was validated by the activity changes of dehydrogenase (MDH) and succinate dehydrogenase (SDH) as well as docking simulation. Homology modeling and docking study revealed that hydrogen bonds and hydrophobic interactions played a vital role in methylaervine-protein stability. This study provided new insight into the antifungal activity of methylaervine, which is important for the development of novel botanical fungicides based on methylaervine.

Recent progress of antibacterial natural products: Future antibiotics candidates.

The discovery of novel antibacterial molecules plays a key role in solving the current antibiotic crisis issue. Natural products have long been an important source of drug discovery. Herein, we reviewed 256 natural products from 11 structural classes in the period of 2016-01/2020, which were selected by SciFinder with new compounds or new structures and MICs lower than 10 µg/mL or 10 µM as criterions. This review will provide some effective antibacterial lead compounds for medicinal chemists, which will promote the antibiotics research based on natural products to the next level.

Cyclooxygenase-2 Facilitates Newcastle Disease Virus Proliferation and Is as a Target for Canthin-6-One Antiviral Activity.

Cyclooxygenase-2 (COX-2), one of the mediators of inflammation in response to viral infection, plays an important role in host antiviral defense system. But its role in Newcastle disease virus (NDV) proliferation process remains unclear. This study revealed that inhibition of COX-2 could benefit NDV proliferation and overexpression of COX-2 dose-dependently suppressed NDV proliferation. Overexpression of COX-2 also showed inhibitory effect on NDV-induced endoplasmic reticulum (ER)-stress and autophagy, also promoted the expression of antiviral genes. However, prostaglandin E2 (PGE2), the major product of COX-2, had indistinctive effects on NDV proliferation. At variant time point post viral infection, a tight regulation pattern of COX-2 by NDV was observed. Using inhibitors and siRNA against signaling molecules, the nuclear factor-κB (NF-κB) and melanoma differentiation-associated gene 5 (MDA5) were identified as critical factors for NDV induced COX-2 expression. Nonetheless, at late stage of NDV proliferation, substantial suppression of COX-2 protein synthesis could be detected, accompanied by a decrease in mRNA half-life. Furthermore, three C ring-truncated canthin-6-one analogs were used to activate COX-2 expression and showed inhibitory effect on NDV proliferation with the effective concentrations on µM level. Taken together, these results illustrated a novel NDV-regulated cellular mechanism and indicated that COX-2 is an important regulator of NDV proliferation which can serve as a potential target for anti-NDV agents.

Recent developments of chalcones as potential antibacterial agents in medicinal chemistry.

Chalcones, containing an α,ß-unsaturated ketone fragment, are an important pharmacologically active agents because of their diverse mechanisms. This review provides an update on the recent developments (2009-2019.3) in the antibacterial activity of natural and synthetic chalcones. Moreover, the structure-activity relationships and mechanisms are also carefully summarized which will provide some important guidance for design and synthesis in future. This comprehensive and critical review will be helpful for medicinal chemists to develop more candidate antibacterial agents.