Catalytic selectivity and evolution of cytochrome P450 enzymes involved in monoterpene indole alkaloids biosynthesis.

Cytochrome P450 enzyme (CYP)-catalyzed functional group transformations are pivotal in the biosynthesis of metabolic intermediates and products, as exemplified by the CYP-catalyzed C7-hydroxylation and the subsequent C7-C8 bond cleavage reaction responsible for the biosynthesis of the well-known antitumor monoterpene indole alkaloid (MIA) camptothecin. To determine the key amino acid residues responsible for the catalytic selectivity of the CYPs involved in MIA biosynthesis, we characterized the enzymes CYP72A728 and CYP72A729 as stereoselective 7-deoxyloganic acid 7-hydroxylases (7DLHs). We then conducted a comparative analysis of the amino acid sequences and the predicted structures of the CYP72A homologs involved in camptothecin biosynthesis, as well as those of the CYP72A homologs implicated in the pharmaceutically significant MIAs biosynthesis in Catharanthus roseus. The crucial amino acid residues for the catalytic selectivity of the CYP72A-catalyzed reactions were identified through fragmental and individual residue replacement, catalytic activity assays, molecular docking, and molecular dynamic simulations analysis. The fragments 1 and 3 of CYP72A565 were crucial for its C7-hydroxylation and C7-C8 bond cleavage activities. Mutating fragments 1 and 2 of CYP72A565 transformed the bifunctional CYP72A565 into a monofunctional 7DLH. Evolutionary analysis of the CYP72A homologs suggested that the bifunctional CYP72A in MIA-producing plants may have evolved into a monofunctional CYP72A. The gene pairs CYP72A728-CYP72A610 and CYP72A729-CYP72A565 may have originated from a whole genome duplication event. This study provides a molecular basis for the CYP72A-catalyzed hydroxylation and C-C bond cleavage activities of CYP72A565, as well as evolutionary insights of CYP72A homologs involved in MIAs biosynthesis.

Kopsileuconines A-D: Bisindole alkaloids with cytotoxic activity from Kopsia hainanensis.

Kopsileuconines A-D (1-4), four monoterpenoid bisindole alkaloids with unprecedented skeletons, along with their biosynthetically related precursors (5-8) were isolated from the roots of Kopsia hainanensis. Compound 1 possessed an undescribed C-6-C-5' dimerization pattern of aspidofractinine-type alkaloids. Compounds 2-4 were rhazinilam-kopsine (2) and rhazinilam-aspidofractinine type (3 and 4) bisindole alkaloids with undescribed skeletons, respectively. Their structures with absolute configurations were fully accomplished by extensive spectroscopic analysis, quantum-chemical calculations, and X-ray crystallography. A plausible biosynthetic pathway for 1-4 was proposed. Compound 2 exhibited a significant inhibitory effect against human lung cancer cell lines PC9 (EGFR mutant), with an IC50 value of 15.07 ± 1.19 µM.

Chromatographic fingerprinting of epiphytic fungal strains isolated from Withania somnifera and biological evaluation of isolated okaramine H.

Medicinal plants are "goldmines" of natural products, and continue to provide key scaffolds for drug development. They have immense therapeutic potential, encapsulating a plethora of metabolites within them, which have yet to be explored. Withania somnifera (L.) Dunal is one such medicinal plant known since time immemorial for its therapeutic activity in the Ayurveda system of medicine. Studies have revealed Nature's marvel of these medicinal plants harbouring endophytic and epiphytic microorganisms from phyllosphere to rhizosphere. Chromatographic fingerprinting was carried out using HPTLC and HPLC on five epiphytic strains isolated from the leaves, stem and fruits of Withania somnifera. Out of five filamentous fungi, one fungus identified as Aspergillus aculeatus S20 was well explored. An indole alkaloid, okaramine H, was isolated using systematic chromatographic investigation at a retention time of 26.278 min showing λmax at 206, 236, 284 and 370 nm. Confirmation was achieved using NMR and mass spectrometry (MS) as analytical techniques. Structure elucidation was done by studying the fragmentation pattern using MS/MS and an accurate mass was determined using HR-ESI-QTOF-MS showing m/z of 521.2546 [M + H]+. The percentage purity of isolated okaramine H was found to be >90. Well known for its insecticidal activity, okaramine H was explored for its antileishmanial activity against the Leishmania donovani parasite for the first time. Under in vitro conditions, the compound showed an inhibitory effect on Leishmania donovani promastigotes with an IC50 of 147 µg mL-1.

Cheminformatics-Guided Exploration of Synthetic Marine Natural Product-Inspired Brominated Indole-3-Glyoxylamides and Their Potentials for Drug Discovery.

Marine natural products (MNPs) continue to be tested primarily in cellular toxicity assays, both mammalian and microbial, despite most being inactive at concentrations relevant to drug discovery. These MNPs become missed opportunities and represent a wasteful use of precious bioresources. The use of cheminformatics aligned with published bioactivity data can provide insights to direct the choice of bioassays for the evaluation of new MNPs. Cheminformatics analysis of MNPs found in MarinLit (n = 39,730) up to the end of 2023 highlighted indol-3-yl-glyoxylamides (IGAs, n = 24) as a group of MNPs with no reported bioactivities. However, a recent review of synthetic IGAs highlighted these scaffolds as privileged structures with several compounds under clinical evaluation. Herein, we report the synthesis of a library of 32 MNP-inspired brominated IGAs (25-56) using a simple one-pot, multistep method affording access to these diverse chemical scaffolds. Directed by a meta-analysis of the biological activities reported for marine indole alkaloids (MIAs) and synthetic IGAs, the brominated IGAs 25-56 were examined for their potential bioactivities against the Parkinson's Disease amyloid protein alpha synuclein (α-syn), antiplasmodial activities against chloroquine-resistant (3D7) and sensitive (Dd2) parasite strains of Plasmodium falciparum, and inhibition of mammalian (chymotrypsin and elastase) and viral (SARS-CoV-2 3CLpro) proteases. All of the synthetic IGAs tested exhibited binding affinity to the amyloid protein α-syn, while some showed inhibitory activities against P. falciparum, and the proteases, SARS-CoV-2 3CLpro, and chymotrypsin. The cellular safety of the IGAs was examined against cancerous and non-cancerous human cell lines, with all of the compounds tested inactive, thereby validating cheminformatics and meta-analyses results. The findings presented herein expand our knowledge of marine IGA bioactive chemical space and advocate expanding the scope of biological assays routinely used to investigate NP bioactivities, specifically those more suitable for non-toxic compounds. By integrating cheminformatics tools and functional assays into NP biological testing workflows, we can aim to enhance the potential of NPs and their scaffolds for future drug discovery and development.

Intranasal LAG3 antibody infusion induces a rapid antidepressant effect via the hippocampal ERK1/2-BDNF signaling pathway in chronically stressed mice.

The decline of microglia in the dentate gyrus is a new phenomenon that may explain the pathogenesis of depression, and reversing this decline has an antidepressant effect. The development of strategies that restore the function of dentate gyrus microglia in under stressful conditions is becoming a new focus. Lymphocyte-activating gene-3 (LAG3) is an immune checkpoint expressed by immune cells including microglia. One of its functions is to suppress the expansion of immune cells. In a recent study, chronic systemic administration of a LAG3 antibody that readily penetrates the brain was reported to reverse chronic stress-induced hippocampal microglia decline and depression-like behaviors. We showed here that a single intranasal infusion of a LAG3 antibody (In-LAG3 Ab) reversed chronic unpredictable stress (CUS)-induced depression-like behaviors in a dose-dependent manner, which was accompanied by an increase in brain-derived neurotrophic factor (BDNF) in the dentate gyrus. Infusion of an anti-BDNF antibody into the dentate gyrus, construction of knock-in mice with the BDNF Val68Met allele, or treatment with the BDNF receptor antagonist K252a abolished the antidepressant effect of In-LAG3 Ab. Activation of extracellular signal-regulated kinase1/2 (ERK1/2) is required for the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and BDNF decrease in the dentate gyrus. Moreover, both inhibition and depletion of microglia prevented the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and impairment of ERK1/2-BDNF signaling in the dentate gyrus. These results suggest that In-LAG3 Ab exhibits an antidepressant effect through microglia-mediated activation of ERK1/2 and synthesis of BDNF in the dentate gyrus.

Design, semi-synthesis and bioevaluation of koumine-like derivatives as potential antitumor agents in vitro and in vivo.

Aims: Five series of novel koumine-like compounds were designed, semi-synthesized and systematically evaluated for antitumor activities.Methods: All compounds were evaluated for antiproliferative activity against four human cancer cell lines, including HT-29, HCT-116, HCT-15 and Caco-2.Results: Most compounds exhibited much higher antiproliferation activities (IC50 <10 µM) than koumine. Two selected compounds A4 and C5 showed comparable antitumor effects to 5-FU in vivo, as well as better safety profiles. Further studies suggested that A4 and C5 could arrest HT-29 cell cycle in G2 phase and raise reactive oxygen species level, thus inducing cell apoptosis related to Erk MAPK and NF-κB signaling pathways inhibition.Conclusion: These results will greatly promote the druggability study of these koumine-like compounds.

[Box: see text].

Biomimetic and Concise Total Syntheses of Prenylated and Bicyclo[2.2.2]diazaoctane-Containing Indole Alkaloids Including Taichunamide A, Notoamide N and Versicolamide B.

Total syntheses of the title prenylated indole alkaloids together with seven others are reported. Biogenetic considerations have been employed in devising the reaction sequences leading to these targets with, in the opening stages, electrochemically-derived indole-3-carboxaldehyde 15 being subject to an aldol-type condensation reaction involving diketopiperazine derivative 19. This led, after prototopic shifts, intramolecular Diels-Alder cycloaddition and hydrolysis/deprotection steps, to the racemic forms of the bicyclo[2.2.2]diazaoctane-containing natural product stephacidin A (2) and its C6 epimer 3. Epoxidation of the last compound afforded, following rearrangement of the primary oxidation products, a mixture of (±)-taichunamide A [(±)-4] and (±)-versicolamide B [(±)-7]. Related protocols allowed for the conversion of (±)-stephacidin A [(±)-2] into (±)-notoamide B [(±)-5]. Analogous aldol condensation, nucleophilic reduction, and epoxidation steps led to the formation of (-)-notoamide E and its conversion into notoamide C as well as the indole fragmentation product amoenamide E. A late-stage chlorination reaction applied to (±)-stephacidin A provided access to the spirocyclic oxindole (±)-notoamide N [(±)-6].

Structure-activity relationship study of Pseudellone C as anti-glioma agents by targeting TNF/TNFR signaling pathway.

Glioma, a common primary brain tumor, is highly infiltrative and invasive, often leading to drug resistance and recurrence. Therefore, the development of novel therapeutic agents is urgently needed. Pseudellone C is a novel marine triindole alkaloid. Screening of its antiproliferative activity against 55 cell lines revealed its anti-CNS cancer potential. A total of 42 derivatives of Pseudellone C were designed and synthesized, and their inhibitory activities against two human glioma cell lines (U-87MG and LN-229) were evaluated using the CCK-8 assay. Ten derivatives exhibited potent antiproliferative activity with IC50 values below 10 µmol, which are 18- to 39- fold more potent than Pseudellone C. Among these, derivative 4o demonstrated favorable blood-brain barrier permeability. Mechanistic studies revealed that 4o induces apoptosis primarily by activating the downstream caspase 3 cascade via the TNF/TNFR pathway. Structure-activity relationship correlations were systematically analyzed, and a pharmacophore model for further rational design was constructed.

New insights into the chemistry and anticancer activity of Ophiocordyceps xuefengensis.

Ophiocordyceps xuefengensis (O. xuefengensis), the sister taxon of Ophiocordyceps sinensis (O. sinensis), is consumed as a "tonic food" due to its health benefits. However, little is known regarding the chemistry and bioactivity of O. xuefengensis. In this study, we characterized 80 indole-based alkaloids in the ethyl acetate fraction of O. xuefengensis by high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-Q-TOF-MS/MS), of which 54 indole-based alkaloids were identified as possibly new compounds. Furthermore, 29 of these compounds were established as potential anti-cancer compounds by ligand fishing combined with HPLC-Q-TOF-MS/MS. Moreover, molecular docking identified the NH- and OH- groups of these compounds as the key active groups. The present study has expanded the knowledge on the characteristic indole-based alkaloids and anti-cancer activity of O. xuefengensis.

Characterization of active alkaloids and metabolites in rats after oral administration of Zuojin Pill using UHPLC-Q-TOF-MS combined with bioinformatics and molecular docking analyses.

Zuojin Pill (ZJP), a traditional Chinese medicine prescription composed of Rhizoma Coptidis and Euodiae Fructus in the ratio of 6:1 (w/w), has been widely used for the treatment of gastric disorders. However, an in-depth understanding of in vivo metabolism and distribution profiles of protoberberine alkaloids (PBAs) and indole alkaloids (IDAs) in ZJP is lacking. In this study, a method using ultra-high performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was developed to systematically screen the alkaloids and their metabolites in rat plasma and various tissues after oral administration of ZJP. Furthermore, bioinformatics and molecular docking analyses were conducted to elucidate the contribution of the alkaloids and metabolites enriched in the stomach to the therapeutic effect of ZJP on gastritis. A total of 33 compounds, including 7 prototype alkaloids and 26 metabolites, were chemically defined or tentatively identified in this work. The metabolic pathways of PBAs (hydroxylation, oxidation, reduction, demethylation, demethylenation, glucuronide conjugation, sulfate conjugation) and IDAs (hydroxylation, glucuronide conjugation) were revealed. Notably, 7 prototype alkaloids and 18 metabolites were detected in the stomach, indicating their propensity for gastric distribution. These alkaloids and metabolites showed strong affinities with the 7 hub targets associated with gastritis, such as CCR7, CXCR4, IL6, IFNG, CCL2, TNF, and PTPRC, and could be considered the potential active substances of ZJP for treating gastritis. In conclusion, this study clarified the gastric distribution propensity of PBAs and IDAs and their metabolites, as well as their favorable binding interactions with gastritis-related targets, which could provide essential data for the further study of the pharmacodynamic material basis and gastroprotective mechanism of ZJP.

Discovery of Uncommon Tryptophan-Containing Diketopiperazines from Aspergillus homomorphus CBS 101889 Using an Aspergillus nidulans Heterologous Expression System.

Fungal secondary metabolite (SM) biosynthetic gene clusters (BGCs) containing dimethylallyltryptophan synthases (DMATSs) produce structurally diverse prenylated indole alkaloids with wide-ranging activities that have vast potential as human therapeutics. To discover new natural products produced by DMATSs, we mined the Department of Energy Joint Genome Institute's MycoCosm database for DMATS-containing BGCs. We found a DMATS BGC in Aspergillus homomorphus CBS 101889, which also contains a nonribosomal peptide synthetase (NRPS). This BGC appeared to have a previously unreported combination of genes, which suggested the cluster might make novel SMs. We refactored this BGC with highly inducible promoters into the model fungus Aspergillus nidulans. The expression of this refactored BGC in A. nidulans resulted in the production of eight tryptophan-containing diketopiperazines, six of which are new to science. We have named them homomorphins A-F (2, 4-8). Perhaps even more intriguingly, to our knowledge, this is the first discovery of C4-prenylated tryptophan-containing diketopiperazines and their derivatives. In addition, the NRPS from this BGC is the first described that has the ability to promiscuously combine tryptophan with either of two different amino acids, in this case, l-valine or l-allo-isoleucine.

Computational Docking as a Tool in Guiding the Drug Design of Rutaecarpine Derivatives as Potential SARS-CoV-2 Inhibitors.

COVID-19 continues to spread around the world. This is mainly because new variants of the SARS-CoV-2 virus emerge due to genomic mutations, evade the immune system and result in the effectiveness of current therapeutics being reduced. We previously established a series of detection platforms, comprising computational docking analysis, S-protein-based ELISA, pseudovirus entry, and 3CL protease activity assays, which allow us to screen a large library of phytochemicals from natural products and to determine their potential in blocking the entry of SARS-CoV-2. In this new screen, rutaecarpine (an alkaloid from Evodia rutaecarpa) was identified as exhibiting anti-SARS-CoV-2 activity. Therefore, we conducted multiple rounds of structure-activity-relationship (SAR) studies around this phytochemical and generated several rutaecarpine analogs that were subjected to in vitro evaluations. Among these derivatives, RU-75 and RU-184 displayed remarkable inhibitory activity when tested in the 3CL protease assay, S-protein-based ELISA, and pseudovirus entry assay (for both wild-type and omicron variants), and they attenuated the inflammatory response induced by SARS-CoV-2. Interestingly, RU-75 and RU-184 both appeared to be more potent than rutaecarpine itself, and this suggests that they might be considered as lead candidates for future pharmacological elaboration.

Conofolidine: A Natural Plant Alkaloid That Causes Apoptosis and Senescence in Cancer Cells.

Natural products contribute substantially to anticancer therapy; the plant kingdom provides an important source of molecules. Conofolidine is a novel Aspidosperma-Aspidosperma bisindole alkaloid isolated from the Malayan plant Tabernaemontana corymbosa. Herein, we report conofolidine's broad-spectrum anticancer activity together with that of three other bisindoles-conophylline, leucophyllidine, and bipleiophylline-against human-derived breast, colorectal, pancreatic, and lung carcinoma cell lines. Remarkably, conofolidine was able to induce apoptosis (e.g., in MDA-MB-468 breast) or senescence (e.g., in HT-29 colorectal) in cancer cells. Annexin V-FITC/PI, caspase activation, and PARP cleavage confirmed the former while positive ß-gal staining corroborated the latter. Cell cycle perturbations were evident, comprising S-phase depletion, accompanied by downregulated CDK2, and cyclins (A2, D1) with p21 upregulation. Confocal imaging of HCT-116 cells revealed an induction of aberrant mitotic phenotypes-membrane blebbing, DNA-fragmentation with occasional multi-nucleation. DNA integrity assessment in HCT-116, MDA-MB-468, MIAPaCa-2, and HT-29 cells showed increased fluorescent γ-H2AX during the G1 cell cycle phase; γ-H2AX foci were validated in HCT-116 and MDA-MB-468 cells by confocal microscopy. Conofolidine increased oxidative stress, preceding apoptosis- and senescence-induction in most carcinoma cell lines as seen by enhanced ROS levels accompanied by increased NQO1 expression. Collectively, we present conofolidine as a putative potent anticancer agent capable of inducing heterogeneous modes of cancerous cell death in vitro, encouraging further preclinical evaluations of this natural product.

Synthesis of Bisindole Alkaloids and Their Mode of Action against Methicillin-Resistant Staphylococcus Aureus.

About 100,000 deaths are attributed annually to infections with methicillin-resistant Staphylococcus aureus (MRSA) despite concerted efforts toward vaccine development and clinical trials involving several preclinically efficacious drug candidates. This necessitates the development of alternative therapeutic options against this drug-resistant bacterial pathogen. Using the Masuda borylation-Suzuki coupling (MBSC) sequence, we previously synthesized and modified naturally occurring bisindole alkaloids, alocasin A, hyrtinadine A and scalaradine A, resulting in derivatives showing potent in vitro and in vivo antibacterial efficacy. Here, we report on a modified one-pot MBSC protocol for the synthesis of previously reported and several undescribed N-tosyl-protected bisindoles with anti-MRSA activities and moderate cytotoxicity against human monocytic and kidney cell lines. In continuation of the mode of action investigation of the previously synthesized membrane-permeabilizing hit compounds, mechanistic studies reveal that bisindoles impact the cytoplasmic membrane of Gram-positive bacteria by promiscuously interacting with lipid II and membrane phospholipids while rapidly dissipating membrane potential. The bactericidal and lipid II-interacting lead compounds 5c and 5f might be interesting starting points for drug development in the fight against MRSA.

Glucoconjugated monoterpene indole alkaloids with xanthine oxidase inhibitory activity from Ophiorrhiza japonica.

Continued interest in the bioactive alkaloids led to the isolation of five undescribed alkaloids (1-5), ophiorglucidines A-E, and seven known analogues (6-12) from the water-soluble fraction of Ophiorrhiza japonica. The structures were elucidated based on spectroscopic data and quantum calculations as well as X-ray crystallographic analysis. The structure of 1 was characterized as a hexacyclic skeleton including a double bridge linking the indole and the monoterpene moieties, which is the first report of a single crystal with this type of structure. Moreover, the inhibitory effect of zwitterionic indole alkaloid glycosides on xanthine oxidase was found for the first time. The alkaloids 2 and 3, both of which have a pentacyclic zwitterionic system, were more active than the reference inhibitor, allopurinol (IC50 = 11.1 µM) with IC50 values of 1.0 µM, and 2.5 µM, respectively. Structure-activity relationships analyses confirmed that the carbonyl group at C-14 was a key functional group responsible for the inhibitory effects of these alkaloids.

Rutaecarpine-inspired scaffold-hopping strategy and Ullmann cross-coupling based synthetic approach: Identification of pyridopyrimidinone-indole based novel anticancer chemotypes.

Natural products as starting templates have shown historically major contribution to development of drugs. Inspired by the structure-function of an anticancer natural alkaloid Rutaecarpine, the Scaffold-hopped Acyclic Analogues of Rutaecarpine (SAAR) with 'N'-atom switch (1°-hop) and ring-opening (2°-hop) were investigated. A new synthetic route was developed for an effective access to the analogues, i.e. 2-indolyl-pyrido[1,2-a]pyrimidinones, which involved preparation of N-Boc-N'-phthaloyltryptamine/mexamine-bromides and pyridopyrmidinon-2-yl triflate, a nickel/palladium-catalysed Ullmann cross-coupling of these bromides and triflate, deprotection of phthalimide followed by N-aroylation, and Boc-deprotection. Fourteen novel SAAR-compounds were prepared, and they showed characteristic antiproliferative activity against various cancer cells. Three most active compounds (11a, 11b, and 11c) exhibited good antiproliferative activity, IC50 7.7-15.8 µM against human breast adenocarcinoma cells (MCF-7), lung cancer cells (A549), and colon cancer cells (HCT-116). The antiproliferative property was also observed in the colony formation assay. The SAAR compound 11b was found to have superior potency than original natural product Rutaecarpine and an anticancer drug 5-FU in antiproliferative activities with relatively lower cytotoxicity towards normal breast epithelial cells (MCF10A) and significantly higher inhibitory effect on cancer cells' migration. The compound 11b was found to possess favourable in silico physicochemical characteristics (lipophilicity-MLOGP, TPSA, and water solubility-ESOL, and others), bioavailability score, and pharmacokinetic properties (GI absorption, BBB non-permeant, P-gp, and CYP2D6). Interestingly, the compound 11b did not show any medicinal chemistry structural alert of PAINS and Brenk filter. The study represents for the first time the successful discovery of new potent anticancer chemotypes using Rutaecarpine natural alkaloid as starting template and reaffirms the significance of natural product-inspired scaffold-hopping technique in drug discovery research.

The alleviatory effects of koumine on MSU-induced gouty arthritis via the TLR4/NF-κB/NLRP3 pathway.

The aim of this study was to validate the preventive effects of koumine (KM), a monoterpene indole alkaloid, on gouty arthritis (GA) and to explore its possible mechanisms. C57BL/6 mice were intraperitoneally administered KM (0.8, 2.4 or 7.2 mg/kg), colchicine (3.0 mg/kg) or sterile saline. One hour later, a monosodium urate (MSU) suspension was injected into the right hind paws of the mice to establish an acute gout model. Inflammation symptoms were evaluated at 0, 3, 6, 12 and 24 h, and the mechanical withdrawal threshold was evaluated at 0, 6 and 24 h. After 24 h, the mice were euthanized, and the joint tissue, kidney and blood were collected for subsequent experiments. Histological examination and antioxidant enzyme, kidney index and serum uric acid (UA) measurements were taken. The expression levels of the signalling pathway components were determined. KM effectively alleviated the symptoms of redness, swelling and pain; counteracted inflammatory cell infiltration; and increased antioxidant enzyme levels, reduced kidney index and serum UA levels through regulating UA excretion in MSU-induced mice. The expression of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB)/nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) signalling pathway proteins and mRNA were reduced in the KM group. These results suggest that KM may be effective in alleviating GA through the TLR4/NF-κB/NLRP3 pathway.

Synthesis and Cheminformatics-Directed Antibacterial Evaluation of Echinosulfonic Acid-Inspired Bis-Indole Alkaloids.

Synthetic efforts toward complex natural product (NP) scaffolds are useful ones, particularly those aimed at expanding their bioactive chemical space. Here, we utilised an orthogonal cheminformatics-based approach to predict the potential biological activities for a series of synthetic bis-indole alkaloids inspired by elusive sponge-derived NPs, echinosulfone A (1) and echinosulfonic acids A-D (2-5). Our work includes the first synthesis of desulfato-echinosulfonic acid C, an α-hydroxy bis(3'-indolyl) alkaloid (17), and its full NMR characterisation. This synthesis provides corroborating evidence for the structure revision of echinosulfonic acids A-C. Additionally, we demonstrate a robust synthetic strategy toward a diverse range of α-methine bis(3'-indolyl) acids and acetates (11-16) without the need for silica-based purification in either one or two steps. By integrating our synthetic library of bis-indoles with bioactivity data for 2048 marine indole alkaloids (reported up to the end of 2021), we analyzed their overlap with marine natural product chemical diversity. Notably, the C-6 dibrominated α-hydroxy bis(3'-indolyl) and α-methine bis(3'-indolyl) analogues (11, 14, and 17) were found to contain significant overlap with antibacterial C-6 dibrominated marine bis-indoles, guiding our biological evaluation. Validating the results of our cheminformatics analyses, the dibrominated α-methine bis(3'-indolyl) alkaloids (11, 12, 14, and 15) were found to exhibit antibacterial activities against methicillin-sensitive and -resistant Staphylococcus aureus. Further, while investigating other synthetic approaches toward bis-indole alkaloids, 16 incorrectly assigned synthetic α-hydroxy bis(3'-indolyl) alkaloids were identified. After careful analysis of their reported NMR data, and comparison with those obtained for the synthetic bis-indoles reported herein, all of the structures have been revised to α-methine bis(3'-indolyl) alkaloids.

Globospiramine Exhibits Inhibitory and Fungicidal Effects against Candida albicans via Apoptotic Mechanisms.

Candidiasis is considered an emerging public health concern because of the occurrence of drug-resistant Candida strains and the lack of an available structurally diverse antifungal drug armamentarium. The indole alkaloid globospiramine from the anticandidal Philippine medicinal plant Voacanga globosa exhibits a variety of biological activities; however, its antifungal properties remain to be explored. In this study, we report the in vitro anticandidal activities of globospiramine against two clinically relevant Candida species (C. albicans and C. tropicalis) and the exploration of its possible target proteins using in silico methods. Thus, the colony-forming unit (CFU) viability assay revealed time- and concentration-dependent anticandidal effects of the alkaloid along with a decrease in the number of viable CFUs by almost 50% at 60 min after treatment. The results of the MIC and MFC assays indicated inhibitory and fungicidal effects of globospiramine against C. albicans (MIC = 8 µg/mL; MFC = 8 µg/mL) and potential fungistatic effects against C. tropicalis at lower concentrations (MIC = 4 µg/mL; MFC > 64 µg/mL). The FAM-FLICA poly-caspase assay showed metacaspase activation in C. albicans cells at concentrations of 16 and 8 µg/mL, which agreed well with the MIC and MFC values. Molecular docking and molecular dynamics simulation experiments suggested globospiramine to bind strongly with 1,3-ß-glucan synthase and Als3 adhesin-enzymes indirectly involved in apoptosis-driven candidal inhibition.

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.