Identification of transcription factor genes responsive to MeJA and characterization of a LaMYC2 transcription factor positively regulates lycorine biosynthesis in Lycoris aurea.

Jasmonates (JAs) are among the main phytohormones, regulating plant growth and development, stress responses, and secondary metabolism. As the major regulator of the JA signaling pathway, MYC2 also plays an important role in plant secondary metabolite synthesis and accumulation. In this study, we performed a comparative transcriptome analysis of Lycoris aurea seedlings subjected to methyl jasmonate (MeJA) at different treatment times. A total of 31,193 differentially expressed genes (DEGs) were identified by RNA sequencing. Among them, 732 differentially expressed transcription factors (TFs) comprising 51 TF families were characterized. The most abundant TF family was WRKY proteins (80), followed by AP2/ERF-EFR (67), MYB (59), bHLH (52), and NAC protein (49) families. Subsequently, by calculating the Pearson's correlation coefficient (PCC) between the expression level of TF DEGs and the lycorine contents, 41 potential TF genes (|PCC| >0.8) involved in lycorine accumulation were identified, including 36 positive regulators and 5 negative regulators. Moreover, a MeJA-inducible MYC2 gene (namely LaMYC2) was cloned on the basis of transcriptome sequencing. Bioinformatic analyses revealed that LaMYC2 proteins contain the bHLH-MYC_N domain and bHLH-AtAIB_like motif. LaMYC2 protein is localized in the cell nucleus, and can partly rescue the MYC2 mutant in Arabidopsis thaliana. LaMYC2 protein could interact with most LaJAZs (especially LaJAZ3 and LaJAZ4) identified previously. Transient overexpression of LaMYC2 increased lycorine contents in L. aurea petals, which might be associated with the activation of the transcript levels of tyrosine decarboxylase (TYDC) and phenylalanine ammonia lyase (PAL) genes. By isolating the 887-bp-length promoter fragment upstream of the start codon (ATG) of LaTYDC, we found several different types of E-box motifs (CANNTG) in the promoter of LaTYDC. Further study demonstrated that LaMYC2 was indeed able to bind the E-box (CACATG) present in the LaTYDC promoter, verifying that the pathway genes involved in lycorine biosynthesis could be regulated by LaMYC2, and that LaMYC2 has positive roles in the regulation of lycorine biosynthesis. These findings demonstrate that LaMYC2 is a positive regulator of lycorine biosynthesis and may facilitate further functional research of the LaMYC2 gene, especially its potential regulatory roles in Amaryllidaceae alkaloid accumulation in L. aurea.

Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50 = 20 µM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.

Preparation and Evaluation of Folate Modified PEG-PLLA Nanoparticles Loaded with Lycorine for Glioma Treatment.

Lycorine is a kind of natural active ingredient with a strong antitumor effect. In this study, folate ligand-conjugated polyethylene glycol-block-poly(l-lactide) (PEG-PLLA) nanoparticles (FA-PEG-PLLA NPs) were designed to deliver lycorine to enhance its anti-glioma activity. The successful preparation of the FA-PEG-PLLA polymer was confirmed by 1H-NMR, FT-IR, XRD, TGA, and DSC. The optimal formulation for LYC@FA-PEG-PLLA NPs was determined by response surface analysis as follows: sodium dodecyl sulfate (SDS) of 1%, carrier material of 0.03 g, organic phase volume of 3 mL, and ultrasonic power of 20%. The LYC@FA-PEG-PLLA NPs exhibited an encapsulation efficiency of 83.58% and a particle size of 49.71 nm, demonstrating good stability. Hemolysis experiments, MTT assays, and cell scratch assays revealed excellent biocompatibility of FA-PEG-PLLA and superior anti-glioma activity of LYC@FA-PEG-PLLA NPs compared to the raw drug. Additionally, cell apoptosis assays, ROS experiments, and western blot analysis demonstrated that LYC@FA-PEG-PLLA NPs contributed to cell apoptosis by inducing ROS generation and increasing the expression of NF-κB inhibitory protein IκBα. These results suggested that LYC@FA-PEG-PLLA NPs hold promise for glioma treatment.

Molecular Docking and GC/MS-Based Approach for Identification of Anxiolytic Alkaloids from Griffinia (Amaryllidaceae) Species in a Zebrafish Model.

Griffinia gardneriana Ravenna, Griffinia liboniana Morren and Griffinia nocturna Ravenna (Amarillydaceae) are bulbous plants found in tropical regions of Brazil. Our work aimed to determine the alkaloid profiles of Griffinia spp. and evaluate their anxiolytic potential through in vivo and in silico assays. The plants grown in greenhouses were dried and their ground bulbs were subjected to liquid-liquid partitions, resulting in alkaloid fractions that were analyzed by gas chromatography coupled to mass spectrometry (GC-MS). Anxiolytic activity was evaluated in zebrafish (Danio rerio) through intraperitoneal injection at doses of 40, 100 and 200 mg/kg in light-dark box test. GC-MS analyses revealed 23 alkaloids belonging to different skeleton types: lycorine, homolychorine, galanthamine, crinine, haemanthamine, montanine and narcisclasine. The chemical profiles were relatively similar, presenting 8 alkaloids common to the three species. The major component for G. gardneriana and G. liboniana was lycorine, while G. nocturna consisted mainly of anhydrolycorine. All three alkaloid fractions demonstrated anxiolytic effect. Furthermore, pre-treatment with diazepam and pizotifen drugs was able to reverse the anxiolytic action, indicating involving the GABAergic and serotonergic receptors. Molecular docking showed that the compounds vittatine, lycorine and 11,12-dehydro-2-methoxyassoanine had high affinity with both receptors, suggesting them to be responsible for the anxiolytic effect.

Lycorine and homolycorine derivatives for chemo-sensitizing resistant human ovarian adenocarcinoma cells.

BACKGROUND: Multidrug resistance is the major obstacle to cancer chemotherapy. Modulation of P-glycoprotein and drug combination approaches have been considered important strategies to overcome drug resistance. PURPOSE: Aiming at generating a small library of Amaryllidaceae-type alkaloids to overcome drug resistance, two major alkaloids, isolated from Pancratium maritimum, lycorine (1), and 2α-10bα-dihydroxy-9-O-demethylhomolycorine (2), were derivatized, giving rise to nineteen derivatives (3 - 21). METHODS: The main chemical transformation of lycorine resulted from the cleavage of ring E of the diacetylated lycorine derivative (3) to obtain compounds that have carbamate and amine functions (5 - 16), while acylation of compound 2 provided derivatives 17 - 21. Compounds 1 - 21 were evaluated for their effects on cytotoxicity, and drug resistance reversal, using resistant human ovarian carcinoma cells (HOC/ADR), overexpressing P-glycoprotein (P-gp/ABCB1), as model. RESULTS: Excluding lycorine (1) (IC50 values of 1.2- 2.5 µM), the compounds were not cytotoxic or showed moderate/weak cytotoxicity. Chemo-sensitization assays were performed by studying the in vitro interaction between the compounds and the anticancer drug doxorubicin. Most of the compounds have shown synergistic interactions with doxorubicin. Compounds 5, 6, 9 - 14, bearing both carbamate and aromatic amine moieties, were found to have the highest sensitization rate, reducing the dose of doxorubicin 5-35 times, highlighting their potential to reverse drug resistance in combination chemotherapy. Selected compounds (4 - 6, 9 - 14, and 21), able of re-sensitizing resistant cancer cells, were further evaluated as P-gp inhibitors. Compound 11, which has a para­methoxy-N-methylbenzylamine moiety, was the strongest inhibitor. In the ATPase assay, compounds 9-11 and 13 behaved as verapamil, suggesting competitive inhibition of P-gp. At the same time, none of these compounds affected P-gp expression at the mRNA or protein level. CONCLUSIONS: This study provided evidence of the potential of Amaryllidaceae alkaloids as lead candidates for the development of MDR reversal agents.

Bruceine D and Narclasine inhibit the proliferation of breast cancer cells and the prediction of potential drug targets.

BACKGROUND: Breast cancer is one of the most common female malignancies. This study explored the underlying mechanism through which the two plant compounds (Brucaine D and Narclasine) inhibited the proliferation of breast cancer cells. OBJECTIVE: The purpose of this study was to explore the effect of Brucaine D and Narclasine on breast cancer development and their potential drug targets. METHODS: GSE85871 dataset containing 212 samples and the hallmark gene set "h.all.v2023.1.Hs.symbols.gmt" were downloaded from the Gene Expression Omnibus (GEO) database and the Molecular Signatures Database (MSigDB) database, respectively. Principal component analysis (PCA) was applied to classify clusters showing similar gene expression pattern. Single sample gene set enrichment analysis (ssGSEA) was used to calculate the hallmark score for different drug treatment groups. The expressions of genes related to angiogenesis, glycolysis and cell cycle were detected. Protein-protein interaction (PPI) network analysis was performed to study the interaction of the hub genes. Then, HERB database was employed to identify potential target genes for Narclasine and Bruceine D. Finally, in vitro experiments were conducted to validate partial drug-target pair. RESULTS: PCA analysis showed that the significant changes in gene expression patterns took place in 6 drugs treatment groups (Narciclasine, Bruceine D, Japonicone A, 1beta-hydroxyalatolactone, Britanin, and four mixture drugs) in comparison to the remaining drug treatment groups. The ssGSEA pathway enrichment analysis demonstrated that Narciclasine and Bruceine treatments had similar enriched pathways, for instance, suppressed pathways related to angiogenesis, Glycolysis, and cell cycle, etc.. Further gene expression analysis confirmed that Narciclasine and Bruceine had a strong ability to inhibit these cell cycle genes, and that MYC, CHEK2, MELK, CDK4 and EZH2 were closely interacted with each other in the PPI analysis. Drug target prediction revealed that Androgen Receptor (AR) and Estrogen Receptor 1 (ESR1) were the targets for Bruceine D, and Cytochrome P450 3A4 enzyme (CYP3A4) was the target for Narciclasine. Cell experiments also confirmed the connections between Narciclasine and CYP3A4. CONCLUSION: The present study uncovered that Narciclasine and Bruceine D could inhibit the growth of breast cancer and also predicted the potential targets for these two drugs, providing a new therapeutic direction for breast cancer patients.

Ligand fishing approach to explore Amaryllidaceae alkaloids as potential antiviral candidates targeting SARS-CoV-2 Nsp4.

Ligand fishing, also described as affinity-based assay, represents a convenient and efficient approach to separate potential ligands from complex matrixes or chemical libraries. This approach contributes to the identification of lead compounds that can bind to a specific target. In the context of COVID-19, the search for novel therapeutic agents is crucial. Small molecule-based antiviral drugs, such as Amaryllidaceae alkaloids, have been described as potential candidates because they can inhibit RNA viruses. Among various SARS-CoV-2 proteins, Nsp3, Nsp4, and Nsp6 play a crucial role in the pathogenicity of the virus and are attractive targets for developing COVID-19 treatments. These proteins are responsible for the replication/transcription complex (RTC) within double-membrane vesicles (DMVs), and their inhibition disrupts the virus's infectious cycle. Herein, we have successfully expressed and immobilized the SARS-CoV-2 Nsp4 protein on magnetic beads (Nsp4-MBs) and employed a ligand fishing assay to screen a collection of ten Amaryllidaceae-based alkaloids and applied to Hippeastrum aulicum extract. Remarkably, four out of ten alkaloids, namely 2-α-7-dimethoxyhomolycorine (6), haemanthamine (5), albomaculine (8), and tazettine (9), exhibited selective affinities for Nsp4. Albomaculine (8) and haemanthamine (5) were also identified from extract by the affinity assay. These findings highlight the potential of these alkaloids as model compounds for future drug discovery studies aimed at developing therapeutic interventions against SARS-CoV-2 infections.

Molecular mechanism of lycorine in the treatment of glioblastoma based on network pharmacology and molecular docking.

Lycorine is a naturally active alkaloid that has been shown to have inhibitory effects on a variety of cancers. However, the underlying mechanism of lycorine in the treatment of glioblastoma (GBM) is unclear. In this study, we investigated the mechanism of lycorine in the treatment of GBM based on network pharmacology and molecular docking. Lycorine-related targets overlapped with GBM-related targets to obtain intersections that represent potential anti-GBM targets for lycorine. The protein-protein interaction (PPI) network was constructed using the STRING online database and analyzed by Cytoscape software, and 10 key target genes (AKT1, SRC, HSP90AA1, HRAS, MMP9, BCL2L1, IGF1, MAPK14, STAT1, and KDR) were obtained, which played an important role in the therapeutic effect of lycorine on GBM. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that lycorine acts on GBM by multiple pathways, including inducing apoptosis and reactive oxygen species production. The molecular docking results showed that lycorine had strong binding efficiency with the 10 key genes. In addition, we found that the use of lycorine-induced apoptosis in U-87 MG glioblastoma cells. Here, the mechanism of action of lycorine against GBM was elucidated and verified by experiments, which provided evidence support for its clinical application.

Amaryllidaceae alkaloids from the bulbs of Crinum latifolium L. and their cholinesterase inhibitory activities.

Eleven previously undescribed Amaryllidaceae alkaloids, crinalatifolines A-K (1-11), and two first naturally occurring alkaloids, dihydroambelline (12) and N-demethyldihydrogalanthamine (13), were isolated from the bulbs of Crinum latifolium L. Additionally, thirty-seven known alkaloids and one alkaloid artifact were also isolated from this plant species. Their structures and absolute configurations were elucidated using extensive spectroscopic techniques, including IR, NMR, MS, and ECD. Evaluations of the cholinesterase inhibitory activities of most of these compounds were conducted. Among the tested compounds, ungeremine exhibited the highest potency against acetylcholinesterase and butyrylcholinesterase, with the IC50 values of 0.10 and 1.21 µM, respectively. These values were 9.4- and 2.4-fold more potent than the reference drug galanthamine.

Interplay between Amaryllidaceae alkaloids, the bacteriome and phytopathogens in Lycoris radiata.

Alkaloids are a large group of plant secondary metabolites with various structures and activities. It is important to understand their functions in the interplay between plants and the beneficial and pathogenic microbiota. Amaryllidaceae alkaloids (AAs) are unique secondary metabolites in Amaryllidaceae plants. Here, we studied the interplay between AAs and the bacteriome in Lycoris radiata, a traditional Chinese medicinal plant containing high amounts of AAs. The relationship between AAs and bacterial composition in different tissues of L. radiata was studied. In vitro experiments revealed that AAs have varying levels of antimicrobial activity against endophytic bacteria and pathogenic fungi, indicating the importance of AA synthesis in maintaining a balance between plants and beneficial/pathogenic microbiota. Using bacterial synthetic communities with different compositions, we observed a positive feedback loop between bacteria insensitive to AAs and their ability to increase accumulation of AAs in L. radiata, especially in leaves. This may allow insensitive bacteria to outcompete sensitive ones for plant resources. Moreover, the accumulation of AAs enhanced by insensitive bacteria could benefit plants when challenged with fungal pathogens. This study highlights the functions of alkaloids in plant-microbe interactions, opening new avenues for designing plant microbiomes that could contribute to sustainable agriculture.

Auxin and light-mediated regulation of growth, morphogenesis, and alkaloid biosynthesis in Crinum x powellii 'Album' callus.

Crinum x powellii 'Album' belongs to the Amaryllidaceae medicinal plant family that produces a range of structurally diverse alkaloids with potential therapeutic properties. The optimal conditions for in vitro tissue growth, morphogenesis, and alkaloid biosynthesis remain unclear. Auxin and light play critical roles in regulating plant growth, development, and alkaloid biosynthesis in several Amaryllidaceae plants. Here, we have succeeded in showing, for the first time, that the combination of auxin and light significantly influence C. x powellii "Album" in vitro tissue growth, survival, and morphogenesis compared to individual treatments. Furthermore, this combination also upregulates the expression of alkaloid biosynthetic genes and led to an increase in the content of certain alkaloids, suggesting a positive impact on the defense and therapeutic potential of the calli. Our findings provide insights into the regulation of genes involved in alkaloid biosynthesis in C. x powellii "Album" callus and underline the potential of auxin and light as tools for enhancing their production in plants. This study provides a foundation for further exploration of C. x powellii "Album" calli as a sustainable source of bioactive alkaloids for pharmaceutical and agricultural applications. Furthermore, this study paves the way to the discovery of the biosynthetic pathway of specialized metabolites from C. x powellii "Album", such as cherylline and lycorine.

Lycorine inhibits pancreatic cancer cell growth and neovascularization by inducing Notch1 degradation and downregulating key vasculogenic genes.

Pancreatic cancer is highly metastatic and lethal with an increasing incidence globally and a 5-year survival rate of only 8%. One of the factors contributing to the high mortality is the lack of effective drugs in the clinical setting. We speculated that effective compounds against pancreatic cancer exist in natural herbs and explored active small molecules among traditional Chinese medicinal herbs. The small molecule lycorine (MW: 323.77) derived from the herb Lycoris radiata inhibited pancreatic cancer cell growth with an IC50 value of 1 µM in a concentration-dependent manner. Lycorine markedly reduced pancreatic cancer cell viability, migration, invasion, neovascularization, and gemcitabine resistance. Additionally, lycorine effectively suppressed tumor growth in mouse xenograft models without obvious toxicity. Pharmacological studies revealed that the levels and half-life of Notch1 oncoprotein in the pancreatic cancer cells Panc-1 and Patu8988 were notably reduced. Moreover, the expression of the key vasculogenic genes Semaphorin 4D (Sema4D) and angiopoietin-2 (Ang-2) were also significantly inhibited by lycorine. Mechanistically, lycorine strongly triggered the degradation of Notch1 oncoprotein through the ubiquitin-proteasome system. In conclusion, lycorine effectively inhibits pancreatic cancer cell growth, migration, invasion, neovascularization, and gemcitabine resistance by inducing degradation of Notch1 oncoprotein and downregulating the key vasculogenic genes Sema4D and Ang-2. Our findings provide a new therapeutic candidate and treatment strategy against pancreatic cancer.

Antiproliferative activity and apoptosis-inducing mechanism of Amaryllidaceae alkaloid montanine on A549 and MOLT-4 human cancer cells.

The isoquinoline alkaloids found in Amaryllidaceae are attracting attention due to attributes that can be harnessed for the development of new drugs. The possible molecular mechanisms by which montanine exerts its inhibitory effects against cancer cells have not been documented. In the present study, montanine, manthine and a series of 15 semisynthetic montanine analogues originating from the parent alkaloid montanine were screened at a single test dose of 10 µM to explore their cytotoxic activities against a panel of eight cancer cell lines and one non-cancer cell line. Among montanine and its analogues, montanine and its derivatives 12 and 14 showed the highest cytostatic activity in the initial single-dose screening. However, the native montanine exhibited the greatest antiproliferative activity against cancer cells, with a lower mean IC50 value of 1.39 µM, compared to the displayed mean IC50 values of 2.08 µM for 12 and 3.57 µM for 14. Montanine exhibited the most potent antiproliferative activity with IC50 values of 1.04 µM and 1.09 µM against Jurkat and A549 cell lines, respectively. We also evaluated montanine's cytotoxicity and cell death mechanisms. Our results revealed that montanine triggered apoptosis of MOLT-4 cells via caspase activation, mitochondrial depolarisation and Annexin V/PI double staining. The Western blot results of MOLT-4 cells showed that the protein levels of phosphorylated Chk1 Ser345 were upregulated with increased montanine concentrations. Our findings provide new insights into the mechanisms underlying the cytostatic, cytotoxic and pro-apoptotic activities of montanine alkaloids in lung adenocarcinoma A549 and leukemic MOLT-4 cancer cell types.

Regioselective SmI2-Mediated Radical ipso-Substitution Cyclization for the Construction of Pyrrolophenanthridinone Skeletons: The Synthesis of Amaryllidaceae Alkaloids.

Here, we report a regioselective, samarium(II) diiodide mediated intramolecular radical ipso-substitution cyclization. Through the use of a methoxy group as a leaving group, it was possible to regulate the regioselectivity of the reaction by changing the temperature and additives. We applied the developed reaction to the synthesis of four Amaryllidaceae alkaloids and have shown that the present reaction successfully overcomes regioselectivity issues encountered with other cyclization methods.

Chemical and Biological Aspects of Different Species of the Genus Clinanthus Herb. (Amaryllidaceae) from South America.

The genus Clinanthus Herb. is found in the Andes Region (South America), mainly in Peru, Ecuador, and Bolivia. These plants belong to the Amaryllidaceae family, specifically the Amaryllidoideae subfamily, which presents an exclusive group of alkaloids known as Amaryllidaceae alkaloids that show important structural diversity and pharmacological properties. It is possible to find some publications in the literature regarding the botanical aspects of Clinanthus species, although there is little information available about their chemical and biological activities. The aim of this work was to obtain the alkaloid profile and the anti-cholinesterase activity of four different samples of Clinanthus collected in South America: Clinanthus sp., Clinanthus incarnatus, and Clinanthus variegatus. The alkaloid extract of each sample was analyzed by gas chromatography coupled with mass spectrometry (GC-MS), and their potential against the enzymes acetyl- and butyrylcholinesterase were evaluated. Thirteen alkaloids have been identified among these species, while six unidentified structures have also been detected in these plants. The alkaloid extract of the C. variegatus samples showed the highest structural diversity as well as the best activity against AChE, which was likely due to the presence of the alkaloid sanguinine. The results suggest this genus as a possible interesting new source of Amaryllidaceae alkaloids, which could contribute to the development of new medicines.

Advances on the Amaryllidacea Alkaloids Collected in South Africa, Andean South America and the Mediterranean Basin.

The alkaloids are one of the most represented family of natural occurring biological active compounds. Amaryllidaceae are also very well known for their beautiful flower and are thus used as ornamental plants in historic and public gardens. The Amaryllidacea alkaloids constitute an important group that is subdivided into different subfamilies with different carbon skeletons. They are well known from ancient times for their long application in folk medicine, and in particular, Narcissus poeticus L. was known to Hippocrates of Cos (ca. B.C. 460-370), who treated uterine tumors with a formulate prepared from narcissus oil. To date, more than 600 alkaloids of 15 chemical groups exhibiting various biological activities have been isolated from the Amaryllidaceae plants. This plant genus is diffused in regions of Southern Africa, Andean South America and the Mediterranean basin. Thus, this review describes the chemical and biological activity of the alkaloids collected in these regions in the last two decades as weel those of isocarbostyls isolated from Amaryllidaceae in the same regions and same period.

ERK Inhibition Promotes Engraftment of Allografts by Reprogramming T-Cell Metabolism.

Extracellular regulated protein kinases (ERK) signaling is a master regulator of cell behavior, life, and fate. Although ERK pathway is shown to be involved in T-cell activation, little is known about its role in the development of allograft rejection. Here, it is reported that ERK signaling pathway is activated in allograft-infiltrating T cells. On the basis of surface plasmon resonance technology, lycorine is identified as an ERK-specific inhibitor. ERK inhibition by lycorine significantly prolongs allograft survival in a stringent mouse cardiac allotransplant model. As compared to untreated mice, lycorine-treated mice show a decrease in the number and activation of allograft-infiltrated T cells. It is further confirmed that lycorine-treated mouse and human T cells are less responsive to stimulation in vitro, as indicated by their low proliferative rates and decreased cytokine production. Mechanistic studies reveal that T cells treated with lycorine exhibit mitochondrial dysfunction, resulting in metabolic reprogramming upon stimulation. Transcriptome analysis of lycorine-treated T cells reveals an enrichment in a series of downregulated terms related to immune response, the mitogen-activated protein kinase cascade, and metabolic processes. These findings offer new insights into the development of immunosuppressive agents by targeting the ERK pathway involved in T-cell activation and allograft rejection.

Amaryllidaceae alkaloids in skin cancer management: Photoprotective effect on human keratinocytes and anti-proliferative activity in melanoma cells.

Skin cancer has high rates of mortality and therapeutic failure. In this study, to develop a multi-agent strategy for skin cancer management, the selective cytotoxicity of several alkaloid fractions and pure alkaloids isolated from Amaryllidaceae species was evaluated in melanoma cells. In addition, UVB-stimulated keratinocytes (HaCaT) were exposed to seven alkaloid fractions characterized by GC-MS, and the production of intracellular reactive oxygen species (ROS) and IL-6, were measured to evaluate their photoprotection effects. The Eucharis caucana (bulb) alkaloid fraction (20 µg/ml) had a clear effect on the viability of melanoma cells, reducing it by 45.7% without affecting healthy keratinocytes. This alkaloid fraction and tazettine (both at 2.5 µg/ml) suppressed UVB-induced ROS production by 31.6% and 29.4%, respectively. The highest anti-inflammatory potential was shown by the Zephyranthes carinata (bulb) alkaloid fraction (10 µg/ml), which reduced IL-6 production by 90.8%. According to the chemometric analysis, lycoramine and tazettine had a photoprotective effect on the UVB-exposed HaCaT cells, attenuating the production of ROS and IL-6. These results suggest that Amaryllidaceae alkaloids have photoprotective and therapeutic potential in skin cancer management, especially at low concentrations.

Anti-SARS-CoV-2 Activity and Cytotoxicity of Amaryllidaceae Alkaloids from Hymenocallis littoralis.

The Amaryllidaceae species are well-known as a rich source of bioactive compounds in nature. Although Hymenocallis littoralis has been studied for decades, its polar components were rarely explored. The current phytochemical investigation of Amaryllidaceae alkaloids from H. littoralis led to the identification of three previously undescribed compounds: O-demethyl-norlycoramine (1), (-)-2-epi-pseudolycorine (2) and (+)-2-epi-pseudolycorine (3), together with eight known compounds: 6α-hydroxyhippeastidine (4), 6ß-hydroxyhippeastidine (5), lycorine (6), 2-epi-lycorine (7), zephyranthine (8), ungeremine (9), pancratistatin (10) and 9-O-demethyl-7-O-methyllycorenine (11). Among the eight previously reported compounds, five were isolated from H. littoralis for the first time (compounds 4, 5, 7, 8, and 9). Compounds 1, 4, 5, 7, 8, and 11 exhibited weak anti-SARS-CoV-2 activity (EC50 = 40-77 µM) at non-cytotoxic concentrations. Assessment of cytotoxicity on the Vero-E6 cell line revealed lycorine and pancratistatin as cytotoxic substances with CC50 values of 1.2 µM and 0.13 µM, respectively. The preliminary structure-activity relationship for the lycorine-type alkaloids in this study was further investigated, and as a result ring C appears to play a crucial role in their anti-SARS-CoV-2 activity.

Cripowellins Pause Plasmodium falciparum Intraerythrocytic Development at the Ring Stage.

Cripowellins from Crinum erubescens are known pesticidal and have potent antiplasmodial activity. To gain mechanistic insights to this class of natural products, studies to determine the timing of action of cripowellins within the asexual intraerythrocytic cycle of Plasmodium falciparum were performed and led to the observation that this class of natural products induced reversible cytostasis in the ring stage within the first 24 h of treatment. The transcriptional program necessary for P. falciparum to progress through the asexual intraerythrocytic life cycle is well characterized. Whole transcriptome abundance analysis showed that cripowellin B "pauses" the transcriptional program necessary to progress through the intraerythrocytic life cycle coinciding with the lack of morphological progression of drug treated parasites. In addition, cripowellin B-treated parasites re-enter transcriptional progression after treatment was removed. This study highlights the use of cripowellins as chemical probes to reveal new aspects of cell cycle progression of the asexual ring stage of P. falciparum which could be leveraged for the generation of future antimalarial therapeutics.