Mass spectrometry-based metabolomics for the elucidation of alkaloid biosynthesis and function in invasive Vincetoxicum rossicum populations.

The genus Vincetoxicum includes a couple of highly invasive vines in North America that threaten biodiversity and challenge land management strategies. Vincetoxicum species are known to produce bioactive phenanthroindolizidine alkaloids that might play a role in the invasiveness of these plants via chemical interactions with other organisms. Untargeted, high-resolution mass spectrometry-based metabolomics approaches were used to explore specialized metabolism in Vincetoxicum plants collected from invaded sites in Ontario, Canada. All metabolites corresponding to alkaloids in lab and field samples of V. rossicum and V. nigrum were identified, which collectively contained 25 different alkaloidal features. The biosynthesis of these alkaloids was investigated by the incorporation of the stable isotope-labelled phenylalanine precursor providing a basis for an updated biosynthetic pathway accounting for the rapid generation of chemical diversity in invasive Vincetoxicum. Aqueous extracts of aerial Vincetoxicum rossicum foliage had phytotoxic activity against seedlings of several species, resulting in identification of tylophorine as a phytotoxin; tylophorine and 14 other alkaloids from Vincetoxicum accumulated in soils associated with full-sun and a high-density of V. rossicum. Using desorption-electrospray ionization mass spectrometry, 15 alkaloids were found to accumulate at wounded sites of V. rossicum leaves, a chemical cocktail that would be encountered by feeding herbivores. Understanding the specialized metabolism of V. rossicum provides insight into the roles and influences of phenanthroindolizidine alkaloids in ecological systems and enables potential, natural product-based approaches for the control of invasive Vincetoxicum and other weedy species.

Benzoindolizidine Alkaloids Tylophorine and Lycorine and Their Analogues with Antiviral, Anti-Inflammatory, and Anticancer Properties: Promises and Challenges.

Ongoing viral research, essential for public health due to evolving viruses, gains significance owing to emerging viral infections such as the SARS-CoV-2 pandemic. Marine and plant alkaloids show promise as novel potential pharmacological strategies. In this narrative review, we elucidated the potential of tylophorine and lycorine, two naturally occurring plant-derived alkaloids with a shared benzoindolizidine scaffold, as antiviral agents to be potentially harnessed against respiratory viral infections. Possible structure-activity relationships have also been highlighted. The substances and their derivatives were found to be endowed with powerful and broad-spectrum antiviral properties; moreover, they were able to counteract inflammation, which often underpins the complications of viral diseases. At last, their anticancer properties hold promise not only for advancing cancer research but also for mitigating the oncogenic effects of viruses. This evidence suggests that tylophorine and lycorine could effectively counteract the pathogenesis of respiratory viral disease and its harmful effects. Although common issues about the pharmacologic development of natural substances remain to be addressed, the collected evidence highlights a possible interest in tylophorine and lycorine as antiviral and/or adjuvant strategies and encourages future more in-depth pre-clinical and clinical investigations to overcome their drawbacks and harness their power for therapeutic purposes.

Bioactive natural products in COVID-19 therapy.

The devastating COVID-19 pandemic has caused more than six million deaths worldwide during the last 2 years. Effective therapeutic agents are greatly needed, yet promising magic bullets still do not exist. Numerous natural products (cordycepin, gallinamide A, plitidepsin, telocinobufagin, and tylophorine) have been widely studied and play a potential function in treating COVID-19. In this paper, we reviewed published studies (from May 2021 to April 2022) relating closely to bioactive natural products (isolated from medicinal plants, animals products, and marine organisms) in COVID-19 therapy in vitro to provide some essential guidance for anti-SARS-CoV-2 drug research and development.

The Synergistic Inhibition of Coronavirus Replication and Induced Cytokine Production by Ciclesonide and the Tylophorine-Based Compound Dbq33b.

Ciclesonide is an inhaled corticosteroid used to treat asthma and has been repurposed as a treatment for mildly ill COVID-19 patients, but its precise mechanism of action is unclear. Herein, we report that ciclesonide blocks the coronavirus-induced production of the cytokines IL-6, IL-8, and MCP-1 by increasing IκBα protein levels and significantly decreasing p65 nuclear translocation. Furthermore, we found that the combination of ciclesonide and dbq33b, a potent tylophorine-based coronavirus inhibitor that affects coronavirus-induced NF-κB activation a little, additively and synergistically decreased coronavirus-induced IL-6, IL-8, and MCP-1 cytokine levels, and synergistically inhibited the replication of both HCoV-OC43 and SARS-CoV-2. Collectively, the combination of ciclesonide and dbq33b merits consideration as a treatment for COVID-19 patients who may otherwise be overwhelmed by high viral loads and an NF-κB-mediated cytokine storm.

Target-Directed Design, Synthesis, Antiviral Activity, and SARs of 9-Substituted Phenanthroindolizidine Alkaloid Derivatives.

On the basis of our previous studies on the antiviral mechanism against tobacco mosaic virus (TMV) and structure-activity relationship of phenanthroindolizidine alkaloids, a series of 9-substituted tylophorine derivatives targeting TMV RNA were designed, synthesized, and assessed for their anti-TMV activities. The bioassay results indicated that most of these compounds showed good in vivo anti-TMV activities, and some of them displayed higher activity than that of commercial ribavirin. Especially, the anti-TMV activities of compound 3b, 4, and 6 are 2-3 times higher than that of commercial ribavirin, according to EC50 values. In this work, we have demonstrated an effective way to design new inhibitors against plant virus and developed 9-ethoxy methyl tylophorine (4) with excellent anti-TMV activity (in vitro activity, 70.2%/500 µg/mL and 27.1%/100 µg/mL; inactivation activity, 67.7%/500 µg/mL and 30.5%/100 µg/mL; curative activity, 65.3%/500 µg/mL and 30.8%/100 µg/mL; and protection activity, 65.9%/500 µg/mL and 36.0%/100 µg/mL) as a potential plant viral inhibitor.

Design, Synthesis and In-Vitro Biological Evaluation of Antofine and Tylophorine Prodrugs as Hypoxia-Targeted Anticancer Agents.

Phenanthroindolizidines, such as antofine and tylophorine, are a family of natural alkaloids isolated from different species of Asclepiadaceas. They are characterized by interesting biological activities, such as pronounced cytotoxicity against different human cancerous cell lines, including multidrug-resistant examples. Nonetheless, these derivatives are associated with severe neurotoxicity and loss of in vivo activity due to the highly lipophilic nature of the alkaloids. Here, we describe the development of highly polar prodrugs of antofine and tylophorine as hypoxia-targeted prodrugs. The developed quaternary ammonium salts of phenanthroindolizidines showed high chemical and metabolic stability and are predicted to have no penetration through the blood-brain barrier. The designed prodrugs displayed decreased cytotoxicity when tested under normoxic conditions. However, their cytotoxic activity considerably increased when tested under hypoxic conditions.

Design, Synthesis and in Vitro Evaluation of Tylophorine Derivatives as Possible Antitumor Agents.

Structural simplification and modification of natural products are always very important resources to antitumor drugs. By introducing various aminomethyl groups and amide groups into the phenanthrene ring of tylophorine, a novel series of tylophorine derivatives have been designed and synthesized, and their antiproliferative activities against MCF-7, A549 and HepG-2 cells have been evaluated, too. The results indicated that most of the prepared compounds exhibited good antitumor activities. Especially, one compound with an {ethyl[2-(morpholin-4-yl)ethyl]amino}methyl group at the side chain exhibited the most significant cytotoxic effects.

Inhibition of SARS-CoV-2 by Highly Potent Broad-Spectrum Anti-Coronaviral Tylophorine-Based Derivatives.

Tylophorine-based compounds and natural cardiotonic steroids (cardenolides and bufadienolides) are two classes of transmissible gastroenteritis coronavirus inhibitors, targeting viral RNA and host cell factors, respectively. We tested both types of compounds against two types of coronaviruses, to compare and contrast their antiviral properties, and with view to their further therapeutic development. Examples of both types of compounds potently inhibited the replication of both feline infectious peritonitis virus and human coronavirus OC43 with EC50 values of up to 8 and 16 nM, respectively. Strikingly, the tylophorine-based compounds tested inhibited viral yields of HCoV-OC43 to a much greater extent (7-8 log magnitudes of p.f.u./ml) than the cardiotonic steroids (about 2-3 log magnitudes of p.f.u./ml), as determined by end point assays. Based on these results, three tylophorine-based compounds were further examined for their anti-viral activities on two other human coronaviruses, HCoV-229E and SARS-CoV-2. These three tylophorine-based compounds inhibited HCoV-229E with EC50 values of up to 6.5 nM, inhibited viral yields of HCoV-229E by 6-7 log magnitudes of p.f.u./ml, and were also found to inhibit SARS-CoV-2 with EC50 values of up to 2.5-14 nM. In conclusion, tylophorine-based compounds are potent, broad-spectrum inhibitors of coronaviruses including SARS-CoV-2, and could be used for the treatment of COVID-19.

Tylophorine-based compounds are therapeutic in rheumatoid arthritis by targeting the caprin-1 ribonucleoprotein complex and inhibiting expression of associated c-Myc and HIF-1α.

Interruption of the Warburg effect - the observation that un-stimulated macrophages reprogram their core metabolism from oxidative phosphorylation toward aerobic glycolysis to become pro-inflammatory M1 macrophages upon stimulation - is an emerging strategy for the treatment of cancer and anti-inflammatory diseases such as rheumatoid arthritis. We studied this process with view to the discovery of novel therapeutics, and found that tylophorine-based compounds targeted a ribonucleoprotein complex containing caprin-1 and mRNAs of c-Myc and HIF-1α in LPS/IFN-γ stimulated Raw264.7 cells, diminished the protein levels of c-Myc and HIF-1α, and consequently downregulated their targeted genes that are associated with the Warburg effect, as well as the pro-inflammatory iNOS and COX2. The tylophorine-based compound DBQ 33b significantly meliorated the severity and incidence of type II collagen-monoclonal antibody-induced rheumatoid arthritis and diminished gene expressions of c-Myc, HIF-1α, iNOS, COX2, TNFα, and IL-17A in vivo. Moreover, pharmacological inhibition of either c-Myc or HIF-1α exhibited similar effects as the tylophorine-based compound DBQ 33b, even though inhibition of c-Myc reversed the induction of iNOS and COX2 in LPS/IFN-γ stimulated Raw264.7 cells to a lesser degree. Therefore, simultaneous inhibition of both c-Myc and HIF-1α is efficacious for anti-inflammation in vitro and in vivo and merits further study.

Tylophorine reduces protein biosynthesis and rapidly decreases cyclin D1, inhibiting vascular smooth muscle cell proliferation in vitro and in organ culture.

BACKGROUND: Tylophorine (TYL) is an alkaloid with antiproliferative action in cancer cells. Vascular smooth muscle cell (VSMC) proliferation and neointima formation contribute to restenosis after percutaneous coronary interventions. HYPOTHESIS/PURPOSE: Our goal was to examine the potential of TYL to inhibit VSMC proliferation and migration, and to dissect underlying signaling pathways. STUDY DESIGN AND METHODS: TYL was administered to platelet-derived growth factor (PDGF-BB)-stimulated, serum-stimulated, quiescent and unsynchronized VSMC of rat and human origin. BrdU incorporation and resazurin conversion were used to assess cell proliferation. Cell cycle progression was analyzed by flow cytometry of propidium iodide-stained nuclei. Expression profiles of proteins and mRNAs were determined using western blot analysis and RT-qPCR. The Click-iT OPP Alexa Fluor 488 assay was used to monitor protein biosynthesis. RESULTS: TYL inhibited PDGF-BB-induced proliferation of rat aortic VSMCs by arresting cells in G1 phase of the cell cycle with an IC50 of 0.13 µmol/l. The lack of retinoblastoma protein phosphorylation and cyclin D1 downregulation corroborated a G1 arrest. Inhibition of proliferation and cyclin D1 downregulation were species- and stimulus-independent. TYL also decreased levels of p21 and p27 proteins, although at later time points than observed for cyclin D1. Co-treatment of VSMC with TYL and MG132 or cycloheximide (CHX) excluded proteasome activation by TYL as the mechanism of action. Comparable time-dependent downregulation of cyclin D1, p21 and p27 in TYL- or CHX-treated cells, together with decreased protein synthesis observed in the Click-iT assay, suggests that TYL is a protein synthesis inhibitor. Besides proliferation, TYL also suppressed migration of PDGF-activated VSMC. In a human saphenous vein organ culture model for graft disease, TYL potently inhibited intimal hyperplasia. CONCLUSION: This unique activity profile renders TYL an interesting lead for the treatment of vasculo-proliferative disorders, such as restenosis.

Tylophorine Abrogates G2/M Arrest Induced by Doxorubicine and Promotes Increased Apoptosis in T47D Breast Cancer Cells

Background: The effects of tylophorine, a natural alkaloid found in Tylophora indica, administered as a single compound or in combination with doxorubicin on cell cycling and apoptosis were assessed in T47D breast cancer cells, selected as a model system for breast cancer. Methods: Cell cycle distribution and apoptosis were examined by flow cytometry. Caspase 3 and 9 expression was determined by immunocytochemistry.Result: We found that tylophorine did not significantly influence the cell cycle distribution of T47D cells. However, the alkaloid did prevent accumulation of cells in the G2/M phase. In addition, tylophorine increased the number of apoptotic cells. Expression of proapoptotic proteins (caspases 3 and 9) was up-regulated upon administration of tyloporine alone or in combination with doxorubicin. Conclusions: Tylophorine alone or in combination with doxorubicin induced apoptosis in T47D breast cancer cells through modulation of the cell cycle and affecting the expression of caspases 3 and 9.

Neoteric trends in tissue culture-mediated biotechnology of Indian ipecac [Tylophora indica (Burm. f.) Merrill].

Tylophora indica (Burm. f.) Merrill, an ethno-pharmacologically important perennial climber of Asclepiadaceae, is commonly known as Antamul or Indian ipecac. It is essentially accredited for its medicinal properties owing to its wide range of alkaloids in the form of bioactive secondary metabolites, such as tylophorine, tylophorinine, and tylophorinidine. Accelerated mass propagation of Tylophora is challenging because of its reduced seed germination frequency that consequently headed the pursuit for efficient protocols on in vitro propagation for the large-scale regeneration, conservation as well as sustainable supply of quality propagules. Ample tissue culture-mediated biotechnological investigations have been carried out on this medicinal plant till date and several micropropagation protocols have been standardized as well. The present review compares between several typical methods as well as factors, involving on direct and indirect organogenesis of Tylophora along with various up-to-date and modified techniques such as somatic embryogenesis, protoplast culture, synthetic seed production, genetic transformation, and in vitro interventions for the secondary metabolite production that have been reported in last two decades. This compilation will allow assessing the achievements and trends of Tylophora research so far, as well as will advance the research more rapidly, since many aspects, basic and applied, have yet to be explored.