Addressing the Evolution of Cardenolide Formation in Iridoid-Synthesizing Plants: Site-Directed Mutagenesis of PRISEs (Progesterone-5β-Reductase/Iridoid Synthase-like Enzymes) of Plantago Species.

Enzymes capable of processing a variety of compounds enable plants to adapt to diverse environmental conditions. PRISEs (progesterone-5β-reductase/iridoid synthase-like enzymes), examples of such substrate-promiscuous enzymes, are involved in iridoid and cardenolide pathways and demonstrate notable substrate promiscuity by reducing the activated C=C double bonds of plant-borne and exogenous 1,4-enones. In this study, we identified PRISE genes in () and (), and the corresponding enzymes were determined to share a sequence identity of 95%.

Overexpression and RNAi-mediated Knockdown of Two 3β-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis.

3β-hydroxy-Δ5-steroid dehydrogenases (3βHSDs) are supposed to be involved in -cardenolide biosynthesis. Here, a novel () was isolated from shoot cultures and expressed in . Recombinant 3βHSD1 and 3βHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only 3βHSD2 converted small ketones and secondary alcohols efficiently.

Effects of Lipophilicity and Structural Features on the Antiherpes Activity of Digitalis Cardenolides and Derivatives.

There is growing interest in exploring Digitalis cardenolides as potential antiviral agents. Hence, we herein investigated the influence of structural features and lipophilicity on the antiherpes activity of 65 natural and semisynthetic cardenolides assayed in vitro against HSV-1. The presence of an α,β-unsaturated lactone ring at C-17, a β-hydroxy group at C-14 and C-3β-OR substituents were considered essential requirements for this biological activity.

Knockout of , Encoding a PRISE (Progesterone 5β-Reductase/Iridoid Synthase-Like Enzyme), Leads to Metabolic Changes in Response to Exogenous Methyl Vinyl Ketone (MVK).

Small or specialized natural products (SNAPs) produced by plants vary greatly in structure and function, leading to selective advantages during evolution. With a limited number of genes available, a high promiscuity of the enzymes involved allows the generation of a broad range of SNAPs in complex metabolic networks. Comparative metabolic studies may help to understand why-or why not-certain SNAPs are produced in plants.

RNAi-mediated gene knockdown of progesterone 5β-reductases in Digitalis lanata reduces 5β-cardenolide content.

Studying RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes) in 5β-cardenolide formation. Progesterone 5β-reductases (P5βR) are assumed to catalyze the reduction of progesterone to 5β-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5β-cardenolides. P5βRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes.

21-Hydroxypregnane 21-O-malonylation, a crucial step in cardenolide biosynthesis, can be achieved by substrate-promiscuous BAHD-type phenolic glucoside malonyltransferases from Arabidopsis thaliana and homolog proteins from Digitalis lanata.

Three putative 21-hydroxypregnane 21-O-malonyltransferases (21MaT) from Digitalis lanata were partially purified. Two of them were supposed to be BAHD-type enzymes. We were unable to purify them in quantities necessary for reliable sequencing.

Cytotoxicity of glucoevatromonoside alone and in combination with chemotherapy drugs and their effects on Na,K-ATPase and ion channels on lung cancer cells.

Cardiac glycosides (CGs) are useful drugs to treat cardiac illnesses and have potent cytotoxic and anticancer effects in cultured cells and animal models. Their receptor is the Na,K ATPase, but other plasma membrane proteins might bind CGs as well. Herein, we evaluated the short- and long-lasting cytotoxic effects of the natural cardenolide glucoevatromonoside (GEV) on non-small-cell lung cancer H460 cells.

Semisynthetic Cardenolides Acting as Antiviral Inhibitors of Influenza A Virus Replication by Preventing Polymerase Complex Formation.

Influenza virus infections represent a major public health issue by causing annual epidemics and occasional pandemics that affect thousands of people worldwide. Vaccination is the main prophylaxis to prevent these epidemics/pandemics, although the effectiveness of licensed vaccines is rather limited due to the constant mutations of influenza virus antigenic characteristics. The available anti-influenza drugs are still restricted and there is an increasing viral resistance to these compounds, thus highlighting the need for research and development of new antiviral drugs.

Elucidation of the mechanism of anti-herpes action of two novel semisynthetic cardenolide derivatives.

Human herpesviruses are among the most prevalent pathogens worldwide and have become an important public health issue. Recurrent infections and the emergence of resistant viral strains reinforce the need of searching new drugs to treat herpes virus infections. Cardiac glycosides are used clinically to treat cardiovascular disturbances, such as congestive heart failure and atrial arrhythmias.

Investigation of the cytotoxic activity of two novel digitoxigenin analogues on H460 lung cancer cells.

Cardiac glycosides (CGs) are natural compounds traditionally used for the treatment of heart disorders, and recently new therapeutic possibilities were proposed. Their antitumor reports and clinical trials have notably enhanced, including those targeted for lung cancer, the most lethal type that lacks of new treatment agents, instigating the research of these molecules. The CGs studied here, named C10 {3β-[(N-(2-hydroxyethyl)aminoacetyl]amino-3-deoxydigitoxigenin} and C18 (3β-(aminoacetyl)amino-3-deoxydigitoxigenin), are semisynthetic derivatives prepared from digitoxigenin scaffold.

Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae.

A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ -3β-hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Δ -isomerase gene from Comamonas testosteronii, (c) a mutated steroid-5β-reductase gene from Arabidopsis thaliana, and (d) a steroid 21-hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed.

Potential anti-herpes and cytotoxic action of novel semisynthetic digitoxigenin-derivatives.

In recent years, new therapeutic possibilities were proposed for cardiac glycosides traditionally used to treat heart diseases, such as anticancer and antiviral activities. In this sense, this work aimed to synthesize the readily accessible 3β-azido-3-deoxydigitoxigenin (5) from digitoxigenin (1). Two new series of compounds were obtained from derivative (5): (i) O-glycosyl trizols through click chemistry with propargyl glycosides; and (ii) compounds substituted in the alpha carbonyl position with different residues linked via an amino-group.

Exploiting enzyme promiscuity to shape plant specialized metabolism.

The amazing variability of plant metabolism and its rapid divergence during evolution pose fundamental questions as to the driving forces, mechanisms, and players in metabolic differentiation. This review examines concepts that help us understand adaptive pathway evolution, with a particular emphasis on plant specialized metabolism, previously often termed secondary metabolism. Following a general introduction to pathway and metabolite evolution, the focus is directed to enzyme promiscuity and its classification.

PRISEs (progesterone 5β-reductase and/or iridoid synthase-like 1,4-enone reductases): Catalytic and substrate promiscuity allows for realization of multiple pathways in plant metabolism.

PRISEs (progesterone 5β-reductase and/or iridoid synthase-like 1,4-enone reductases) are involved in cardenolide and iridoid biosynthesis. We here investigated a PRISE (rAtSt5βR) from Arabidopsis thaliana, a plant producing neither cardenolides nor iridoids. The structure of rAtSt5βR was elucidated with X-ray crystallography and compared to the known structures of PRISEs from Catharanthus roseus (rCrISY) and Digitalis lanata (rDlP5βR).

Cytotoxicity of AMANTADIG - a semisynthetic digitoxigenin derivative - alone and in combination with docetaxel in human hormone-refractory prostate cancer cells and its effect on Na/K-ATPase inhibition.

Cardiac glycosides (CGs) are natural compounds used to treat congestive heart failure. They have garnered attention as a potential cancer treatment option, especially because they bind to Na/K-ATPase as a target and activate intracellular signaling pathways leading to a variety of cellular responses. In this study we evaluated AMANTADIG, a semisynthetic cardenolide derivative, for its cytotoxic activity in two human androgen-insensitive prostate carcinoma cell lines, and the potential synergistic effects with docetaxel.

Cardiac Glycoside Glucoevatromonoside Induces Cancer Type-Specific Cell Death.

Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity.

Cytotoxic and cytostatic effects of digitoxigenin monodigitoxoside (DGX) in human lung cancer cells and its link to Na,K-ATPase.

Cardiac glycosides (CGs) are natural compounds widely used to treat several cardiac conditions and more recently have been recognized as potential antitumor agents. They are known as Na,K-ATPases ligands, which is a promising drug target in cancer. In this study, the short and long-lasting cytotoxic effects of the natural cardenolide digitoxigenin monodigitoxoside (DGX) were evaluated against two non-small cell lung cancer lines (A549 and H460 cells).

The Foxgloves (Digitalis) Revisited.

This review provides a renewed look at the genus . Emphasis will be put on those issues that attracted the most attention or even went through paradigmatic changes since the turn of the millennium. PubMed and Google Scholar were used ("" and "Foxglove" were the key words) to identify research from 2000 till 2017 containing data relevant enough to be presented here.

Production of the Cytotoxic Cardenolide Glucoevatromonoside by Semisynthesis and Biotransformation of Evatromonoside by a Digitalis lanata Cell Culture.

Recent studies demonstrate that cardiac glycosides, known to inhibit Na/K-ATPase in humans, have increased susceptibility to cancer cells that can be used in tumor therapy. One of the most promising candidates identified so far is glucoevatromonoside, which can be isolated from the endangered species ssp. .

Cytotoxic effects of the cardenolide convallatoxin and its Na,K-ATPase regulation.

Cardenolides are cardiac glycosides, mostly obtained from natural sources. They are well known for their inhibitory action on the Na,K-ATPase, an effect that regulates cardiovascular alterations such as congestive heart failure and atrial arrhythmias. In recent years, they have also sparked new interest in their anticancer potential.

A new semisynthetic cardenolide analog 3β-[2-(1-amantadine)- 1-on-ethylamine]-digitoxigenin (AMANTADIG) affects G2/M cell cycle arrest and miRNA expression profiles and enhances proapoptotic survivin-2B expression in renal cell carcinoma cell lines.

Cardiac glycosides are well known in the treatment of cardiovascular diseases; however, their application as treatment option for cancer patients is under discussion. We showed that the cardiac glycoside digitoxin and its analog AMANTADIG can inhibit the growth of renal cell carcinoma (RCC) cell lines and increase G2/M cell cycle arrest. To identify the signaling pathways and molecular basis of this G2/M arrest, microRNAs were profiled using microRNA arrays.

Three-Step Test System for the Identification of Novel GABA Receptor Modulating Food Plants.

Potentiation of γ-amino butyric acid (GABA)-induced GABA receptor (GABAR) activation is a common pathway to achieve sedative, sleep-enhancing, anxiolytic, and antidepressant effects. Presently, a three-component test system was established for the identification of novel GABAR modulating food plants. In the first step, potentiation of GABA-induced response of the GABAR was analysed by two-electrode voltage clamp (TEVC) for activity on human α1β2-GABAR expressed in Xenopus laevis oocytes.

Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris.

The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase from T.

Progesterone 5β-reductases/iridoid synthases (PRISE): gatekeeper role of highly conserved phenylalanines in substrate preference and trapping is supported by molecular dynamics simulations.

Vein Patterning 1 (VEP1)-encoded progesterone 5β-reductases/iridoid synthases (PRISE) belong to the short-chain dehydrogenase/reductase superfamily of proteins. They are characterized by a set of highly conserved amino acids in the substrate-binding pocket. All PRISEs are capable of reducing the activated C=C double bond of various enones enantioselectively and therefore have a potential as biocatalysts in bioorganic synthesis.

The New Semisynthetic Cardenolide Analog 3β-[2-(1-Amantadine)-1-on-ethylamine]-digitoxigenin (AMANTADIG) Efficiently Suppresses Cell Growth in Human Leukemia and Urological Tumor Cell Lines.

Background/aim: The use of cardenolides in the treatment of cardiac insufficiency is well-established. However, the potential of cardenolides in tumor therapy has not been comprehensively studied. The aim of the present study was to characterize the cytotoxic effects of the new semisynthetic cardenolide analog AMANTADIG (3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin), and the cardenolide digitoxin on leukemia and urological tumor cell lines.