Thevetia thevetioides Cardenolide and Related Cardiac Glycoside Profile in Mature and Immature Seeds by High-Resolution Thin-Layer Chromatography (HPTLC) and Quadrupole Time of Flight-Tandem Mass Spectrometry (Q-TOF MS/MS) Reveals Insights of the Cardenolide Biosynthetic Pathway.

Thevetia thevetioides is a species within the Apocynaceae family known for containing cardenolide-glycosides, commonly referred to as cardiac glycosides, which are characteristic of this genus. The seeds of the Thevetia species are frequently used as a model source for studying cardiac steroids, as these glycosides can be more readily extracted from the oil-rich seeds than from the plant's green tissues. In this work, the cardenolide profile of ripe and immature seeds was determined and compared to establish the main differences. Ripe seeds contain six related cardenolides and triosides, with thevetin B being the predominant component. In contrast, immature seeds exhibit a total of thirteen cardiac glycosides, including monoglycosides such as neriifolin and peruvosides A, B, and C, as well as diglycosides like thevebiosides A, B, and C. Some of these compounds have previously been identified as degradation products of more complex cardiac glycosides; however, their presence in immature seeds, as described in this study, suggests that they may serve as biosynthetic precursors to the triosides observed in mature seeds. The glycoside patterns observed via HPTLC are associated with specific chemical structures characteristic of this genus, typically featuring thevetose or acetyl-thevetose at the first position, followed by glucose or gentibiose in di- or trisaccharides, independent of the trioside aglycones identified: digitoxigenin, cannogenin, or yccotligenin. Ripe seeds predominantly contain triosides, including thevetin B, C, and A, the latter of which has not been previously reported.

Evaluation of the activity of cardiac glycosides on RORγ and RORγT nuclear receptors.

Cardiac glycosides, derived from plants and animals, have been recognized since ancient times. These substances hinder the function of the sodium-potassium pump within eukaryotic cells. Many reports have shown that these compounds influence the activity of nuclear receptors. Thus, we assessed the effects of various cardiac glycosides at nontoxic concentrations on RORγ and RORγT. RORγT is a crucial protein involved in the differentiation of Th17 lymphocytes. Sixteen analyzed cardiac glycosides exhibited varying toxicities in HepG2 cells, all of which demonstrated agonistic effects on RORγ, as confirmed in the RORγ-HepG2 reporter cell line. The overexpression of both the RORγ and RORγT isoforms intensified the effects of these compounds. Additionally, these glycosides induced the expression of G6PC, a gene regulated by RORγ, in HepG2 cells. Subsequently, the effects of two endogenous cardiac glycosides (marinobufagenin and ouabain) and the three most potent glycosides (bufalin, oleandrin, and telecinobufagenin) were evaluated in Th17 primary lymphocytes. All of these compounds increased the expression of the IL17A, IL17F, IFNG, and CXCL10 genes, but they exhibited varying effects on GZMB and CCL20 expression. Molecular docking analysis revealed the robust binding affinity of cardiac glycosides for the ligand binding domain of the RORγ/RORγT receptors. Thus, we demonstrated that at nontoxic concentrations, cardiac glycosides have agonistic effects on RORγ/RORγT nuclear receptors, augmenting their activity. This potential can be harnessed to modulate the phenotype of IL17-expressing cells (e.g., Th17 or Tc17 lymphocytes) in adoptive therapy for combating various types of cancer.

Cardiac glycosides with cytotoxic activity from the seeds of Thevetia peruviana.

Phytochemical investigation on the extract of the seeds of Thevetia peruviana resulted in the isolation of six new cardiac glycosides, namely theveperosides A-F (1-6), including a rare 19-nor-cardenolide (1), together with seven known analogues (7-13). The chemical structures of these compounds were determined based on detailed spectroscopic analysis. The cytotoxic activities of 1-13 were evaluated against MCF-7, HCT-116, HeLa, and HepG2 cancer cell lines, and their structure-activity relationships (SARs) were investigated. Compound 3 exhibited the significant cytotoxic effects with IC50 values ranging from 0.032 to 0.055 µΜ, which could induce HepG2 cells apoptosis in a dose-dependent manner.

Cardiac glycosides from Streblus asper with potential antiviral activity.

Ten undescribed cardiac glycosides, strasperosides A-J, together with twelve known analogues, were isolated from Streblus asper Lour. Their structures were elucidated on the basis of spectroscopic analysis, electronic circular dichroism data, and chemical methods. These cardiac glycosides showed diversity in steroid skeleton and sugar moiety. Strasperosides A and B are a pair of unusual stereoisomers featuring different orientation of the lactone motif. Ten cardiac glycosides demonstrated potent antiviral effects on HSV-1 in vitro with the IC50 values from 0.19 ± 0.08 to 1.03 ± 0.25 µM and the therapeutic indices from 66.61 ± 5.08 to 326.75 ± 11.75.

Bioassay-Guided Fractionation and Biological Activity of Cardenolides from Streptocaulon juventas.

The discovery that Na/K-ATPase acts as a signal transducer led us to investigate the structural diversity of cardiotonic steroids and study their ligand effects. By applying Na/K-ATPase activity assay-guided fractionation, we isolated a total of 20 cardiotonic steroids from Streptocaulon juventas, including an undescribed juventasoside B (10: ) and 19 known cardiotonic steroids. Their structures have been elucidated. Using our platform of purified Na/K-ATPase and an LLC-PK1 cell model, we found that 10: , at a concentration that induces less than 10% Na/K-ATPase inhibition, can stimulate the Na/K-ATPase/Src receptor complex and selectively activate downstream pathways, ultimately altering prostate cancer cell growth. By assessing the ligand effect of the isolated cardiotonic steroids, we found that the regulation of cell viability by the isolated cardiotonic steroids was not associated with their inhibitory potencies against Na/K-ATPase activity but reflected their ligand-binding affinity to the Na/K-ATPase receptor. Based on this discovery, we identified a unique active cardiotonic steroid, digitoxigenin (1: ), and verified that it can protect LLC-PK1 cells from hypoxic injury, implicating its potential use in ischemia/reperfusion injury and inducing collagen synthesis in primary human dermal fibroblast cells, and implicating that compound 2: is the molecular basis of the wound healing activity of S. juventas.

The Cytotoxic Cardiac Glycoside (-)-Cryptanoside A from the Stems of Cryptolepis dubia and Its Molecular Targets.

A cardiac glycoside epoxide, (-)-cryptanoside A (1), was isolated from the stems of Cryptolepis dubia collected in Laos, for which the complete structure was confirmed by analysis of its spectroscopic and single-crystal X-ray diffraction data, using copper radiation at a low temperature. This cardiac glycoside epoxide exhibited potent cytotoxicity against several human cancer cell lines tested, including HT-29 colon, MDA-MB-231 breast, OVCAR3 and OVCAR5 ovarian cancer, and MDA-MB-435 melanoma cells, with the IC50 values found to be in the range 0.1-0.5 µM, which is comparable with that observed for digoxin. However, it exhibited less potent activity (IC50 1.1 µM) against FT194 benign/nonmalignant human fallopian tube secretory epithelial cells when compared with digoxin (IC50 0.16 µM), indicating its more selective activity toward human cancer versus benign/nonmalignant cells. (-)-Cryptanoside A (1) also inhibited Na+/K+-ATPase activity and increased the expression of Akt and the p65 subunit of NF-κB but did not show any effects on the expression of PI3K. A molecular docking profile showed that (-)-cryptanoside A (1) binds to Na+/K+-ATPase, and thus 1 may directly target Na+/K+-ATPase to mediate its cancer cell cytotoxicity.

The Na+/K+-ATPase: A potential therapeutic target in cardiometabolic diseases.

Cardiometabolic diseases (CMD) are a direct consequence of modern living and contribute to the development of multisystem diseases such as cardiovascular diseases and diabetes mellitus (DM). CMD has reached epidemic proportions worldwide. A sodium pump (Na+/K+-ATPase) is found in most eukaryotic cells' membrane and controls many essential cellular functions directly or indirectly. This ion transporter and its isoforms are important in the pathogenesis of some pathological processes, including CMD. The structure and function of Na+/K+-ATPase, its expression and distribution in tissues, and its interactions with known ligands such as cardiotonic steroids and other suspected endogenous regulators are discussed in this review. In addition, we reviewed recent literature data related to the involvement of Na+/K+-ATPase activity dysfunction in CMD, focusing on the Na+/K+-ATPase as a potential therapeutic target in CMD.

Epistatic Effects Between Amino Acid Insertions and Substitutions Mediate Toxin resistance of Vertebrate Na+,K+-ATPases.

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino acid substitutions in the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work revealed that hystricognath rodents (e.g., chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino acid insertions in the H1-H2 loop, but their functional significance was not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance in homologs of each of the two species that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla NKA unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse NKA, the amino acid insertion and substitution Q111R both contribute to an augmented CTS resistance without compromising ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific mutational effects on CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

Synthesis and Biological Evaluation of Cardiac Glycosides for Cancer Therapy by Targeting the DNA Damage Response.

Cardiac glycosides (CGs) are bioactive compounds originally used to treat heart diseases, but recent studies have demonstrated their anticancer activity. We previously demonstrated that Antiaris toxicaria 2 (AT2) possesses anticancer activity in KRAS mutated lung cancers via impinging on the DNA damage response (DDR) pathway. Toward developing this class of molecules for cancer therapy, herein we report a multistep synthetic route utilizing k-strophanthidin as the initial building block for determination of structure-activity relationships (SARs). A systematic structural design approach was applied that included modifications of the sugar moiety, the glycoside linker, stereochemistry, and lactone ring substitutions to generate a library of O-glycosides and MeON-neoglycosides derivatives. These molecules were screened for their anticancer activities and their impact on DDR signaling in KRAS mutant lung cancer cells. These results demonstrate the ability to chemically synthesize CG derivatives and define the SARs to optimize AT2 as a cancer therapeutic.

The Na+,K+-ATPase in complex with beryllium fluoride mimics an ATPase phosphorylated state.

The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane via a functional cycle that includes various phosphoenzyme intermediates. However, the structure and function of these intermediates and how metal fluorides mimick them require further investigation. Here, we describe a 4.0 Å resolution crystal structure and functional properties of the pig kidney Na+,K+-ATPase stabilized by the inhibitor beryllium fluoride (denoted E2-BeFx). E2-BeFx is expected to mimic properties of the E2P phosphoenzyme, yet with unknown characteristics of ion and ligand binding. The structure resembles the E2P form obtained by phosphorylation from inorganic phosphate (Pi) and stabilized by cardiotonic steroids, including a low-affinity Mg2+ site near ion binding site II. Our anomalous Fourier analysis of the crystals soaked in Rb+ (a K+ congener) followed by a low-resolution rigid-body refinement (6.9-7.5 Å) revealed preocclusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg2+ location indicates a site of initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Furthermore, using binding and activity studies, we find that the BeFx-inhibited enzyme is also able to bind ADP/ATP and Na+. These results relate the E2-BeFx complex to a transient K+- and ADP-sensitive E∗P intermediate of the functional cycle of the Na+,K+-ATPase, prior to E2P.

Cardiac glycosides from the roots of Streblus asper Lour. with activity against Epstein-Barr virus lytic replication.

Cardiac glycosides (CGs) show potential broad-spectrum antiviral activity by targeting cellular host proteins. Herein are reported the isolation of five new (1-5) and eight known (7-13) CGs from the roots of Streblus asper Lour. Of these compounds 1 and 7 exhibited inhibitory action against EBV early antigen (EA) expression, with half-maximal effective concentration values (EC50) being less than 60 nM, and they also showed selectivity, with selectivity index (SI) values being 56.80 and 103.17, respectively. Preliminary structure activity relationships indicated that the C-10 substituent, C-5 hydroxy groups, and C-3 sugar unit play essential roles in the mediation of the inhibitory activity of CGs against EBV. Further enzyme experiments demonstrated that these compounds might inhibit ion pump function and thereby change the intracellular signal transduction pathway by binding to Na+/K+-ATPase, as validated by simulated molecular docking. This study is the first report that CGs can effectively limit EBV lytic replication, and the observations made in this study may be of value for lead compound development.

Cytotoxic cardiac glycosides from the root of Streblus asper.

Bioassay-guided separation of the root of Streblus asper led to the identification of six undescribed cardiac glycosides, including a rare cardiac glycoside dimer, along with twelve previously reported analogues. Their structures were determined on the basis of analyses of spectroscopic methods (1D and 2D-NMR spectroscopy), high-resolution electrospray ionization mass spectrometry (HRESIMS), circular dichroism (CD), and comparison of their spectroscopic data with previously reported data. Regarding their cytotoxic activities, microculture tetrazolium assays showed that all isolated cardiac glycosides strongly inhibited MCC-803, T24, SKOV-3, HepG2, Wi-38, and A549 cancer cell lines, with IC50 values ranging from 0.075 µM to 0.752 µM. One cardiac glycoside, a rare cardiac glycoside dimer, exhibited the strongest activity against the six cancer cell lines, with IC50 values ranging from 0.075 µM to 0.214 µM. In addition, the structure-activity relationships (SARs) of cardiac glycosides were investigated. In summary, S. asper showed marked cytotoxicity to several cancer cell lines, which could be meaningful for discovering new anticancer agents.

Investigation of cardiac glycosides from oleander in a human induced pluripotent stem cells derived cardiomyocyte model.

The ingestion of Nerium oleander and Thevetia peruviana are common causes for poisoning in Southeast Asia. All parts of the oleander shrub contain cardiac glycosides of the cardenolide type. These glycosides act via inhibition of a Na+/K+-ATPase which might cause severe arrhythmia and subsequent death in oleander-poisoned patients. The current study uses human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) in a microelectrode array (MEA) system to assess the cardiac effects of neriifolin, oleandrin, digitoxigenin, peruvoside and thevetin A from the oleander plant. Digoxin was used as established reference compound. All tested compounds showed a corrected field potential duration (FPDc) shortening and was the lowest for 600 nM digitoxigenin with -36.9 ± 1.2 %. Next to the dose-dependent pro-arrhythmic potential, a complete beat arrest of the spontaneously beating hiPSC-CM was observed at a concentration of 300 nM for neriifolin, 600 nM for oleandrin and 1000 nM for digitoxigenin and peruvoside. Thevetin A did not cause arrhythmia up to a final concentration of 1000 nM. Thus, it was possible to establish a cardiac effect rank order of the tested substances: neriifolin > oleandrin > digitoxigenin = peruvoside > digoxin > thevetin A.

Malayoside, a cardenolide glycoside extracted from Antiaris toxicaria Lesch, induces apoptosis in human non-small lung cancer cells via MAPK-Nur77 signaling pathway.

Lung cancer is the leading cause of cancer deaths in the world. Non-small cell lung cancer (NSCLC), with poor prognosis and resistance to chemoradiotherapy, is the most common histological type of lung cancer. Therefore, it is necessary to develop new and more effective treatment strategy for NSCLC. Nur77, an orphan member of the nuclear receptor superfamily, induces apoptosis in cancer cells including NSCLC cells, by high expression and translocation to mitochondria. Small molecules trigger expression and mitochondrial localization of Nur77 may be an ideal anti-cancer drug candidate. Here, we report malayoside, a cardiac glycoside in the extract of Antiaris toxicaria Lesch., had different sensitivities to NSCLC cells. Malayoside induced apoptosis in NCI-H460 cells. Meanwhile, malayoside induced Nur77 expression and mitochondrial localization, and its induction of apoptosis was Nur77-dependent. To investigate the molecular mechanism of malayoside inducing Nur77 and apoptosis, we found that malayoside activated MAPK signaling pathway, including both ERK and p38 phosphorylation. The suppression of MAPK signaling activation inhibited the expression of Nur77 and apoptosis induced by malayoside. Our studies in nude mice showed that malayside potently inhibited the growth of tumor cells in vivo. Furthermore, the anti-cancer effect of malayosidwas in vivo was also related to the elevated expression of Nur77, p-ERK, and p-p38 proteins. Our results suggest that malayoside possesses an anti-NSCLC activity in vitro and in vivo mainly via activation of MAPK-Nur77 signaling pathway, indicating that malayoside is a promising chemotherapeutic candidate for NSCLC.

Structural Analysis of Diastereomeric Cardiac Glycosides and Their Genins Using Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry.

Ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) is an economical and indispensable tool in natural product research to investigate novel metabolites, biomarker discovery, chemical diversity exploration, and structure elucidation. In this study, the structural analysis of 38 naturally occurring cardiac glycosides (CGs) in various tissues of Nerium oleander was achieved by the extensive use of mass spectrometry. The chemical diversity of CGs was described on the basis of characteristic MS/MS fragmentation patterns, accurate mass measurement, and published scientific information on CGs from Nerium oleander. It was observed that only six genins, viz., Δ16anhydrogitoxigenin, Δ16adynerigenin, gitoxigenin, oleandrigenin, digitoxigenin, and adynerigenine, produce 38 diverse chemical structures of CGs. Among them, 20 were identified as diastereomers having a difference in a sugar (l-oleandrose, ß-d-diginose, and ß-d-sarmentose) unit. However, the differentiation of diastereomeric CGs was not possible by only MS/MS fragments. Thus, the diastereomer's chromatographic elution order was assigned on the basis of the relative retention time (RRt) of two reference standards (odoroside A and oleandrin) among their diastereomers. Besides this, the in-source fragmentation of CGs and the MS/MS of m/z 325 and 323 disaccharide daughter ions also exposed the intrinsic structure information on the sugar units. The daughter ions m/z 162, 145, 113, 95, and 85 in MS/MS spectra indicated the abundance of l-oleandrose, ß-d-diginose, and ß-d-sarmentose sugars. At the same time, m/z 161, 143, 129, and 87 product ions confirmed the presence of a ß-d-digitalose unit. As a result, the UPLC-ESI/TQD system was successfully utilized for the structure characterization of CGs in Nerium oleander tissues.

Cardiac Oxidative Signaling and Physiological Hypertrophy in the Na/K-ATPase α1s/sα2s/s Mouse Model of High Affinity for Cardiotonic Steroids.

The Na/K-ATPase is the specific receptor for cardiotonic steroids (CTS) such as ouabain and digoxin. At pharmacological concentrations used in the treatment of cardiac conditions, CTS inhibit the ion-pumping function of Na/K-ATPase. At much lower concentrations, in the range of those reported for endogenous CTS in the blood, they stimulate hypertrophic growth of cultured cardiac myocytes through initiation of a Na/K-ATPase-mediated and reactive oxygen species (ROS)-dependent signaling. To examine a possible effect of endogenous concentrations of CTS on cardiac structure and function in vivo, we compared mice expressing the naturally resistant Na/K-ATPase α1 and age-matched mice genetically engineered to express a mutated Na/K-ATPase α1 with high affinity for CTS. In this model, total cardiac Na/K-ATPase activity, α1, α2, and ß1 protein content remained unchanged, and the cardiac Na/K-ATPase dose-response curve to ouabain shifted to the left as expected. In males aged 3-6 months, increased α1 sensitivity to CTS resulted in a significant increase in cardiac carbonylated protein content, suggesting that ROS production was elevated. A moderate but significant increase of about 15% of the heart-weight-to-tibia-length ratio accompanied by an increase in the myocyte cross-sectional area was detected. Echocardiographic analyses did not reveal any change in cardiac function, and there was no fibrosis or re-expression of the fetal gene program. RNA sequencing analysis indicated that pathways related to energy metabolism were upregulated, while those related to extracellular matrix organization were downregulated. Consistent with a functional role of the latter, an angiotensin-II challenge that triggered fibrosis in the α1r/rα2s/s mouse failed to do so in the α1s/sα2s/s. Taken together, these results are indicative of a link between circulating CTS, Na/K-ATPase α1, ROS, and physiological cardiac hypertrophy in mice under baseline laboratory conditions.

Long-Range Stereodirecting Participation across a Glycosidic Linkage in Glycosylation Reactions.

The formation of an unprecedented 12-membered macrocyclic ketal through the long-range participation of a levulinoyl group across a glycosidic linkage was observed in glycosylation reactions. This finding indicated that stereodirecting participation is not limited to groups within the glycan ring being activated, thus broadening the scope of remote group participation in glycosylation.

Binding of cardiotonic steroids to Na+,K+-ATPase in the E2P state.

The sodium pump (Na+, K+-ATPase, NKA) is vital for animal cells, as it actively maintains Na+ and K+ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF3- complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K+-antagonism and suggest a route to isoform specific CTSs.

Cytotoxicity of Oleandrin Is Mediated by Calcium Influx and by Increased Manganese Uptake in Saccharomyces cerevisiae Cells.

Oleandrin, the main component of Nerium oleander L. extracts, is a cardiotoxic glycoside with multiple pharmacological implications, having potential anti-tumoral and antiviral characteristics. Although it is accepted that the main mechanism of oleandrin action is the inhibition of Na+/K+-ATPases and subsequent increase in cell calcium, many aspects which determine oleandrin cytotoxicity remain elusive. In this study, we used the model Saccharomyces cerevisiae to unravel new elements accounting for oleandrin toxicity. Using cells expressing the Ca2+-sensitive photoprotein aequorin, we found that oleandrin exposure resulted in Ca2+ influx into the cytosol and that failing to pump Ca2+ from the cytosol to the vacuole increased oleandrin toxicity. We also found that oleandrin exposure induced Mn2+ accumulation by yeast cells via the plasma membrane Smf1 and that mutants with defects in Mn2+ homeostasis are oleandrin-hypersensitive. Our data suggest that combining oleandrin with agents which alter Ca2+ or Mn2+ uptake may be a way of controlling oleandrin toxicity.