Screening of Peptides that Specifically Binds to M3-M4 Extracellular Domain of Sodium Pump α1 Subunit and Analysis of Their Bioactivity In Vitro and In Vivo.

Interaction between ouabain (OUA) and Na+/K+-pump remains in the current focus of hypertension research. This study aimed to find an oligopeptide that would antagonize the inhibitory effect of endogenous OUA on Na+/K+-pump and examine its activity at the cellular and organism levels. To this end, Phage Random 12 Peptide Library was employed to screen for specific polypeptide ligands that interact with M3-M4 extracellular domain of Na+/K+-pump α1 subunit known as OUA-binding site. Synthetic sequence ILEYTWLEAGGGS of extracellular domain M3-M4 of Na+/K+-pump α1 subunit was used as the target. The phage positive clones were screened and identified using the phage library and double sandwich ELISA. DNA was extracted and sequenced to synthesize 3 peptide ligands to Na+/K+-pump: P-A, P-B, and P-C. We also studied the effects of the short peptide with the highest potency for countering OUA on proliferation and apoptosis of EA.hy926 vascular endothelial cells and on systolic BP in spontaneously hypertensive rats (SHR). The effect of peptide P-A on proliferation (stimulation with physiological concentrations of OUA) and on apoptosis (stimulation with OUA in high concentrations) of EA.hy926 vascular endothelial cells was assessed by the MTT test and flow cytometry, respectively. In SHR rats, intravenous injection of P-A decreased systolic BP. Oligopeptide P-A competitively antagonized the inhibitory action of OUA on Na+/K+-pump, OUA-induced proliferation, and OUA-provoked apoptosis of cultured EA.hy926 cells. Our findings open vista for the emergence of novel hypertensive drugs.

Na+/K+-ATPase Revisited: On Its Mechanism of Action, Role in Cancer, and Activity Modulation.

Maintenance of Na+ and K+ gradients across the cell plasma membrane is an essential process for mammalian cell survival. An enzyme responsible for this process, sodium-potassium ATPase (NKA), has been currently extensively studied as a potential anticancer target, especially in lung cancer and glioblastoma. To date, many NKA inhibitors, mainly of natural origin from the family of cardiac steroids (CSs), have been reported and extensively studied. Interestingly, upon CS binding to NKA at nontoxic doses, the role of NKA as a receptor is activated and intracellular signaling is triggered, upon which cancer cell death occurs, which lies in the expression of different NKA isoforms than in healthy cells. Two major CSs, digoxin and digitoxin, originally used for the treatment of cardiac arrhythmias, are also being tested for another indication-cancer. Such drug repositioning has a big advantage in smoother approval processes. Besides this, novel CS derivatives with improved performance are being developed and evaluated in combination therapy. This article deals with the NKA structure, mechanism of action, activity modulation, and its most important inhibitors, some of which could serve not only as a powerful tool to combat cancer, but also help to decipher the so-far poorly understood NKA regulation.

Effects of brief chilling and desiccation on ion homeostasis in the central nervous system of the migratory locust, Locusta migratoria.

In insects, chilling, anoxia, and dehydration are cues to trigger rapid physiological responses enhancing stress tolerance within minutes. Recent evidence suggests that responses elicited by different cues are mechanistically distinct from each other, though these differences have received little attention. Further, the effects are not well studied in neural tissue. In this study, we examined how brief exposure to desiccation and chilling affect ion homeostatic mechanisms in metathoracic ganglion of the migratory locust, Locusta migratoria. Both desiccation and chilling enhanced resistance to anoxia, though only chilling hastened recovery from anoxic coma. Similarly, only chilling enhanced resistance to pharmacological perturbation of neuronal ion homeostasis. Our results indicate that chilling and desiccation trigger mechanistically distinct responses and, while both may be important for neuronal ion homeostasis, chilling has a larger effect on this tissue. SUMMARY STATEMENT: This is one of few studies to demonstrate the importance of the central nervous system in rapid acclimatory responses in insects.

Cellular sensing platform with enhanced sensitivity based on optogenetic modulation of cell homeostasis.

We demonstrate a new biosensing concept with impact on the development of rapid, point of need cell based sensing with boosted sensitivity and wide relevance for bioanalysis. It involves optogenetic stimulation of cells stably transfected to express light sensitive protein channels for optical control of membrane potential and of ion homeostasis. Time-lapse impedance measurements are used to reveal cell dynamics changes encompassing cellular responses to bioactive stimuli and optically induced homeostasis disturbances. We prove that light driven perturbations of cell membrane potential induce homeostatic reactions and modulate transduction mechanisms that amplify cellular response to bioactive compounds. This allows cell based biosensors to respond more rapidly and sensitively to low concentrations of bioactive/toxic analytes: statistically relevant impedance changes are recorded in less than 30 min, in comparison with >8 h in the best alternative reported tests for the same low concentration (e.g. a concentration of 25 µM CdCl2, lower than the threshold concentration in classical cellular sensors). Comparative analysis of model bioactive/toxic compounds (ouabain and CdCl2) demonstrates that cellular reactivity can be boosted by light driven perturbations of cellular homeostasis and that this biosensing concept is able to discriminate analytes with different modes of action (i.e. CdCl2 toxicity versus ion pump inhibition by ouabain), a significant advance against state of the art cell based sensors.

The cardenolides ouabain and reevesioside A promote FGF2 secretion and subsequent FGFR1 phosphorylation via converged ERK1/2 activation.

Na+/K+-ATPase α1 was reported to directly interact with and recruit FGF2 (fibroblast growth factor 2), a vital cell signaling protein implicated in angiogenesis, to the inner plasma membrane for subsequent secretion. Cardenolides, a class of cardiac glycosides, were reported to downregulate FGF2 secretion upon binding to Na+/K+-ATPase α1 in a cell system with ectopically expressed FGF2 and Na+/K+-ATPase α1. Herein, we disclose that the cardenolides ouabain and reevesioside A significantly enhance the secretion/release of FGF2 and the phosphorylation of FGFR1 (fibroblast growth factor receptor 1) in a time- and dose-dependent manner, in A549 carcinoma cells. A pharmacological approach was used to elucidate the pertinent upstream effectors. Only the ERK1/2 inhibitor U0126 but not the other inhibitors examined (including those inhibiting the unconventional secretion of FGF2) was able to reduce ouabain-induced FGF2 secretion and FGFR1 activation. ERK1/2 phosphorylation was increased upon ouabain treatment, a process found to be mediated through upstream effectors including ouabain-induced phosphorylated EGFR and a reduced MKP1 protein level. Therefore, at least two independent lines of upstream effectors are able to mediate ouabain-induced ERK1/2 phosphorylation and the subsequent FGF2 secretion and FGFR1 activation. These finding constitute unprecedent insights into the regulation of FGF2 secretion by cardenolides.

Conformational states of the pig kidney Na+/K+-ATPase differently affect bufadienolides and cardenolides: A directed structure-activity and structure-kinetics study.

There is a renewed interest in the Na+/K+-ATPase (NKA, EC 3.6.3.9) either as a target for new therapeutic uses or for understanding the putative pathophysiological role of its mammalian endogenous ligands. Recent data indicate that bufalin binds to the pig kidney NKA in a way different from ouabain and digoxin, raising the question of a putative class difference between bufadienolides and cardenolides. The purpose of this work was to perform a study of the relationship between structure and both activity and kinetics, focusing mainly on the influence of the lactone ring in C17 (5 vs. 6 membered), the effect of C14-15 cyclization and the carbohydrate moiety in C3. We compared the potency of fourteen related cardiotonic steroids (CTS) for inhibition of the cycling pig kidney NKA in two different concentrations of K+, as well as the affinity for binding to the E2P conformation of the enzyme (Mg-Pi medium) and the potency for inhibiting the E2[2K] conformation of the NKA (K+-pNPPase activity). Cardenolides were clearly sensitive to the antagonistic effect of high K+ concentrations whereas bufadienolides were not or less sensitive. The C14-15 cyclization observed in some bufadienolides, such as resibufogenin and marinobufagin, caused a drastic fall in the affinity for binding to the NKA in the E2P conformation and increased the velocity of K+-pNPPase inhibition. The absence of a carbohydrate moiety in C3 increased the velocity of inhibition. Cardenolides were much more dependent on the E2P conformation for binding than bufadienolides since their ratios of E2[2K] IC50 to E2P Ki were higher than for bufadienolides. Therefore, the present data established the remarkable influence of C14-15 cyclization and of the carbohydrate moiety in C3 on both affinity and kinetics of CTS and indicate that, as a class, bufadienolides would harbor qualitative differences from cardenolides with respect to the NKA conformations to which they can bind.

Cardiotonic steroids as potential Na+/K+-ATPase inhibitors - a computational study.

Cardiotonic steroids (CTS) are steroidal drugs, processed from the seeds and dried leaves of the genus Digitalis as well as from the skin and parotid gland of amphibians. The most commonly known CTS are ouabain, digoxin, digoxigenin and bufalin. CTS can be used for safer medication of congestive heart failure and other related conditions due to promising pharmacological and medicinal properties. Ouabain isolated from plants is widely utilized in in vitro studies to specifically block the sodium potassium (Na+/K+-ATPase) pump. For checking, whether ouabain derivatives are robust inhibitors of Na+/K+-ATPase pump, molecular docking simulation was performed between ouabain and its derivatives using YASARA software. The docking energy falls within the range of 8.470 kcal/mol to 7.234 kcal/mol, in which digoxigenin was found to be the potential ligand with the best docking energy of 8.470 kcal/mol. Furthermore, pharmacophore modeling was applied to decipher the electronic features of CTS. Molecular dynamics simulation was also employed to determine the conformational properties of Na+/K+-ATPase-ouabain and Na+/K+-ATPase-digoxigenin complexes with the plausible structural integrity through conformational ensembles for 100 ns which promoted digoxigenin as the most promising CTS for treating conditions of congestive heart failure patients.

Modular Total Synthesis and Cell-Based Anticancer Activity Evaluation of Ouabagenin and Other Cardiotonic Steroids with Varying Degrees of Oxygenation.

A Cu(II)-catalyzed diastereoselective Michael/aldol cascade approach is used to accomplish concise total syntheses of cardiotonic steroids with varying degrees of oxygenation including cardenolides ouabagenin, sarmentologenin, 19-hydroxysarmentogenin, and 5- epi-panogenin. These syntheses enabled the subsequent structure activity relationship (SAR) studies on 37 synthetic and natural steroids to elucidate the effect of oxygenation, stereochemistry, C3-glycosylation, and C17-heterocyclic ring. Based on this parallel evaluation of synthetic and natural steroids and their derivatives, glycosylated steroids cannogenol-l-α-rhamnoside (79a), strophanthidol-l-α-rhamnoside (92), and digitoxigenin-l-α-rhamnoside (97) were identified as the most potent steroids demonstrating broad anticancer activity at 10-100 nM concentrations and selectivity (nontoxic at 3 µM against NIH-3T3, MEF, and developing fish embryos). Further analyses indicate that these molecules show a general mode of anticancer activity involving DNA-damage upregulation that subsequently induces apoptosis.

Quantum Modeling: A Bridge between the Pumping and Signaling Functions of Na/K-ATPase.

Although the signaling function of Na/K-ATPase has been studied for decades, the chasm between the pumping function and the signaling function of Na/K-ATPase is still an open issue. This article explores the relationship between ion pumping and signaling with attention to the amplification of oxidants through this signaling function. We specifically consider the Na/K-ATPase with respect to its signaling function as a superposition of different states described for its pumping function. We then examine how alterations in the relative amounts of these states could alter signaling through the Src-EGFR-ROS pathway. Using assumptions based on some experimental observations published by our laboratories and others, we develop some predictions regarding cellular oxidant stress.

The Na,K-ATPase-Dependent Src Kinase Signaling Changes with Mesenteric Artery Diameter.

Inhibition of the Na,K-ATPase by ouabain potentiates vascular tone and agonist-induced contraction. These effects of ouabain varies between different reports. In this study, we assessed whether the pro-contractile effect of ouabain changes with arterial diameter and the molecular mechanism behind it. Rat mesenteric small arteries of different diameters (150⁻350 µm) were studied for noradrenaline-induced changes of isometric force and intracellular Ca2+ in smooth muscle cells. These functional changes were correlated to total Src kinase and Src phosphorylation assessed immunohistochemically. High-affinity ouabain-binding sites were semi-quantified with fluorescent ouabain. We found that potentiation of noradrenaline-sensitivity by ouabain correlates positively with an increase in arterial diameter. This was not due to differences in intracellular Ca2+ responses but due to sensitization of smooth muscle cell contractile machinery to Ca2+. This was associated with ouabain-induced Src activation, which increases with increasing arterial diameter. Total Src expression was similar in arteries of different diameters but the density of high-affinity ouabain binding sites increased with increasing arterial diameters. We suggested that ouabain binding induces more Src kinase activity in mesenteric small arteries with larger diameter leading to enhanced sensitization of the contractile machinery to Ca2+.

The cardenolides strophanthidin, digoxigenin and dihydroouabain act as activators of the human RORγ/RORγT receptors.

Two isoforms of a ligand-activated nuclear receptor, RORγ and RORγT, have been implicated in various physiological functions, including energy metabolism, circadian rhythm and immune system development. Using a stably transfected reporter cell line, we screened two chemical libraries and identified three cardenolides (natural, plant-derived pesticides) as activators of RORγ-dependent transcription. These compounds increased G6PC and NPAS2 expression in HepG2 cells, accompanied by increased occupancy of RORγ within the promoters of these genes. Further, strophanthidin, digoxigenin and dihydroouabain upregulated IL17A and IL17F expression and enhanced IL17 secretion in Th17 human lymphocytes. Molecular docking analyses of these compounds to the RORγ LBD showed favorable docking scores, suggesting that cardenolides may act as agonists of the receptor. Thus, our results provide new chemical structures for further development of RORγ-selective modulators with virtual therapeutic potential.

Comparative Action of Cardiotonic Steroids on Intracellular Processes in Rat Cortical Neurons.

Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein-protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.

Spin-labeled derivatives of cardiotonic steroids as tools for characterization of the extracellular entrance to the binding site on Na+ ,K+ -ATPase.

The information obtained from crystallized complexes of the Na+ ,K+ -ATPase with cardiotonic steroids (CTS) is not sufficient to explain differences in the inhibitory properties of CTS such as stereoselectivity of CTS binding or effect of glycosylation on the preference to enzyme isoforms. The uncertainty is related to the spatial organization of the hydrophilic cavity at the entrance of the CTS-binding site. Therefore, there is a need to supplement the crystallographic description with data obtained in aqueous solution, where molecules have significant degree of flexibility. This work addresses the applicability of the electron paramagnetic resonance (EPR) method for the purpose. We have designed and synthesized spin-labeled compounds based on the cinobufagin steroid core. The length of the spacer arms between the steroid core and the nitroxide group determines the position of the reporting group (N-O) confined to the binding site. High affinity to Na+ ,K+ -ATPase is inferred from their ability to inhibit enzymatic activity. The differences between the EPR spectra in the absence and presence of high ouabain concentrations identify the signature peaks originating from the fraction of the spin labels bound within the ouabain site. The degree of perturbations of the EPR spectra depends on the length of the spacer arm. Docking of the compounds into the CTS site suggests which elements of the protein structure might be responsible for interference with the spin label (e.g., steric clashes or immobilization). Thus, the method is suitable for gathering information on the cavity leading to the CTS-binding site in Na+ ,K+ -ATPase in all conformations with high affinity to CTS.

Changes in [3H]-ouabain and [3H]-neurotensin binding to rat cerebral cortex membranes after administration of antipsychotic drugs haloperidol and clozapine.

Evidences indicate the relationship between neurotensinergic and dopaminergic systems. Neurotensin inhibits synaptosomal membrane Na+, K+-ATPase activity, an effect blocked by SR 48692, antagonist for high affinity neurotensin receptor (NTS1) type. Assays of high affinity [3H]-ouabain binding (to analyze K+ site of Na+, K+-ATPase) show that in vitro addition of neurotensin decreases binding. Herein potential interaction between NTS1 receptor, dopaminergic D2 receptor and Na+, K+-ATPase was studied. To test the involvement of dopaminergic D2 receptors in [3H]-ouabain binding inhibition by neurotensin, Wistar rats were administered i.p.with antipsychotic drugs haloperidol (2mg/kg) and clozapine (3, 10 and 30mg/kg). Animals were sacrificed 18h later, cerebral cortices harvested, membrane fractions prepared and high affinity [3H]-ouabain binding assayed in the absence or presence of neurotensin at a 10 micromolar concentration. No differences versus controls for basal binding or for binding inhibition by neurotensin were recorded, except after 10mg/kg clozapine. Rats were administered with neurotensin (3, 10y 30µg, i.c.v.) and 60min later, animals were sacrificed, cerebral cortices harvested and processed to obtain membrane fractions for high affinity [3H]-ouabain binding assays. Results showed a slight but statistically significant decrease in binding with the 30µg neurotensin dose. To analyze the interaction between dopaminergic D2 and NTS1 receptors, [3H]-neurotensin binding to cortical membranes from rats injected with haloperidol (2mg/kg, i.p.) or clozapine (10mg/kg) was assayed. Saturation curves and Scatchard transformation showed that the only statistically significant change occurred in Bmax after haloperidol administration. Hill number was close to the unit in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modulate the interaction between neurotensin and Na+, K+-ATPase. At the same time, support the notion of an interaction among dopaminergic and neurotensinergic systems and Na+, K+-ATPase at central synapses.

Purification of Na,K-ATPase from Pig Kidney.

The method of purification of Na,K-ATPase from pig kidney is based on a differential centrifugation and SDS-treatment of a microsomal preparation. The yield is 0.4 mg protein per 1 g tissue with the specific (ouabain-sensitive) activity of 25-28 µmol Pi/min per mg protein and nucleotide binding capacity of 3 nmol/mg. The protein/lipid ratio is 1/1 (mg/mg) with a protein purity of ~80 %.

Epi-reevesioside F inhibits Na+/K+-ATPase, causing cytosolic acidification, Bak activation and apoptosis in glioblastoma.

Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC50 values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na+/K+-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na+ but not Ca2+, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na+/K+-ATPase, leading to overload of intracellular Na+ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.

Role of the Na(+)/K(+)-ATPase beta-subunit in peptide-mediated transdermal drug delivery.

In this work, we discovered that the Na(+)/K(+)-ATPase beta-subunit (ATP1B1) on epidermal cells plays a key role in the peptide-mediated transdermal delivery of macromolecular drugs. First, using a yeast two-hybrid assay, we screened candidate proteins that have specific affinity for the short peptide TD1 (ACSSSPSKHCG) identified in our previous work. Then, we verified the specific binding of TD1 to ATP1B1 in yeast and mammalian cells by a pull-down ELISA and an immunoprecipitation assay. Finally, we confirmed that TD1 mainly interacted with the C-terminus of ATP1B1. Our results showed that the interaction between TD1 and ATP1B1 affected not only the expression and localization of ATP1B1, but also the epidermal structure. In addition, this interaction could be antagonized by the exogenous competitor ATP1B1 or be inhibited by ouabain, which results in the decreased delivery of macromolecular drugs across the skin. The discovery of a critical role of ATP1B1 in the peptide-mediated transdermal drug delivery is of great significance for the future development of new transdermal peptide enhancers.

A structural rearrangement of the Na+/K+-ATPase traps ouabain within the external ion permeation pathway.

With the use of the energy of ATP hydrolysis, the Na+/K+-ATPase is able to transport across the cell membrane Na+ and K+ against their electrochemical gradients. The enzyme is strongly inhibited by ouabain and its derivatives, some that are therapeutically used for patients with heart failure (cardiotonic steroids). Using lanthanide resonance energy transfer, we trace here the conformational changes occurring on the external side of functional Na+/K+-ATPases induced by the binding of ouabain. Changes in donor/acceptor pair distances are mainly observed within the α subunit of the enzyme. To derive a structural model matching the experimental lanthanide resonance energy transfer distances measured with bound ouabain, we carried out molecular dynamics simulations with energy restraints applied simultaneously using a novel methodology with multiple non-interacting fragments. The restrained simulation, initiated from the X-ray structure of the E2(2K+) state, became strikingly similar to the X-ray structure of the sodium-bound state. The final model shows that ouabain is trapped within the external ion permeation pathway of the pump.

Ligand orientation in a membrane-embedded receptor site revealed by solid-state NMR with paramagnetic relaxation enhancement.

NMR relaxation enhancement by paramagnetic metals provides powerful restraints on the three-dimensional structures of proteins in solution, and this approach has recently been utilized in several NMR structural investigations of proteins in the solid-state. Here we utilize paramagnetic relaxation enhancement (PRE) by Mn(2+) with cross-polarization magic-angle spinning (CP-MAS) solid-state NMR to investigate the interaction of a membrane-embedded protein the Na,K-ATPase (NKA) with a cardiotonic steroid inhibitor. The inhibitor, a diacetonide derivate of the cardiac glycoside ouabain, with (13)C labelled acetonide groups in the rhamnose sugar and steroid moieties ([(13)C2]ODA), is 1000-fold less potent than the parent compound. It is shown that the (13)C CP-MAS solid-state NMR spectra of the NKA-[(13)C2]ODA complex exhibit distinct signals for the two (13)C labels of the inhibitor when bound to the ouabain site of membrane-embedded NKA. Recent crystal structures of NKA indicate that the catalytic α-subunit binds a single Mn(2+) in a transmembrane site close to the high-affinity ouabain site. Here, complexation of NKA with Mn(2+) broadens the resonance line from the rhamnose group substantially more than the steroid peak, indicating that the rhamnose group is closer to the Mn(2+) site than is the steroid group. These observations agree with computational molecular docking simulations and are consistent with ODA adopting an inverted orientation compared to ouabain in the cardiac glycoside site, with the modified rhamnose group drawn toward the transmembrane centre of the protein. This work demonstrates that PRE can provide unique information on the positions and orientations of ligands within their binding pockets of transmembrane proteins.

Development of a concise synthesis of ouabagenin and hydroxylated corticosteroid analogues.

The natural product ouabagenin is a complex cardiotonic steroid with a highly oxygenated skeleton. This full account describes the development of a concise synthesis of ouabagenin, including the evolution of synthetic strategy to access hydroxylation at the C19 position of a steroid skeleton. In addition, approaches to install the requisite butenolide moiety at the C17 position are discussed. Lastly, methodology developed in this synthesis has been applied in the generation of novel analogues of corticosteroid drugs bearing a hydroxyl group at the C19 position.