-
Journal of Fluorescence Sep 2023The interaction of orphenadrine hydrochloride (ORD) with the model protein, bovine serum albumin (BSA), was investigated using a variety of spectroscopic techniques such...
The interaction of orphenadrine hydrochloride (ORD) with the model protein, bovine serum albumin (BSA), was investigated using a variety of spectroscopic techniques such as steady-state fluorescence, ultraviolet-visible, Fourier transform infrared, 3-D spectroscopy, and electrochemical methods under physiological conditions. Stern-Volmer plots were used to calculate fluorescence quenching at various temperatures. The findings point to a static quenching mechanism between ORD and BSA. At various reaction times, the binding sites (n) and binding constants (K) of ORD to BSA were recorded. Thermodynamic parameters ∆H, ∆S and ∆G between ORD and BSA were calculated and reported. The average binding distance (r) between the donor (BSA) and acceptor (ORD) molecules was predicted using Förster's theory. Three-dimensional fluorescence spectra, Fourier transform infrared spectra, and synchronous fluorescence studies all supported the alternations in protein structure following the interaction with ORD. A displacement study using site probes such as warfarin, ibuprofen, and digitoxin confirmed ORD binding at Sudlow's site I of BSA. The effect of common metal ions such as Cu, Ni, Ca, Co, and Zn on binding constant values was investigated and reported.
Topics: Serum Albumin, Bovine; Orphenadrine; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Binding Sites; Thermodynamics; Protein Binding; Circular Dichroism
PubMed: 36976401
DOI: 10.1007/s10895-023-03199-y -
Drugs - Real World Outcomes Jun 2023Cardiac glycosides such as digoxin, digitoxin and ouabain are still used around the world to treat patients with chronic heart failure with reduced ejection fraction...
BACKGROUND
Cardiac glycosides such as digoxin, digitoxin and ouabain are still used around the world to treat patients with chronic heart failure with reduced ejection fraction (HFrEF) and/or atrial fibrillation (AF). However, in the US, only digoxin is licensed for treating these illnesses, and the use of digoxin for this group of patients is increasingly being replaced in the US by a new standard of care with groups of more expensive drugs. However, ouabain and digitoxin, and less potently digoxin, have also recently been reported to inhibit SARS-CoV-2 virus penetration into human lung cells, thus blocking COVID-19 infection. COVID-19 is known to be a more aggressive disease in patients with cardiac comorbidities, including heart failure.
OBJECTIVE
We therefore considered the possibility that digoxin might provide at least a measure of relief from COVID-19 in digoxin-treated heart failure patients. To this end, we hypothesized that treatment with digoxin rather than standard of care might equivalently protect heart failure patients with regard to diagnosis of COVID-19, hospitalization and death.
METHODS
To test this hypothesis, we conducted a cross-sectional study by using the US Military Health System (MHS) Data Repository to identify all MHS TRICARE Prime and Plus beneficiaries aged 18-64 years with a heart failure (HF) diagnosis during the period April 2020 to August 2021. In the MHS, all patients receive equal, optimal care without regard to rank or ethnicity. Analyses included descriptive statistics on patient demographics and clinical characteristics, and logistic regressions to determine likelihood of digoxin use.
RESULTS
We identified 14,044 beneficiaries with heart failure in the MHS during the study period. Of these, 496 were treated with digoxin. However, we found that both digoxin-treated and standard-of-care groups were equivalently protected from COVID-19. We also noted that younger active duty service members and their dependents with HF were less likely to receive digoxin compared with older, retired beneficiaries with more comorbidities.
CONCLUSION
The hypothesis of equivalent protection by digoxin treatment of HF patients in terms of susceptibility to COVID-19 infection appears to be supported by the data.
PubMed: 36933173
DOI: 10.1007/s40801-023-00360-8 -
The Journal of Membrane Biology Jun 2023The nature of odoroside A, a cardiac glycoside (CG) extracted from Nerium oleander, as well as its chemical structure is quite similar to a well-known CG, ouabain...
The nature of odoroside A, a cardiac glycoside (CG) extracted from Nerium oleander, as well as its chemical structure is quite similar to a well-known CG, ouabain possessing a steroid skeleton, a five-membered unsaturated lactone ring, and a sugar moiety as a common structure. Like ouabain, odoroside A inhibits the activity of Na/K-ATPase (NKA) and shows significant anticancer activity, however its inhibitory mechanism remains unknown. CGs show various physiological activities, including cardiotonic and anticancer activities, through the inhibition of NKA by direct interaction. Additionally, X-ray crystallographic analysis revealed the inhibitory mechanism of ouabain and digoxin in relation to NKA. By using different molecular modeling techniques, docking simulation of odoroside A and NKA was conducted based on the results of these X-ray crystallographic analyses. Furthermore, a comparison of the results with the binding characteristics of three known CGs (ouabain, digoxin, and digitoxin) was also conducted. Odoroside A fitted into the CG binding pocket on the α-subunit of NKA revealed by X-ray crystallography. It had key interactions with Thr797 and Phe783. Also, three known CGs showed similar interactions with Thr797 and Phe783. Interaction modes of odoroside A were quite similar to those of ouabain, digoxin, and digitoxin. Docking simulations indicated that the sugar moiety enhanced the interaction between NKA and CGs, but did not show enhanced NKA inhibitory activity because the sugar moiety was placed outside the entrance of active site. Thus, these results suggest that the inhibitory mechanism of odoroside A to NKA is the same as the known CGs.
Topics: Cardiac Glycosides; Ouabain; Sodium-Potassium-Exchanging ATPase; Digoxin; Digitoxin; Sugars
PubMed: 36840763
DOI: 10.1007/s00232-023-00281-1 -
Organic Letters Feb 2023This letter describes the development of an α-selective glycosylation using l-oleandrose, a 2-deoxysugar that is frequently found in natural products, and its...
This letter describes the development of an α-selective glycosylation using l-oleandrose, a 2-deoxysugar that is frequently found in natural products, and its application to the total synthesis of the natural cardiotonic steroids oleandrin and beaumontoside. To improve the reaction diastereoselectivity and to minimize side-product formation, an extensive evaluation and optimization of the conditions leading to α-selective glycosylation of digitoxigenin with l-oleandrose-based donors was conducted. These studies led to the exploration of 8 different phosphine·acid complexes or salts and yielded HBr·PPh as the optimal catalyst, which provided in the cleanest α-glycosylation and produced protected beaumontoside in 67% yield. Subsequent application of these conditions to synthetic oleandrigenin afforded the desired α-product in 69% isolated yield─enabling the completion of the first synthesis of oleandrin in 17 steps (1.2% yield) from testosterone.
Topics: Glycosylation; Cardiac Glycosides; Digitoxigenin
PubMed: 36739571
DOI: 10.1021/acs.orglett.2c04358 -
Methods in Enzymology 2023The biosynthesis of cardiac glycosides, broadly classified as cardenolides and bufadienolides, has evolved repeatedly among flowering plants. Individual species can...
The biosynthesis of cardiac glycosides, broadly classified as cardenolides and bufadienolides, has evolved repeatedly among flowering plants. Individual species can produce dozens or even hundreds of structurally distinct cardiac glycosides. Although all cardiac glycosides exhibit biological activity by inhibiting the function of the essential Na/K-ATPase in animal cells, they differ in their level of inhibitory activity. For within- and between-species comparisons of cardiac glycosides to address ecological and evolutionary questions, it is necessary to not only quantify their relative abundance, but also their effectiveness in inhibiting the activity of different animal Na/K-ATPases. Here we describe protocols for characterizing the amount and toxicity of cardenolides from plant samples and the degree of insect Na/K-ATPase tolerance to inhibition: (1) an HPLC-based assay to quantify the abundance of individual cardenolides in plant extracts, (2) an assay to quantify inhibition of Na/K-ATPase activity by plant extracts, and (3) extraction of insect Na/K-ATPases for inhibition assays.
Topics: Animals; Cardenolides; Chromatography, High Pressure Liquid; Sodium-Potassium-Exchanging ATPase; Cardiac Glycosides; Plant Extracts
PubMed: 36710014
DOI: 10.1016/bs.mie.2022.08.003 -
Frontiers in Immunology 2022Osteoarthritis (OA) is a common orthopedic degenerative disease, leading to high disability in activities of daily living. There remains an urgent need to identify the...
Osteoarthritis (OA) is a common orthopedic degenerative disease, leading to high disability in activities of daily living. There remains an urgent need to identify the underlying mechanisms and identify new therapeutic targets in OA diagnosis and treatment. Circular RNAs (circRNAs) play a role in the development of multiple diseases. Many studies have reported that circRNAs regulate microRNAs (miRNAs) through an endogenous competitive mechanism. However, it remains unclear if an interplay between circRNAs, miRNAs, and target genes plays a deeper regulatory role in OA. Four datasets were downloaded from the GEO database, and differentially expressed circRNAs (DECs), differentially expressed miRNAs (DEMs), and differentially expressed genes (DEGs) were identified. Functional annotation and pathway enrichment analysis of DEGs and DECs were carried out to determine the main associated mechanism in OA. A protein-protein network (PPI) was constructed to analyze the function of, and to screen out, hub DEGs in OA. Based on the artificial intelligence prediction of protein crystal structures of two hub DEGs, TOP2A and PLK1, digitoxin and oxytetracycline were found to have the strongest affinity, respectively, with molecular docking. Subsequently, overlapping DEMs and miRNAs targeted by DECs obtained target DEMs (DETMs). Intersection of DEGs and genes targeted by DEMs obtained target DEGs (DETGs). Thus, a circRNA-miRNA-mRNA regulatory network was constructed from 16 circRNAs, 32 miRNAs, and 97 mRNAs. Three hub DECs have the largest number of regulated miRNAs and were verified through experiments. In addition, the expression level of 16 DECs was validated by RT-PCR. In conclusion, we constructed a circRNA-miRNA-mRNA regulatory network in OA and three new hub DECs, hsa_circ_0027914, hsa_circ_0101125, and hsa_circ_0102564, were identified as novel biomarkers for OA.
Topics: Humans; RNA, Messenger; MicroRNAs; RNA, Circular; Molecular Docking Simulation; Artificial Intelligence; Activities of Daily Living; Gene Regulatory Networks; Gene Expression Profiling; Osteoarthritis
PubMed: 36700234
DOI: 10.3389/fimmu.2022.1050743 -
Medicine International 2022The alpha subunits (ATP1A1-3) of Na/K-ATPase binds digitoxin with varying affinity. The expression levels of these subunits dictate the anticancer effects of digitoxin....
The alpha subunits (ATP1A1-3) of Na/K-ATPase binds digitoxin with varying affinity. The expression levels of these subunits dictate the anticancer effects of digitoxin. In the present study, three pancreatic cancer cell lines, AsPC-1, Panc-1 and CFPAC-1, were used to investigate the effects of digitoxin in relation to the expression of the subunits ATP1A1 and ATP1A3. Cell viability and intracellular calcium concentrations was measured in relation to the gene and protein expression of ATP1A1 and ATP1A3. Digitoxin was used to treat the cells at concentrations of 1-100 nM, and the intracellular calcium concentrations increased in a concentration-dependent manner in the Panc-1 and in the CFPAC-1 cells with treatment at 100 nM. In the AsPC-1 cells only the supraphysiological concentration of digitoxin (100 nM) resulted in a decrease in the number of viable cells (unviable cells increased to 22%), whereas it had no effect on intracellular calcium levels. The number of viable Panc-1 and CFPAC-1 cells decreased after digitoxin treatment at 25-100 nM (unviable Panc-1 cells increased to 33-59%; unviable CFPAC-1 cells increased to 22-56%). Digitoxin treatment also affected the transcriptional expression of the and subunits. In Panc-1 cells, gene expression was negatively associated with the digitoxin concentration (25-100 nM). In the AsPC-1 and CFPAC-1 cells, the expression of the gene increased in the cells treated with the 100 nM digitoxin concentration. The protein expression of ATP1A1 and ATP1A3 was not altered with digitoxin treatment. The basal protein expression of ATP1A1 was high in the AsPC-1 and CFPAC-1 cells, compared to the Panc-1 cells, in contrast to the basal expression of ATP1A3, which was higher in the Panc-1 cells, compared to the other pancreatic cancer cells used. On the whole, the present study demonstrates that the high expression of ATP1A3 renders pancreatic cancer cells more susceptible to digitoxin-induced cell death. The findings suggest that the expression of ATP1A3 may be used as a marker for tumor sensitivity to digitoxin treatment, where a high expression of ATP1A3 is favorable for the anticancer effects of digitoxin.
PubMed: 36698913
DOI: 10.3892/mi.2022.52 -
Genes Dec 2022L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient...
L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85-90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%-95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants.
Topics: Digitalis; Codon, Initiator; Regeneration; DNA; Ploidies
PubMed: 36553602
DOI: 10.3390/genes13122335 -
Pharmacological Research Jan 2023Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various...
Increasing studies have suggested that some cardiac glycosides, such as conventional digoxin (DIG) and digitoxin, can induce immunogenic cell death (ICD) in various tumors. We previously found that 3'-epi-12β-hydroxyfroside (HyFS), a novel cardenolide compound isolated by our group, could induce cytoprotective autophagy through inactivation of the Akt/mTOR pathway. However, whether HyFS can induce ICD remains unknown. In this study, we extend our work to further investigate whether HyFS could induce both autophagy and ICD, and we investigated the relationship between autophagy and ICD in three TNBC cell lines. Unexpectedly, compared to DIG, we found that HyFS could induce complete autophagy flux but not ICD in three human triple-negative breast cancer (TNBC) cell lines and one murine TNBC model. Inhibition of HyFS-induced autophagy resulted in the production of ICD in TNBC MDA-MB-231, MDA-MB-436, and HCC38 cells. A further mechanism study showed that formation of RIPK1/RIPK3 necrosomes was necessary for ICD induction in DIG-treated TNBC cells, while HyFS treatment led to receptor-interacting serine-threonine kinase (RIPK)1/3 necrosome degradation via an autophagy process. Additionally, inhibition of HyFS-induced autophagy by the autophagy inhibitor chloroquine resulted in the reoccurrence of ICD and reversion of the tumor microenvironment, leading to more significant antitumor effects in immunocompetent mice than in immunodeficient mice. These findings indicate that HyFS-mediated autophagic degradation of RIPK1/RIPK3 necrosomes leads to inactivation of ICD in TNBC cells. Moreover, combined treatment with HyFS and an autophagy inhibitor may enhance the antitumor activities, suggesting an alternative therapeutic for TNBC treatment.
Topics: Animals; Humans; Mice; Apoptosis; Autophagy; Cell Line, Tumor; Immunogenic Cell Death; Receptor-Interacting Protein Serine-Threonine Kinases; Triple Negative Breast Neoplasms; Tumor Microenvironment
PubMed: 36535569
DOI: 10.1016/j.phrs.2022.106613 -
Journal of Chromatography. B,... Jan 2023Therapeutic drug monitoring (TDM) of cardiovascular drugs is essential to improve treatment efficacy and minimize toxicity because of the usage of multiple drugs with a...
Development and validation of an analytical method using liquid chromatography-tandem mass spectrometry for the therapeutic drug monitoring of seven cardiovascular drugs in clinical usage.
Therapeutic drug monitoring (TDM) of cardiovascular drugs is essential to improve treatment efficacy and minimize toxicity because of the usage of multiple drugs with a very limited therapeutic range and the high pharmacokinetic variation in patients. We developed and validated a reliable and economical liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of seven cardiovascular drugs-procainamide, lidocaine, quinidine, deslanoside, digoxin, atorvastatin, and digitoxin-for clinical usage. Serum samples were prepared by simple protein precipitation with an organic solvent consisting of acetonitrile and methanol (2:1 v/v) and analyzed under optimized LC-MS/MS conditions. The chromatographic separations were accomplished within 15 min on a reversed-phase C18 column with a gradient elution of aqueous solvent and acetonitrile while maintaining 0.1 (v/v) % formic acid and 2 mM ammonium formate. The optimized MS/MS conditions in ESI-positive mode offered sufficient sensitivity for the seven cardiovascular drugs (LOQs between 0.5 and 1 ng/mL). This method was fully validated including linearity, selectivity, accuracy, precision, carry-over, and matrix effects. Additionally, stability under several conditions was tested to determine how to handle the standard solutions and serum samples. The seven cardiovascular drugs, simultaneously, were precisely and accurately analyzed in intra- and inter-day assays (RSD < 6 % and recovery between 96.3 and 102.8 %) using only two isotope-labeled internal standards (lidocaine-(diethyl-d10) and digoxin-21, 21, 22-d3). The presented method also showed good accuracy in analyzing the seven drugs in hyperlipidemia, hyperalbuminemia, and hyperglycemia serum, allowing it to be recommended as a common and routine analysis method for cardiovascular drugs in clinical practice.
Topics: Humans; Chromatography, Liquid; Cardiovascular Agents; Tandem Mass Spectrometry; Drug Monitoring; Solvents; Digoxin; Lidocaine; Chromatography, High Pressure Liquid; Reproducibility of Results
PubMed: 36469961
DOI: 10.1016/j.jchromb.2022.123552