Digitoxigenin

The identification of small molecule inhibitors with anthelmintic activities that target conserved proteins among ruminant gastrointestinal nematodes.[Pubmed:38358246]

mBio. 2024 Mar 13;15(3):e0009524.

Gastrointestinal nematode (GIN) infections are a major concern for the ruminant industry worldwide and result in significant production losses. Naturally occurring polyparasitism and increasing drug resistance that potentiate disease outcomes are observed among the most prevalent GINs of veterinary importance. Within the five major taxonomic clades, clade Va represents a group of GINs that predominantly affect the abomasum or small intestine of ruminants. However, the development of effective broad-spectrum anthelmintics against ruminant clade Va GINs has been challenged by a lack of comprehensive druggable genome resources. Here, we first assembled draft genomes for three clade Va species (Cooperia oncophora, Trichostrongylus colubriformis, and Ostertagia ostertagi) and compared them with closely related ruminant GINs. Genome-wide phylogenetic reconstruction showed a relationship among ruminant GINs structured by taxonomic classification. Orthogroup (OG) inference and functional enrichment analyses identified 220 clade Va-specific and Va-conserved OGs, enriched for functions related to cell cycle and cellular senescence. Further transcriptomic analysis identified 61 taxonomically and functionally conserved clade Va OGs that may function as drug targets for new broad-spectrum anthelmintics. Chemogenomic screening identified 11 compounds targeting homologs of these OGs, thus having potential anthelmintic activity. In in vitro phenotypic assays, three kinase inhibitors (Digitoxigenin, K-252a, and staurosporine) exhibited broad-spectrum anthelmintic activities against clade Va GINs by obstructing the motility of exsheathed L3 (xL3) or molting of xL3 to L4. These results demonstrate valuable applications of the new ruminant GIN genomes in gaining better insights into their life cycles and offer a contemporary approach to discovering the next generation of anthelmintics.IMPORTANCEGastrointestinal nematode (GIN) infections in ruminants are caused by parasites that inhibit normal function in the digestive tract of cattle, sheep, and goats, thereby causing morbidity and mortality. Coinfection and increasing drug resistance to current therapeutic agents will continue to worsen disease outcomes and impose significant production losses on domestic livestock producers worldwide. In combination with ongoing therapeutic efforts, advancing the discovery of new drugs with novel modes of action is critical for better controlling GIN infections. The significance of this study is in assembling and characterizing new GIN genomes of Cooperia oncophora, Ostertagia ostertagi, and Trichostrongylus colubriformis for facilitating a multi-omics approach to identify novel, biologically conserved drug targets for five major GINs of veterinary importance. With this information, we were then able to demonstrate the potential of commercially available compounds as new anthelmintics.

Determination of Two Wound Healing Components in Streptocaulon juventas (Lour.) Merr.: Periplogenin and Digitoxigenin.[Pubmed:38061998]

Chem Biodivers. 2024 Jan;21(1):e202301585.

Streptocaulon juventas (Lour.) Merr. (SJ) is a herbal medicine can promote wound healing. Cardiac glycosides, especially periplogenin, Digitoxigenin, and their glycosides were the main constituents of SJ. We aim to establish a method for the simultaneous determination of periplogenin and Digitoxigenin in SJ and evaluate the wound healing activities of these two components. UPLC-QqQ-MS/MS was used for the determination of periplogenin and Digitoxigenin. Meanwhile, rats were subjected to full-thickness skin resection on the back to investigate the wound healing effects of periplogenin and Digitoxigenin. The content of periplogenin and Digitoxigenin in 13 batches of SJ extracts ranged from 43.26 to 97.15 mug/g and 18.04 to 55.55 mug/g, respectively. Periplogenin and Digitoxigenin significantly increased the rate of wound healing in rats, increased the content of hydroxyproline in wound tissue, and improved the pathological state of wound skin tissue.

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status.[Pubmed:37828578]

Cell Commun Signal. 2023 Oct 12;21(1):283.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)-the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored. METHODS: Digoxin, a user-designed Digitoxigenin-alpha-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation-one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery. RESULTS: Primitive AML cells containing CD34(+) LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation. CONCLUSIONS: Our data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery. Video Abstract.

Bioassay-Guided Fractionation and Biological Activity of Cardenolides from Streptocaulon juventas.[Pubmed:37709286]

Planta Med. 2023 Dec;89(15):1444-1456.

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.

Evidence That Binding of Cyclic GMP to the Extracellular Domain of NKA (Sodium-Potassium ATPase) Mediates Natriuresis.[Pubmed:36919600]

Circ Res. 2023 Apr 28;132(9):1127-1140.

BACKGROUND: Extracellular renal interstitial guanosine cyclic 3',5'-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium (Na(+)) reabsorption via Src (Src family kinase) activation. Through which target extracellular cGMP acts to induce natriuresis is unknown. We hypothesized that cGMP binds to the extracellular alpha1-subunit of NKA (sodium-potassium ATPase) on RPT basolateral membranes to inhibit Na(+) transport similar to ouabain-a cardiotonic steroid. METHODS: Urine Na(+) excretion was measured in uninephrectomized 12-week-old female Sprague-Dawley rats that received renal interstitial infusions of vehicle (5% dextrose in water), cGMP (18, 36, and 72 mug/kg per minute; 30 minutes each), or cGMP+rostafuroxin (12 ng/kg per minute) or were subjected to pressure-natriuresis+/-rostafuroxin infusion. Rostafuroxin is a Digitoxigenin derivative that displaces ouabain from NKA. RESULTS: Renal interstitial cGMP and raised renal perfusion pressure induced natriuresis and increased phosphorylated Src(Tyr416) and Erk 1/2 (extracellular signal-regulated protein kinase 1/2)(Thr202/Tyr204); these responses were abolished with rostafuroxin coinfusion. To assess cGMP binding to NKA, we performed competitive binding studies with isolated rat RPTs using bodipy-ouabain (2 muM)+cGMP (10 microM) or rostafuroxin (10 microM) and 8-biotin-11-cGMP (2 muM)+ouabain (10 muM) or rostafuroxin (10 microM). cGMP or rostafuroxin reduced bodipy-ouabain fluorescence intensity, and ouabain or rostafuroxin reduced 8-biotin-11-cGMP staining. We cross-linked isolated rat RPTs with 4-N(3)-PET-8-biotin-11-cGMP (2 muM); 8-N(3)-6-biotin-10-cAMP served as negative control. Precipitation with streptavidin beads followed by immunoblot analysis showed that RPTs after cross-linking with 4-N(3)-PET-8-biotin-11-cGMP exhibited a significantly stronger signal for NKA than non-cross-linked samples and cross-linked or non-cross-linked 8-N(3)-6-biotin-10-cAMP RPTs. Ouabain (10 muM) reduced NKA in cross-linked 4-N(3)-PET-8-biotin-11-cGMP RPTs confirming fluorescence staining. 4-N(3)-PET-8-biotin-11-cGMP cross-linked samples were separated by SDS gel electrophoresis and slices corresponding to NKA molecular weight excised and processed for mass spectrometry. NKA was the second most abundant protein with 50 unique NKA peptides covering 47% of amino acids in NKA. Molecular modeling demonstrated a potential cGMP docking site in the ouabain-binding pocket of NKA. CONCLUSIONS: cGMP can bind to NKA and thereby mediate natriuresis.

Development of alpha-Selective Glycosylation with l-Oleandral and Its Application to the Total Synthesis of Oleandrin.[Pubmed:36739571]

Org Lett. 2023 Feb 17;25(6):966-971.

This letter describes the development of an alpha-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 alpha-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(3) as the optimal catalyst, which provided in the cleanest alpha-glycosylation and produced protected beaumontoside in 67% yield. Subsequent application of these conditions to synthetic oleandrigenin afforded the desired alpha-product in 69% isolated yield horizontal line enabling the completion of the first synthesis of oleandrin in 17 steps (1.2% yield) from testosterone.

17beta-neriifolin from unripe fruits of Cerbera manghas suppressed cell proliferation via the inhibition of HOXA9-dependent transcription and the induction of apoptosis in the human AML cell line THP-1.[Pubmed:36266527]

J Nat Med. 2023 Jan;77(1):180-187.

Homeobox A9 (HOXA9) is a transcription factor that is overexpressed in acute myeloid leukemia (AML). It is associated with the pathogenesis and progression of AML, and is a factor responsible for a poor prognosis. Therefore, the development of HOXA9-targeting molecules may contribute to not only better understanding of the mechanism of HOXA9 regulation, but also the development of therapeutic applications. We constructed a reporter assay system using the promoter region of the KBTBD10 gene, to which HOXA9 directly binds and regulates transcription, in the human acute monocytic leukemia cell line THP-1. Using this luciferase gene assay, we screened 1120 plant extracts and a methanol extract of the unripe fruits of Cerbera manghas was found to suppress the reporter gene expression mediated by the KBTBD10 promoter. From the extract, five steroid-type compounds were identified as the active constituents: 7alpha-neriifolin (1), 17beta-neriifolin (2), 17alpha-Digitoxigenin beta-D-glucosyl-(1 --> 4)-alpha-L-thevetoside (3), 17beta-Digitoxigenin beta-D-glucosyl-(1 --> 4)-alpha-L-thevetoside (4), and acetylthevetin B (5). Among the five compounds, 17beta-neriifolin most potently inhibited HOXA9-dependent gene expression without affecting the HOXA9 mRNA levels, and suppressed cell proliferation by inducing apoptosis. The findings on the structure-activity relationships of the compounds from C. manghas may contribute to the development of small molecule inhibitors of HOXA9.

Common cardiac medications potently inhibit ACE2 binding to the SARS-CoV-2 Spike, and block virus penetration and infectivity in human lung cells.[Pubmed:34773067]

Sci Rep. 2021 Nov 12;11(1):22195.

To initiate SARS-CoV-2 infection, the Receptor Binding Domain (RBD) on the viral spike protein must first bind to the host receptor ACE2 protein on pulmonary and other ACE2-expressing cells. We hypothesized that cardiac glycoside drugs might block the binding reaction between ACE2 and the Spike (S) protein, and thus block viral penetration into target cells. To test this hypothesis we developed a biochemical assay for ACE2:Spike binding, and tested cardiac glycosides as inhibitors of binding. Here we report that ouabain, digitoxin, and digoxin, as well as sugar-free derivatives Digitoxigenin and digoxigenin, are high-affinity competitive inhibitors of ACE2 binding to the Original [D614] S1 and the alpha/beta/gamma [D614G] S1 proteins. These drugs also inhibit ACE2 binding to the Original RBD, as well as to RBD proteins containing the beta [E484K], Mink [Y453F] and alpha/beta/gamma [N501Y] mutations. As hypothesized, we also found that ouabain, digitoxin and digoxin blocked penetration by SARS-CoV-2 Spike-pseudotyped virus into human lung cells, and infectivity by native SARS-CoV-2. These data indicate that cardiac glycosides may block viral penetration into the target cell by first inhibiting ACE2:RBD binding. Clinical concentrations of ouabain and digitoxin are relatively safe for short term use for subjects with normal hearts. It has therefore not escaped our attention that these common cardiac medications could be deployed worldwide as inexpensive repurposed drugs for anti-COVID-19 therapy.

Cytotoxic effect of carbohydrate derivatives of digitoxigenin involves modulation of plasma membrane Ca(2+) -ATPase.[Pubmed:34553411]

J Cell Biochem. 2021 Dec;122(12):1903-1914.

Cardiac glycosides, such as digoxin and digitoxin, are compounds that interact with Na(+) /K(+) -ATPase to induce anti-neoplastic effects; however, these cardiac glycosides have narrow therapeutic index. Thus, semi-synthetic analogs of digitoxin with modifications in the sugar moiety has been shown to be an interesting approach to obtain more selective and more effective analogs than the parent natural product. Therefore, the aim of this study was to assess the cytotoxic potential of novel Digitoxigenin derivatives, Digitoxigenin-alpha-L-rhamno-pyranoside (1) and Digitoxigenin-alpha-L-amiceto-pyranoside (2), in cervical carcinoma cells (HeLa) and human diploid lung fibroblasts (Wi-26-VA4). In addition, we studied the anticancer mechanisms of action of these compounds by comparing its cytotoxic effects with the potential to modulate the activity of three P-type ATPases; Na(+) /K(+) -ATPase, sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA), and plasma membrane Ca(2+) -ATPase (PMCA). Briefly, the results showed that compounds 1 and 2 were more cytotoxic and selectivity for HeLa tumor cells than the nontumor cells Wi-26-VA4. While the anticancer cytotoxicity in HeLa cells involves the modulation of Na(+) /K(+) -ATPase, PMCA and SERCA, the modulation of these P-type ATPases was completely absent in Wi-26-VA4 cells, which suggest the importance of their role in the cytotoxic effect of compounds 1 and 2 in HeLa cells. Furthermore, the compound 2 inhibited directly erythrocyte ghosts PMCA and both compounds were more cytotoxic than digitoxin in HeLa cells. These results provide a better understanding of the mode of action of the synthetic cardiac glycosides and highlights 1 and 2 as potential anticancer agents.

Authentication of tejocote (Crataegus mexicana) dietary supplements based on DNA barcoding and chemical profiling.[Pubmed:34415825]

Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2021 Dec;38(12):1985-1994.

Tejocote (Crataegus mexicana, Mexican hawthorn), known as a weight-loss supplement, has been marketed online and is easily available for overseas direct purchase. Alipotec (brand name) is known as one of the most popular products containing tejocote in Mexico and other countries. However, adverse effects have been reported by users of these supplements. Therefore it is necessary to find the reason for the side effect. Dietary supplement samples labelled as containing tejocote were analysed using mass spectrometry and DNA barcoding analysis. Our results demonstrate that Alipotec samples contained ingredients from different species, yellow oleander instead of tejocote. The rpoB barcode region was able to differentiate between tejocote and yellow oleander species. Moreover, it was also observed that three compounds, including thevetin B, neriifolin, and Digitoxigenin, clearly distinguish between tejocote and yellow oleander samples. This is the first and preliminary investigation to use an integrated approach of both chemical and genomic profiling for the authentication of dietary supplement containing tejocote.

Investigation of cardiac glycosides from oleander in a human induced pluripotent stem cells derived cardiomyocyte model.[Pubmed:34371141]

Toxicol Lett. 2021 Oct 10;350:261-266.

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.

Multiple analysis of mitochondrial metabolism, autophagy and cell death.[Pubmed:34225921]

Methods Cell Biol. 2021;164:95-112.

In the perspective to evaluate the toxicity of drug candidates or the exploration of intracellular signaling pathways of cell stress response and pathophysiological conditions, we propose to evaluate cell death, autophagy, mitochondrial network and energetic metabolism by a series of optimized joint protocols for neonatal primary rat cardiomyocytes or H9c2 cardiac cell line in 96 well microtiter plates. We used Digitoxigenin and Digoxin, two cardiac glycosides, and Rapamycin as control drugs, for inhibition of oxidative stress-induced cell death and autophagy induction, respectively.