Cardiotonic steroids have long been in clinical use for treatment of heart failure and are now emerging as promising agents in various diseases, especially cancer. Their main target is Na(+)/K(+)-ATPase, a membrane protein involved in cellular ion homeostasis. Na(+)/K(+)-ATPase has been implicated in cancer biology by affecting several cellular events and signaling pathways in both sensitive and drug-resistant cancer cells. Hence, we investigated the cytotoxic activities of 66 cardiotonic steroids and cardiotonic steroid derivatives in sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Data were then subjected to quantitative structure-activity relationship analysis (QSAR) and molecular docking into Na(+)/K(+)-ATPase, which both indicated a possible differential expression of the pump in the mentioned cell lines. This finding was confirmed by western blotting, intracellular potassium labeling and next generation sequencing which showed that Na(+)/K(+)-ATPase was less expressed in multidrug-resistant than in sensitive cells.

Cardiogenic shock (CS) is an acute complex condition leading to morbidity and mortality. Vasoactive medications, such as vasopressors and inotropes are considered the cornerstone of pharmacological treatment of CS to improve end-organ perfusion by increasing cardiac output (CO) and blood pressure (BP), thus preventing multiorgan failure.

To present a snapshot of experimental cardiovascular research with a focus on geographical and temporal patterns of early termination due to poor accrual.

Recently, we showed that some new synthetic compounds structurally related to cilostamide (4-(1,2-dihydro-2-oxoquinolin-6-hydroxy)- N-cyclohexyl-N-methylbutanamide), a selective phosphodiesterase 3 (PDE3) inhibitor, produce inotropic effect comparable to that of IBMX (3-isobutyl-1-methylxanthine), a non-selective PDE inhibitor, but with differential chronotropic effect. In this investigation, we compared the pharmacological effects of these compounds as potential cardiotonic agents using the spontaneously beating atria model.

Clinical use of selective PDE3 inhibitors as cardiotonic agents is limited because of their chronotropic and lipolytic side effects. In our previous work, we synthesized a new PDE3 inhibitor named MC2 (6-[4-(4-methylpiperidin-1-yl)-4-oxobutoxy]-4-methylquinolin-2(1H)-one) which produced a high positive inotropic action with a negative chronotropic effect. This work was done to evaluate the effects of MC2 on adipocytes and compare its effects with those of amrinone and cilostamide.

Cardiotonic steroids (CTS) are agents traditionally known for their capacity to bind to the Na,K-ATPase (NKA), affecting the ion transport and the contraction of the heart. Natural CTS have been shown to also have effects on cell signaling pathways. With the goal of developing a new CTS derivative, we synthesized a new digoxin derivative, 21-benzylidene digoxin (21-BD). Previously, we have shown that this compound binds to NKA and has cytotoxic actions on cancer, but not on normal cells. Here, we further studied the mechanisms of actions of 21-BD. Working with HeLa cells, we found that 21-BD decreases the basal, as well as the insulin stimulated proliferation. 21-BD reduces phosphorylation of the epidermal growth factor receptor (EGFR) and extracellular-regulated kinase (ERK), which are involved in pathways that stimulate cell proliferation. In addition, 21-BD promotes apoptosis, which is mediated by the translocation of Bax from the cytosol to mitochondria and the release of mitochondrial cytochrome c to the cytosol. 21-BD also activated caspases-8, -9 and -3, and induced the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). Altogether, these results show that the new compound that we have synthesized exerts cytotoxic actions on HeLa cells by inhibition of cell proliferation and the activation of both the extrinsic and intrinsic apoptotic pathways. These results support the relevance of the cardiotonic steroid scaffold as modulators of cell signaling pathways and potential agents for their use in cancer.

Zika virus (ZIKV) is an emerging arbovirus associated with neurological disorders. Currently, no specific vaccines or antivirals are available to treat the ZIKV infection. Ouabain, a cardiotonic steroid known as Na+/K+-ATPase inhibitor, has been previously described as an immunomodulatory substance by our group. Here, we evaluated for the first time the antiviral activity of this promising substance against a Brazilian ZIKV strain. Vero cells were treated with different concentrations of ouabain before and after the infection with ZIKV. The antiviral effect was evaluated by the TCID50 method and RT-qPCR. Ouabain presented a dose-dependent inhibitory effect against ZIKV, mainly when added post infection. The reduction of infectious virus was accompanied by a decrease in ZIKV RNA levels, suggesting that the mechanism of ZIKV inhibition by ouabain occurred at the replication step. In addition, our in silico data demonstrated a conformational stability and favorable binding free energy of ouabain in the biding sites of the NS5-RdRp and NS3-helicase proteins, which could be related to its mechanism of action. Taken together, these data demonstrate the antiviral activity of ouabain against a Brazilian ZIKV strain and evidence the potential of cardiotonic steroids as promising antiviral agents.

Human adenovirus (HAdV) can cause severe disease and death in both immunocompromised and immunocompetent patients. The current standards of treatment are often ineffective, and no approved antiviral therapy against HAdV exists. We report here the design and validation of a fluorescence-based high-content screening platform for the identification of novel anti-HAdV compounds. The screen was conducted using a wildtype-like virus containing the red fluorescent protein (RFP) gene under the regulation of the HAdV major late promoter. Thus, RFP expression allows monitoring of viral late gene expression (a surrogate marker for virus replication), and compounds affecting virus growth can be easily discovered by quantifying RFP intensity. We used our platform to screen ~1200 FDA-approved small molecules, and identified several cardiotonic steroids, corticosteroids and chemotherapeutic agents as anti-HAdV compounds. Our screening platform provides the stringency necessary to detect compounds with varying degrees of antiviral activity, and facilitates drug discovery/repurposing to combat HAdV infections.

Therapeutic options for coronaviruses remain limited. To address this unmet medical need, we screened 5406 compounds, including United States Food and Drug Administration (FDA)-approved drugs and bioactives, for activity against a South Korean Middle East respiratory syndrome coronavirus (MERS-CoV) clinical isolate. Among 221 identified hits, 54 had therapeutic indexes (TI) greater than 6, representing effective drugs. The time-of-addition studies with selected drugs demonstrated eight and four FDA-approved drugs which acted on the early and late stages of the viral life cycle, respectively. Confirmed hits included several cardiotonic agents (TI > 100), atovaquone, an anti-malarial (TI > 34), and ciclesonide, an inhalable corticosteroid (TI > 6). Furthermore, utilizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we tested combinations of remdesivir with selected drugs in Vero-E6 and Calu-3 cells, in lung organoids, and identified ciclesonide, nelfinavir, and camostat to be at least additive in vitro. Our results identify potential therapeutic options for MERS-CoV infections, and provide a basis to treat coronavirus disease 2019 (COVID-19) and other coronavirus-related illnesses.

Melanoma is the most common cancer in young adults, with a constantly increasing incidence. Metastatic melanoma is a very aggressive cancer with a 5-year survival rate of about 22-25%. This is, in most cases, due to a lack of therapies which are effective on the long term. Hence, it is crucial to find new therapeutic agents to increase patient survival. Toad venoms are a rich source of potentially pharmaceutically active compounds and studies have highlighted their possible effect on cancer cells. We focused on the venoms of two different toad species: Bufo bufo and Rhinella marina. We screened the venom crude extracts, the fractions from crude extracts and isolated biomolecules by studying their antiproliferative properties on melanoma cells aiming to determine the compound or the combination of compounds with the highest antiproliferative effect. Our results indicated strong antiproliferative capacities of toad venoms on melanoma cells. We found that these effects were mainly due to bufadienolides that are cardiotonic steroids potentially acting on the Na+/K+ ATPase pump which is overexpressed in melanoma. Finally, our results indicated that bufalin alone was the most interesting compound among the isolated bufadienolides because it had the highest antiproliferative activity on melanoma cells.

Bufadienolides are cytotoxic drugs that may form the basis for anticancer agents. Due to structural and functional similarity to cardiotonic glycosides, application is restricted. We, therefore, investigated correlation of their putative anticancer effects with inhibition of Na+,K+pumps. The natural bufalin and three derivatives were tested. The anticancer effects of the drugs were checked by observing their inhibitory effects on proliferation of rat liver cancer cells using MTT assay. Inhibition of Na+,K+-pump was determined by measuring pump-mediated current of rat α1/β1 and α2/β1 Na+,K+pumps expressed in Xenopus oocytes. All tested bufadienolides inhibited cell proliferation and Na+,K+pump activity. An activity coefficient A=100xIC50Na,K pump/IC50proliferation was used to describe drug effectivity as anticancer drug. Natural bufalin exhibited lowest effectivity on cell proliferation, and also the A value for rat α1 isoform was the lowest (0.08), the α2 isoform was much less sensitive (A=1.00). The highest A values were obtained for the BF238 derivative with A=0.88 and 2.64 for the α1 and α2 isoforms, respectively. Therefore, we suggest that search for bufalin derivatives with high anticancer effect and low affinity for both Na+,K+pump isoforms may be a promising strategy for development of anticancer drugs.

A new coronavirus causing severe acute respiratory syndrome (SARS-CoV-2) has emerged and with it, a global investigation of new antiviral treatments and supportive care for organ failure due to this life-threatening viral infection. Traditional Persian Medicine (TPM) is one of the most ancient medical doctrines mostly known with the manuscripts of Avicenna and Rhazes. In this paper, we first introduce a series of medicinal plants that would potentially be beneficial in treating SARS-CoV-2 infection according to TPM textbooks. Then, we review medicinal plants based on the pharmacological studies obtained from electronic databases and discuss their mechanism of action in SARS-CoV-2 infection. There are several medicinal plants in TPM with cardiotonic, kidney tonic, and pulmonary tonic activities, protecting the lung, heart, and kidney, the three main vulnerable organs in SARS-CoV-2 infection. Some medicinal plants can prevent "humor infection", a situation described in TPM which has similar features to SARS-CoV-2 infection. Pharmacological evaluations are in line with the therapeutic activities of several plants mentioned in TPM, mostly through antiviral, cytoprotective, anti-inflammatory, antioxidant, and anti-apoptotic mechanisms. Amongst the primarily-introduced medicinal plants from TPM, rhubarb, licorice, garlic, saffron, galangal, and clove are the most studied plants and represent candidates for clinical studies. The antiviral compounds isolated from these plants provide novel molecular structures to design new semisynthetic antiviral agents. Future clinical studies in healthy volunteers as well as patients suffering from pulmonary infections are necessary to confirm the safety and efficacy of these plants as complementary and integrative interventions in SARS-CoV-2 infection.

The interaction of Cardiac Troponin C (cTnC) and Cardiac Troponin I (cTnI) plays a critical role in transmitting the Ca (2+) signal to the other myofilament proteins in the activation of cardiac muscle contraction. As such, the cTnC-cTnI interface is a logical target for cardiotonic agents such as levosimendan that can modulate the Ca (2+) sensitivity of the myofilaments. Evidence indicates that drug candidates may exert their effects by targeting a site formed by binding of the switch region of cTnI to the regulatory N domain of cTnC (cNTnC). In this study, we utilized two-dimensional (1)H- (15)N HSQC NMR spectroscopy to monitor the binding of levosimendan and its analogues, CMDP, AMDP, CI-930, imazodan, and MPDP, to cNTnC.Ca (2+) in complex with two versions of the switch region of cTnI (cTnI 147-163 and cTnI 144-163). Levosimendan, CMDP, AMDP, and CI-930 were found to bind to both cNTnC.Ca (2+).cTnI 147-163 and cNTnC.Ca (2+).cTnI 144-163 complexes. These compounds contain a methyl group that is absent in MPDP or imazodan. Thus, the methyl group is one of the pharmacophores responsible for the action of these pyridazinone drugs on cTnC. Furthermore, the results showed that the cNTnC.Ca (2+).cTnI 144-163 complex presents a higher-affinity binding site for these compounds than the cNTnC.Ca (2+).cTnI 147-163 complex. This is consistent with our observation that the affinity of cTnI 144-163 for cNTnC.Ca (2+) is approximately 10-fold stronger than that of cTnI 147-163, likely a result of electrostatic forces between the N-terminal RRV extension in cTnI 144-163 and the acidic residues in the C and D helices of cNTnC. These results will help in the delineation of the mode of action of levosimendan on the important functional unit of cardiac troponin that constitutes the regulatory domain of cTnC and the switch region of cTnI.