MASTER-NAADP: a membrane permeable precursor of the Ca2+ mobilizing second messenger NAADP.

Upon stimulation of membrane receptors, nicotinic acid adenine dinucleotide phosphate (NAADP) is formed as second messenger within seconds and evokes Ca2+ signaling in many different cell types. Here, to directly stimulate NAADP signaling, MASTER-NAADP, a Membrane permeAble, STabilized, bio-rEversibly pRotected precursor of NAADP is synthesized and release of its active NAADP mimetic, benzoic acid C-nucleoside, 2'-phospho-3'F-adenosine-diphosphate, by esterase digestion is confirmed. In the presence of NAADP receptor HN1L/JPT2 (hematological and neurological expressed 1-like protein, HN1L, also known as Jupiter microtubule-associated homolog 2, JPT2), this active NAADP mimetic releases Ca2+ and increases the open probability of type 1 ryanodine receptor. When added to intact cells, MASTER-NAADP initially evokes single local Ca2+ signals of low amplitude. Subsequently, also global Ca2+ signaling is observed in T cells, natural killer cells, and Neuro2A cells. In contrast, control compound MASTER-NADP does not stimulate Ca2+ signaling. Likewise, in cells devoid of HN1L/JPT2, MASTER-NAADP does not affect Ca2+ signaling, confirming that the product released from MASTER-NAADP is a bona fide NAADP mimetic.

Moxidectin and Ivermectin Inhibit SARS-CoV-2 Replication in Vero E6 Cells but Not in Human Primary Bronchial Epithelial Cells.

Antiviral therapies are urgently needed to treat and limit the development of severe COVID-19 disease. Ivermectin, a broad-spectrum anti-parasitic agent, has been shown to have anti-SARS-CoV-2 activity in Vero cells at a concentration of 5 µM. These limited in vitro results triggered the investigation of ivermectin as a treatment option to alleviate COVID-19 disease. However, in April 2021, the World Health Organization stated the following: "The current evidence on the use of ivermectin to treat COVID-19 patients is inconclusive." It is speculated that the in vivo concentration of ivermectin is too low to exert a strong antiviral effect. Here, we performed a head-to-head comparison of the antiviral activity of ivermectin and the structurally related, but metabolically more stable moxidectin in multiple in vitro models of SARS-CoV-2 infection, including physiologically relevant human respiratory epithelial cells. Both moxidectin and ivermectin exhibited antiviral activity in Vero E6 cells. Subsequent experiments revealed that these compounds predominantly act on the steps following virus cell entry. Surprisingly, however, in human-airway-derived cell models, both moxidectin and ivermectin failed to inhibit SARS-CoV-2 infection, even at concentrations of 10 µM. These disappointing results call for a word of caution in the interpretation of anti-SARS-CoV-2 activity of drugs solely based on their activity in Vero cells. Altogether, these findings suggest that even using a high-dose regimen of ivermectin, or switching to another drug in the same class, is unlikely to be useful for treatment of SARS-CoV-2 in humans.

Resveratrol and Pterostilbene Inhibit SARS-CoV-2 Replication in Air-Liquid Interface Cultured Human Primary Bronchial Epithelial Cells.

The current COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has an enormous impact on human health and economy. In search for therapeutic options, researchers have proposed resveratrol, a food supplement with known antiviral, anti-inflammatory, and antioxidant properties as an advantageous antiviral therapy for SARS-CoV-2 infection. Here, we provide evidence that both resveratrol and its metabolically more stable structural analog, pterostilbene, exhibit potent antiviral properties against SARS-CoV-2 in vitro. First, we show that resveratrol and pterostilbene antiviral activity in African green monkey kidney cells. Both compounds actively inhibit virus replication within infected cells as reduced virus progeny production was observed when the compound was added at post-inoculation conditions. Without replenishment of the compound, antiviral activity was observed up to roughly five rounds of replication, demonstrating the long-lasting effect of these compounds. Second, as the upper respiratory tract represents the initial site of SARS-CoV-2 replication, we also assessed antiviral activity in air-liquid interface (ALI) cultured human primary bronchial epithelial cells, isolated from healthy volunteers. Resveratrol and pterostilbene showed a strong antiviral effect in these cells up to 48 h post-infection. Collectively, our data indicate that resveratrol and pterostilbene are promising antiviral compounds to inhibit SARS-CoV-2 infection. Because these results represent laboratory findings in cells, we advocate evaluation of these compounds in clinical trials before statements are made whether these drugs are advantageous for COVID-19 treatment.

Recent advances in antiviral drug development towards dengue virus.

Despite the high disease burden of dengue virus, there is no approved antiviral treatment or broadly applicable vaccine to treat or prevent dengue virus infection. In the last decade, many antiviral compounds have been identified but only few have been further evaluated in pre-clinical or clinical trials. This review will give an overview of the direct-acting and host-directed antivirals identified to date. Furthermore, important parameters for further development that is, drug properties including efficacy, specificity and stability, pre-clinical animal testing, and combinational drug therapy will be discussed.

Tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro.

In recent decades, chikungunya virus (CHIKV) has re-emerged, leading to outbreaks of chikungunya fever in Africa, Asia and Central and South America. The disease is characterized by a rapid onset febrile illness with (poly)arthralgia, myalgia, rashes, headaches and nausea. In 30 to 40% of the cases, CHIKV infection causes persistent (poly)arthralgia, lasting for months or even years after initial infection. Despite the drastic re-emergence and clinical impact there is no vaccine nor antiviral compound available to prevent or control CHIKV infection. Here, we evaluated the antiviral potential of tomatidine towards CHIKV infection. We demonstrate that tomatidine potently inhibits virus particle production of multiple CHIKV strains. Time-of -addition experiments in Huh7 cells revealed that tomatidine acts at a post-entry step of the virus replication cycle. Furthermore, a marked decrease in the number of CHIKV-infected cells was seen, suggesting that tomatidine predominantly acts early in infection yet after virus attachment and cell entry. Antiviral activity was still detected at 24 hours post-infection, indicating that tomatidine controls multiple rounds of CHIKV replication. Solasodine and sarsasapogenin, two structural derivatives of tomatidine, also showed strong albeit less potent antiviral activity towards CHIKV. In conclusion, this study identifies tomatidine as a novel compound to combat CHIKV infection in vitro.

Tomatidine, a novel antiviral compound towards dengue virus.

Dengue is the most common arboviral disease worldwide with 96 million symptomatic cases annually. Despite its major impact on global human health and huge economic burden there is no antiviral drug available to treat the disease. The first tetravalent dengue virus vaccine was licensed in 2015 for individuals aged 9 to 45, however, most cases are reported in infants and young children. This, together with the limited efficacy of the vaccine to dengue virus (DENV) serotype 2, stresses the need to continue the search for compounds with antiviral activity to DENV. In this report, we describe tomatidine as a novel compound with potent antiviral properties towards all DENV serotypes and the related Zika virus. The strongest effect was observed for DENV-2 with an EC50 and EC90 value of 0.82 and 1.61 µM, respectively, following infection of Huh7 cells at multiplicity of infection of 1. The selectivity index is 97.7. Time-of-drug-addition experiments revealed that tomatidine inhibits virus particle production when added pre, during and up to 12 h post-infection. Subsequent experiments show that tomatidine predominantly acts at a step after virus-cell binding and membrane fusion but prior to the secretion of progeny virions. Tomatidine was found to control the expression of the cellular protein activating transcription factor 4 (ATF4), yet, this protein is not solely responsible for the observed antiviral effect. Here, we propose tomatidine as a candidate for the treatment of dengue given its potent antiviral activity.

Dietary Fiber Pectin Directly Blocks Toll-Like Receptor 2-1 and Prevents Doxorubicin-Induced Ileitis.

Dietary carbohydrate fibers are known to prevent immunological diseases common in Western countries such as allergy and asthma but the underlying mechanisms are largely unknown. Until now beneficial effects of dietary fibers are mainly attributed to fermentation products of the fibers such as anti-inflammatory short-chain fatty acids (SCFAs). Here, we found and present a new mechanism by which dietary fibers can be anti-inflammatory: a commonly consumed fiber, pectin, blocks innate immune receptors. We show that pectin binds and inhibits, toll-like receptor 2 (TLR2) and specifically inhibits the proinflammatory TLR2-TLR1 pathway while the tolerogenic TLR2-TLR6 pathway remains unaltered. This effect is most pronounced with pectins having a low degree of methyl esterification (DM). Low-DM pectin interacts with TLR2 through electrostatic forces between non-esterified galacturonic acids on the pectin and positive charges on the TLR2 ectodomain, as confirmed by testing pectin binding on mutated TLR2. The anti-inflammatory effect of low-DM pectins was first studied in human dendritic cells and mouse macrophages in vitro and was subsequently tested in vivo in TLR2-dependent ileitis in a mouse model. In these mice, ileitis was prevented by pectin administration. Protective effects were shown to be TLR2-TLR1 dependent and independent of the SCFAs produced by the gut microbiota. These data suggest that low-DM pectins as a source of dietary fiber can reduce inflammation through direct interaction with TLR2-TLR1 receptors.