Given the continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of new inhibitors is necessary to enhance clinical efficacy and increase the options for combination therapy for the coronavirus disease 2019. Because marine organisms have been a resource for the discovery of numerous bioactive molecules, we constructed an extract library of marine invertebrates collected from the Okinawa Islands. In this study, the extracts were used to identify antiviral molecules against SARS-CoV-2. Using a cytopathic effect (CPE) assay in VeroE6/TMPRSS2 cells, an extract from the marine sponge Theonella swinhoei was found to reduce virus-induced CPE. Eventually, onnamide A was identified as an antiviral compound in the extract using column chromatography and NMR analysis. Onnamide A inhibited several SARS-CoV-2 variant-induced CPEs in VeroE6/TMPRSS2 cells as well as virus production in the supernatant of infected cells. Moreover, this compound blocked the entry of SARS-CoV-2 pseudo-virions. Taken together, these results demonstrate that onnamide A suppresses SARS-CoV-2 infection, which may be partially related to entry inhibition, and is expected to be a candidate lead compound for the development of anti-SARS-CoV-2 drugs.

Leishmaniasis is a major public health problem, especially affecting vulnerable populations in tropical and subtropical regions. The disease is endemic in 90 countries, and with millions of people at risk, it is seen as one of the ten most neglected tropical diseases. Current treatments face challenges such as high toxicity, side effects, cost, and growing drug resistance. There is an urgent need for safer, affordable treatments, especially for cutaneous leishmaniasis (CL), the most common form. Marine invertebrates have long been resources for discovering bioactive compounds such as sesterterpenoids. Using bioassay-guided fractionations against cutaneous-type leishmaniasis promastigotes, we identified a novel furanosesterterpenoid, petrosaspongin from Okinawan marine sponges and a nudibranch, along with eight known sesterterpenoids, hippospongins and manoalides. The elucidated structure of petrosaspongin features a β-substituted furane ring, a tetronic acid, and a conjugated triene. The sesterterpenoids with a γ-butenolide group exhibited leishmanicidal activity against Leishmania major promastigotes, with IC₅₀ values ranging from 0.69 to 53 μM. The structure–activity relationship and molecular docking simulation suggest that γ-lactone is a key functional group for leishmanicidal activity. These findings contribute to the ongoing search for more effective treatments against CL.

Spices and herbs, which are derived from natural botanical sources, contain many bioactive compounds and play an important role in human health. The general and specific health benefits of these spices and herbs include anti-inflammatory, antioxidant, and anti-tumorigenic activities. Previously, we showed that cathepsin G, which is a neutrophil-derived serine protease localized in human breast cancer tissues, promotes cancer metastasis via induction of platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) expression in MCF-7 human breast cancer cells. Therefore, although regulation of cathepsin G activity is thought to be important in human breast cancer progression, no compounds that inhibit the activity have been identified for therapeutic purposes. In this study, we screened 50 spice and herb extracts. Peppermint, clove, Sichuan pepper, and fenugreek exhibited strong inhibitory effects on cathepsin G activity and suppressed cathepsin G-induced MCF-7 cell aggregation.; importantly, fenugreek suppressed the increase in PAFAH1B2 expression. The IC₅₀ of 37.38 μg/mL of fenugreek extract that showed inhibitory effect on cathepsin G-induced malignant progression was 5.87 times lower than the concentration that exerted cytotoxic effect. Interestingly, quercetin and trigonelline contained in fenugreek inhibited cathepsin G activity and suppressed the induction of cell aggregation and PAFAH1B2 expression in human breast cancer cells. These results suggest that quercetin and trigonelline are partly responsible for the inhibitory effect of fenugreek on cathepsin G-induced malignant progression of human breast cancer cells. Our findings provide a new breast cancer treatment strategy targeting cathepsin G, and fenugreek may have synergistic effects when combined with therapeutic drugs.

We recently reported that onnamide A, a marine-derived natural compound isolated from the sponge Theonella sp., inhibits the entry process of SARS-CoV-2 infection. However, its antiviral activity against other viruses remains largely unexplored. Here, we investigated the effects of onnamide A and its structurally related analog, onnamide B, on hepatitis B virus (HBV) infection. Using iNTCP cells, a hepatoblastoma-derived cell line permissive to HBV infection, we found that onnamides A and B exhibited cytotoxicity, with CC₅₀ values of 0.53 ± 0.10 μM and 2.37 ± 0.25 μM, respectively. Following HBV infection, the levels of total HBV RNA were significantly reduced by onnamide A (IC₅₀ = 0.06 ± 0.01 μM) and onnamide B (IC₅₀ = 0.23 ± 0.06 μM). Notably, both compounds markedly decreased the levels of HBV pregenomic RNA. Furthermore, significant inhibition was particularly evident when onnamide treatment was initiated after HBV infection. Consistent with these observations, onnamides did not affect HBV binding, entry, or covalently closed circular DNA formation, but they significantly suppressed HBV RNA transcription. In particular, the transcriptional activities driven by the core and X promoters were markedly inhibited by onnamide treatment. Taken together, our findings demonstrate that onnamides possess potent anti-HBV activity and highlight their potential as candidate compounds targeting HBV RNA transcription.

Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9–mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.

N-(4-hydroxyphenyl)-retinamide (4-HPR) inhibits the dihydroceramide Δ4-desaturase 1 (DEGS1) enzymatic activity. We previously reported that 4-HPR suppresses the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein-mediated membrane fusion through a decrease in membrane fluidity in a DEGS1-independent manner. However, the precise mechanism underlying the inhibition of viral entry by 4-HPR remains unclear. In this study, we examined the role of reactive oxygen species (ROS) in the inhibition of membrane fusion by 4-HPR, since 4-HPR is a well-known ROS-inducing agent. Intracellular ROS generation was found to be increased in the target cells in a cell–cell fusion assay following 4-HPR treatment, which was attenuated by the addition of the antioxidant, α-tocopherol (TCP). The reduction in membrane fusion susceptibility by 4-HPR treatment in the cell–cell fusion assay was alleviated by TCP addition. Furthermore, fluorescence recovery after photobleaching analysis showed that the lateral diffusion of glycosylphosphatidylinositol-anchored protein and SARS CoV-2 receptor was reduced by 4-HPR treatment and restored by TCP addition. These results indicate that the decrease in SARS-CoV-2 spike protein-mediated membrane fusion and membrane fluidity by 4-HPR was due to ROS generation. Taken together, these results demonstrate that ROS production is associated with the 4-HPR inhibitory effect on SARS-CoV-2 entry.