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BACKGROUND: Since the onset of the coronavirus disease 2019 (COVID-19) pandemic, remarkable advances have been made in vaccine development to reduce mortality. However, therapeutic interventions for COVID-19 are comparatively limited despite these intensive efforts. Furthermore, the rapid mutation capability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a characteristic of its RNA structure, has led to the emergence of multiple variants, necessitating a shift from a predominantly vaccine-centric approach to one that encompasses therapeutic strategies. 6'-Hydroxy justicidin B (6'-HJB), an arylnaphthalene lignan isolated from Justicia procumbens, a traditional Chinese medicine, is known for its antiviral properties. HYPOTHESIS/PURPOSE: The aim of the present study was to assess the effectiveness and safety of 6'-HJB against SARS-CoV-2 in order to determine its potential as a therapeutic agent against COVID-19. METHODS: The efficacy of 6'-HJB was evaluated both in vitro using Vero and Calu-3 cell lines and in vivo using ferrets. The safety assessment included toxicokinetics, safety pharmacology, and Good Laboratory Practice (GLP)-compliant toxicity evaluations following single- and repeated-dose toxicity studies in dogs. RESULTS: The anti-SARS-CoV-2 efficacy of 6'-HJB was evaluated through dose-response curve (DRC) analysis using immunofluorescence; 6'-HJB demonstrated superior inhibition of SARS-CoV-2 growth and lower cytotoxicity than remdesivir. In SARS-CoV-2-infected ferret, 6'-HJB showed efficacy comparable to that of the positive control, Truvada. Further GLP toxicity studies corroborated the safety profile of 6'-HJB. Single-dose and 4-week repeated oral toxicity studies in Beagle dogs demonstrated minimal harmful effects at the highest dosages. The lethal dose of 6'-HJB exceeded 2,000 mg kg-1 in Beagle dogs. Toxicokinetic and GLP safety pharmacology studies demonstrated no adverse effects of 6'-HJB on metabolic processes, respiratory or central nervous systems, or cardiac functions. CONCLUSION: This research highlights both the antiviral efficacy and safety profile of 6'-HJB, underscoring its potential as a novel COVID-19 treatment option. The potential of 6'-HJB was demonstrated using modern scientific methodologies and standards.

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IMPORTANCE: Endochondral ossification plays an important role in skeletal development. Recent studies have suggested a link between increased intracellular reactive oxygen species (ROS) and skeletal disorders. Moreover, previous studies have revealed that increasing the levels of myeloperoxidase (MPO) and osteopontin (OPN) while inhibiting NADPH oxidase 4 (NOX4) can enhance bone growth. This investigation provides further evidence by showing a direct link between NOX4 and MPO, OPN in bone function. OBJECTIVE: This study investigates NOX4, an enzyme producing hydrogen peroxide, in endochondral ossification and bone remodeling. NOX4's role in osteoblast formation and osteogenic signaling pathways is explored. METHODS: Using NOX4-deficient (NOX4-/-) and ovariectomized (OVX) mice, we identify NOX4's potential mediators in bone maturation. RESULTS: NOX4-/- mice displayed significant differences in bone mass and structure. Compared to the normal Control and OVX groups. Hematoxylin and eosin staining showed NOX4-/- mice had the highest trabecular bone volume, while OVX had the lowest. Proteomic analysis revealed significantly elevated MPO and OPN levels in bone marrow-derived cells in NOX4-/- mice. Immunohistochemistry confirmed increased MPO, OPN, and collagen II (COLII) near the epiphyseal plate. Collagen and chondrogenesis analysis supported enhanced bone development in NOX4-/- mice. CONCLUSIONS AND RELEVANCE: Our results emphasize NOX4's significance in bone morphology, mesenchymal stem cell proteomics, immunohistochemistry, collagen levels, and chondrogenesis. NOX4 deficiency enhances bone development and endochondral ossification, potentially through increased MPO, OPN, and COLII expression. These findings suggest therapeutic implications for skeletal disorders.

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The bromodomain and extraterminal domain (BET) family proteins recognize acetyl-lysine (Kac) at the histone tail through two tandem bromodomains, i.e., BD1 and BD2, to regulate gene expression. BET proteins are attractive therapeutic targets in cancer due to their involvement in oncogenic transcriptional activation, and bromodomains have defined Kac-binding pockets. Here, we present DW-71177, a potent BET inhibitor that selectively interacts with BD1 and exhibits strong antileukemic activity. X-ray crystallography, isothermal titration calorimetry, and molecular dynamic studies have revealed the robust and specific binding of DW-71177 to the Kac-binding pocket of BD1. DW-71177 effectively inhibits oncogenes comparable to the pan-BET inhibitor OTX-015, but with a milder impact on housekeeping genes. It efficiently blocks cancer-associated transcriptional changes by targeting genes that are highly enriched with BRD4 and histone acetylation marks, suggesting that BD1-selective targeting could be an effective and safe therapeutic strategy against leukemia.

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Our previous study revealed that the ethanolic extract of Justicia procumbens ameliorates ovalbumin-induced airway inflammation and airway hyper-responsiveness in a mouse model of asthma. However, the mechanism of action of the extract remains unknown. In this study, we prepared DW2008S, an optimized and standardized powder extracted from J. procumbens using anhydrous ethanol, and investigated its anti-asthmatic effect and mechanism of action. Our results showed that DW2008S contains two major ingredients, justicidin A (JA) and justicidin B (JB), which selectively inhibit T helper 2 (Th2) cell responses in concanavalin A-activated spleen cells and polarized Th2 cells. Blockade of T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT) using a neutralizing antibody also selectively inhibited Th2 cell responses. Furthermore, DW2008S regulated TIGIT expression in the mice and cultured cells. Additionally, DW2008S and JA antagonized human adenosine receptor A3 (A3 AR), which mediates mast cell-dependent inflammation and bronchoconstriction. DW2008S and JB inhibited human phosphodiesterase 4 (PDE4), which is known to cause bronchoconstriction; however, the required concentrations were higher than those needed to affect TIGIT . These findings suggest that DW2008S can potentially ameliorate Th2-driven airway inflammation and bronchoconstriction through negative regulation of TIGIT and blockade of A3 AR and PDE4 activities.

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DW2008 is an anhydrous ethanol extract of Justicia procumbens produced by Dong-Wha Pharmaceutical, Inc., Co. as a candidate anti-asthmatic drug. In this study, DW2008 selectively reduced T helper 2 (Th2) cytokines in mouse splenocytes and ameliorated ovalbumin-induced airway inflammation by downregulating pulmonary infiltration of differential inflammatory cells and Th2 cytokines more than a decoction or ethanol extract of J. procumbens did in a mouse asthma model. DW2008 also significantly inhibited airway hyperresponsiveness and reduced the thickness of the airway epithelium. HPLC analysis showed that the major peaks (justicidin A and B) of DW2008 were higher than those of the other extracts. Justicidin A and B significantly suppressed Th2 cytokine levels in mouse spleen cells and exhibited a protective effect in ovalbumin-induced airway inflammation. Our findings indicate that DW2008 effectively inhibits allergic airway inflammatory reactions and airway hyperresponsiveness in a mouse model of asthma, suggesting its potential as an anti-asthmatic agent.

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