Ginsenoside Rg5, a potent agonist of Nrf2, inhibits HSV-1 infection-induced neuroinflammation by inhibiting oxidative stress and NF-κB activation.

Background: Herpes simplex virus type 1 (HSV-1), known to latently infect the host's trigeminal ganglion, can lead to severe herpes encephalitis or asymptomatic infection, potentially contributing to neurodegenerative diseases like Alzheimer's. The virus generates reactive oxygen species (ROS) that significantly impact viral replication and induce chronic inflammation through NF-κB activation. Nuclear factor E2-related factor 2 (Nrf2), an oxidative stress regulator, can prevent and treat HSV-1 infection by activating the passive defense response in the early stages of infection. Methods and results: Our study investigated the antiviral effects of ginsenoside Rg5, an Nrf2 activator, on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection inhibited Nrf2 activity in host cells, induced ROS/NF-κB signaling, and triggered inflammatory cytokines. However, treatment with ginsenoside Rg5 inhibited ROS/NF-κB signaling and reduced inflammatory cytokines through NRF2 induction. Interestingly, the Nrf2 inhibitor ML385 suppressed the expression of NAD(P)H quinone oxidoreductase 1(NQO1) and enhanced the expression of KEAP1 in HSV-1 infected cells. This led to the reversal of VP16 expression inhibition, a protein factor associated with HSV-1 infection, thereby promoting HSV-1 replication. Conclusion: These findings suggest for the first time that ginsenoside Rg5 may serve as an antiviral against HSV-1 infection and could be a novel therapeutic agent for HSV-1-induced neuroinflammation.

Uncovering the colorectal cancer immunotherapeutic potential: Evening primrose (Oenothera biennis) root extract and its active compound oenothein B targeting the PD-1/PD-L1 blockade.

BACKGROUND: The emergence of immune checkpoint inhibitors, a novel class of immunotherapy drugs, represents a major breakthrough in cancer immunotherapy, substantially improving patient survival post-treatment. Blocking programmed death-ligand 1 (PD-L1) and programmed death protein-1 (PD-1) has demonstrated promising clinical results in various human cancer types. The US FDA has recently permitted only monoclonal antibody (mAb)-based PD-L1 or PD-1 blockers. Although these antibodies exhibit high antitumor efficacy, their size- and affinity-induced side effects limit their applicability. PURPOSE: As small-molecule-based PD-1/PD-L1 blockers capable of reducing the side effects of antibody therapies are needed, this study focuses on exploring natural ingredient-based small molecules that can target hPD-L1/PD-1 using herbal medicines and their components. METHODS: The antitumor potential of evening primrose (Oenothera biennis) root extract (EPRE), a globally utilized traditional herbal medicine, folk remedy, and functional food, was explored. A coculture system was established using human PD-L1-expressed murine MC38 cells (hPD-L1-MC38s) and CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) expressing humanized PD-1. The in vivo experiments utilized a colorectal cancer (CRC) C57BL/6 J mouse model bearing MC38 cells expressing humanized PD-L1 and PD-1 proteins. RESULTS: EPRE and its active compound oenothein B effectively hindered the molecular interaction between hPD-L1 and hPD-1. EPRE stimulated tumor-specific T lymphocytes of a hPD-L1/PD-1 CRC mice. This action resulted in the elevated infiltration of cytotoxic CD8+T lymphocytes and subsequent tumor growth reduction. Moreover, the combined therapy of oenothein B, a PD-1/PD-L1 blocker, and FOLFOX (5-fluorouracil plus oxaliplatin) cooperatively suppressed hPD-L1-MC38s growth in the ex vivo model through activated CD8+ TIL antitumor immune response. Oenothein B exhibited a high binding affinity for hPD-L1 and hPD-1. We believe that this study is the first to uncover the inhibitory effects of EPRE and its component, oenothein B, on PD-1/PD-L1 interactions. CONCLUSION: This study identified a promising small-molecule candidate from natural products that blocks the hPD-L1/PD-1 signaling pathway. These findings emphasize the potential of EPRE and oenothein B as effective anticancer drugs.

Natural Product-Based Glycolysis Inhibitors as a Therapeutic Strategy for Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor-Resistant Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide. Targeted therapy against the epidermal growth factor receptor (EGFR) is a promising treatment approach for NSCLC. However, resistance to EGFR tyrosine kinase inhibitors (TKIs) remains a major challenge in its clinical management. EGFR mutation elevates the expression of hypoxia-inducible factor-1 alpha to upregulate the production of glycolytic enzymes, increasing glycolysis and tumor resistance. The inhibition of glycolysis can be a potential strategy for overcoming EGFR-TKI resistance and enhancing the effectiveness of EGFR-TKIs. In this review, we specifically explored the effectiveness of pyruvate dehydrogenase kinase inhibitors and lactate dehydrogenase A inhibitors in combating EGFR-TKI resistance. The aim was to summarize the effects of these natural products in preclinical NSCLC models to provide a comprehensive understanding of the potential therapeutic effects. The study findings suggest that natural products can be promising inhibitors of glycolytic enzymes for the treatment of EGFR-TKI-resistant NSCLC. Further investigations through preclinical and clinical studies are required to validate the efficacy of natural product-based glycolytic inhibitors as innovative therapeutic modalities for NSCLC.

Cosmosiin Induces Apoptosis in Colorectal Cancer by Inhibiting PD-L1 Expression and Inducing ROS.

Immunotherapies, particularly those concerning immune checkpoint inhibitors, have transformed cancer treatment in recent years. Programmed death-ligand 1 (PD-L1) is a key target for immunotherapy that is overexpressed in the cells of colorectal cancer, a widespread malignant cancer that poses a significant healthcare challenge. This study investigated the effects of cosmosiin treatment on colorectal cancer cell lines. Cosmosiin is a naturally occurring flavone glycoside compound that has potential health benefits, including antioxidant and immunomodulatory effects. This study showed that cosmosiin effectively suppresses the expression of PD-L1 and triggers apoptosis, which is facilitated through pathways that are related to reactive oxygen species. These outcomes suggest that cosmosiin could be a promising candidate for an immune checkpoint inhibitor in the treatment of colorectal cancer.

Natural compounds as lactate dehydrogenase inhibitors: potential therapeutics for lactate dehydrogenase inhibitors-related diseases.

Lactate dehydrogenase (LDH) is a crucial enzyme involved in energy metabolism and present in various cells throughout the body. Its diverse physiological functions encompass glycolysis, and its abnormal activity is associated with numerous diseases. Targeting LDH has emerged as a vital approach in drug discovery, leading to the identification of LDH inhibitors among natural compounds, such as polyphenols, alkaloids, and terpenoids. These compounds demonstrate therapeutic potential against LDH-related diseases, including anti-cancer effects. However, challenges concerning limited bioavailability, poor solubility, and potential toxicity must be addressed. Combining natural compounds with LDH inhibitors has led to promising outcomes in preclinical studies. This review highlights the promise of natural compounds as LDH inhibitors for treating cancer, cardiovascular, and neurodegenerative diseases.

Single-Cell RNA Sequencing Reveals Immuno-Oncology Characteristics of Tumor-Infiltrating T Lymphocytes in Photodynamic Therapy-Treated Colorectal Cancer Mouse Model.

Photodynamic therapy (PDT) has shown promise in reducing metastatic colorectal cancer (CRC); however, the underlying mechanisms remain unclear. Modulating tumor-infiltrating immune cells by PDT may be achieved, which requires the characterization of immune cell populations in the tumor microenvironment by single-cell RNA sequencing (scRNA-seq). Here, we determined the effect of Chlorin e6 (Ce6)-mediated PDT on tumor-infiltrating T cells using scRNA-seq analysis. We used a humanized programmed death-1/programmed death ligand 1 (PD-1/PD-L1) MC38 cell allograft mouse model, considering its potential as an immunogenic cancer model and in combination with PD-1/PD-L1 immune checkpoint blockade. PDT treatment significantly reduced tumor growth in mice containing hPD-1/PD-L1 MC38 tumors. scRNA-seq analysis revealed that the PDT group had increased levels of CD8+ activated T cells and CD8+ cytotoxic T cells, but decreased levels of exhausted CD8+ T cells. PDT treatment also enhanced the infiltration of CD8+ T cells into tumors and increased the production of key effector molecules, including granzyme B and perforin 1. These findings provide insight into immune-therapeutic modulation for CRC patients and highlight the potential of PDT in overcoming immune evasion and enhancing antitumor immunity.

Vitisin B inhibits influenza A virus replication by multi-targeting neuraminidase and virus-induced oxidative stress.

The development of drug-resistant influenza and new pathogenic virus strains underscores the need for antiviral therapeutics. Currently, neuraminidase (NA) inhibitors are commonly used antiviral drugs approved by the US Food and Drug Administration (FDA) for the prevention and treatment of influenza. Here, we show that vitisin B (VB) inhibits NA activity and suppresses H1N1 viral replication in MDCK and A549 cells. Reactive oxygen species (ROS), which frequently occur during viral infection, increase virus replication by activating the NF-κB signaling pathway, downmodulating glucose-6-phosphate dehydrogenase (G6PD) expression, and decreasing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response activity. VB decreased virus-induced ROS generation by increasing G6PD expression and Nrf2 activity, and inhibiting NF-κB translocation to the nucleus through IKK dephosphorylation. In addition, VB reduced body weight loss, increased survival, decreased viral replication and the inflammatory response in the lungs of influenza A virus (IAV)-infected mice. Taken together, our results indicate that VB is a promising therapeutic candidate against IAV infection, complements existing drug limitations targeting viral NA. It modulated the intracellular ROS by G6PD, Nrf2 antioxidant response pathway, and NF-κB signaling pathway. These results demonstrate the feasibility of a multi-targeting drug strategy, providing new approaches for drug discovery against IAV infection.

Antitumor Effect of Korean Red Ginseng through Blockade of PD-1/PD-L1 Interaction in a Humanized PD-L1 Knock-In MC38 Cancer Mouse Model.

Blocking immune checkpoints, programmed death-1 (PD-1) and its ligand PD-L1, has proven a promising anticancer strategy for enhancing cytotoxic T cell activity. Although we previously demonstrated that ginsenoside Rg3, Rh2, and compound K block the interaction of PD-1 and PD-L1, the antitumor effect through blockade of this interaction by Korean Red Ginseng alone is unknown. Therefore, we determined the effects of Korean Red Ginseng extract (RGE) on the PD-1/PD-L1 interaction and its antitumor effects using a humanized PD-1/PD-L1-expressing colorectal cancer (CRC) mouse model. RGE significantly blocked the interaction between human PD-1 and PD-L1 in a competitive ELISA. The CD8+ T cell-mediated tumor cell killing effect of RGE was evaluated using murine hPD-L1-expressing MC38 cells and tumor-infiltrating hPD-1-expressing CD8+ T cells isolated from hPD-L1 MC38 tumor-bearing hPD-1 mice. RGE also reduced the survival of hPD-L1 MC38 cells in a cell co-culture system using tumor-infiltrating CD8+ T cells as effector cells combined with hPD-L1 MC38 target cells. RGE or Keytruda (positive control) treatment markedly suppressed the growth of hPD-L1 MC38 allograft tumors, increased CD8+ T cell infiltration into tumors, and enhanced the production of Granzyme B. RGE exhibits anticancer effects through the PD-1/PD-L1 blockade, which warrants its further development as an immunotherapy.

Mulberrofuran G, a Mulberry Component, Prevents SARS-CoV-2 Infection by Blocking the Interaction between SARS-CoV-2 Spike Protein S1 Receptor-Binding Domain and Human Angiotensin-Converting Enzyme 2 Receptor.

Despite the recent development of RNA replication-targeted COVID-19 drugs by global pharmaceutical companies, their prescription in clinical practice is limited by certain factors, including drug interaction, reproductive toxicity, and drug resistance. COVID-19 drugs with multiple targets for the SARS-CoV-2 life cycle may lead to a successful reduction in drug resistance as well as enhanced therapeutic efficacy, and natural products are a potential source of molecules with therapeutic effects against COVID-19. In this study, we investigated the inhibitory efficacy of mulberrofuran G (MG), a component of Morus alba L., also known as mulberry, which has been used as food and traditional medicine, on the binding of the spike S1 receptor-binding domain (RBD) protein to the angiotensin-converting enzyme 2 (ACE2) receptor, which is the initial stage of the SARS-CoV-2 infection. In competitive enzyme-linked immunosorbent assays, MG effectively blocked the spike S1 RBD: ACE2 receptor molecular binding, and investigations using the BLItz system and in silico modeling revealed that MG has high affinity for both proteins. Finally, we confirmed that MG inhibits the entry of SARS-CoV-2 spike pseudotyped virus and a clinical isolate of SARS-CoV-2 into cells, suggesting that MG might be a promising therapeutic candidate for preventing SARS-CoV-2 binding to the cell surface during early infection.

Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction.

There has been an immense effort by global pharmaceutical companies to develop anti-COVID-19 drugs, including small molecule-based RNA replication inhibitors via drug repositioning and antibody-based spike protein blockers related to cell entry by SARS-CoV-2. However, several limitations to their clinical use have emerged in addition to a lack of progress in the development of small molecule-based cell entry inhibitors from natural products. In this study, we tested the effectiveness of kuwanon C (KC), which has mainly been researched using in silico docking simulation and can serve as an effective building block for developing anti-COVID-19 drugs, in blocking the spike S1 RBD:ACE2 receptor interaction. KC is a natural product derived from Morus alba L., commonly known as mulberry, which has known antiviral efficacy. Molecular interaction studies using competitive ELISA and the BLItz system revealed that KC targets both the spike S1 RBD and the ACE2 receptor, successfully disrupting their interaction, as supported by the in silico docking simulation. Furthermore, we established a mechanism of action by observing how KC prevents the infection of SARS-CoV-2 spike pseudotyped virus in ACE2/TPRSS2-overexpressing HEK293T cells. Finally, we demonstrated that KC inhibits clinical isolates of SARS-CoV-2 in Vero cells. Future combinations of small molecule-based cell entry inhibitors, such as KC, with the currently prescribed RNA replication inhibitors are anticipated to significantly enhance the efficacy of COVID-19 therapies.

Augmented Antitumor Effect of Unripe Rubus coreanus Miquel Combined with Oxaliplatin in a Humanized PD-1/PD-L1 Knock-In Colorectal Cancer Mouse Model.

Immune checkpoint inhibitors (ICIs) have been shown to be extraordinarily effective in patients with colorectal cancer (CRC). However, the current ICIs still have adverse effects and limited efficacy of ICI monotherapy. We used a natural product to overcome the vulnerability of ICIs and tried a combination therapy with oxaliplatin to enhance the programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) blockade anticancer effect. In the present study, we evaluated the T cell-mediated antitumor immunity with Unripe Rubus coreanus Miquel extract (RCE), which exerts anticancer properties via PD-1/PD-L1 blockade, combined with oxaliplatin in a co-culture cell model and allograft tumor humanized PD-1 mice. We found that RCE plus oxaliplatin apparently activates hPD-1 tumor-infiltrating CD8+ T cells, resulting in elevations of released interleukin-2 (IL-2) and granzyme B (GrB), and kills hPD-L1 MC38 CRC cells. RCE plus oxaliplatin considerably reduced tumor growth in humanized PD-1/PD-L1-expressing mouse MC38 CRC allograft. Moreover, RCE plus oxaliplatin remarkably increased the infiltration of CD8+ T cells in tumor tissues, as well as increasingly produced GrB of tumor-infiltrating CD8+ T cells in the tumor microenvironment. Our study delineated combination therapy with RCE as a PD-1/PD-L1 blockade and oxaliplatin to improve the response to immune checkpoint blockade therapy in conjunction with standard chemotherapy regimens in CRC.

Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model.

Immune checkpoints such as programmed death-1 (PD-1) have been proven as antitumor targets by enhancing cytotoxic T cell activity. All immune checkpoint blockades are antibody therapeutics that have large size and high affinity, as well as known immune-related side effects and low responses. To overcome the limitation of antibody therapeutics, we have explored PD-1/PD-L1 (programmed death-ligand 1) blockades in traditional oriental medicine, which has a long history but has not yet studied PD-1/PD-L1 blockades. Sanguisorbae Radix extract (SRE) blocked PD-1 and PD-L1 binding in competitive ELISA. SRE effectively inhibited the PD-1/PD-L1 interaction, thereby improving T cell receptor (TCR) signaling and the NFAT-mediated luciferase activity of T cells. SRE treatment reduced tumor growth in the humanized PD-L1 MC38 cell allograft humanized PD-1 mouse model. Additionally, the combination of SRE and pembrolizumab (anti-PD-1 antibody) suppressed tumor growth and increased infiltrated cytotoxic T cells to a greater extent did either agent alone. This study showed that SRE alone has anticancer effects via PD-1/PD-L1 blockade and that the combination therapy of SRE and pembrolizumab has enhanced immuno-oncologic effects.

Geraniin Inhibits the Entry of SARS-CoV-2 by Blocking the Interaction between Spike Protein RBD and Human ACE2 Receptor.

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite the development of vaccines, the emergence of SARS-CoV-2 variants and the absence of effective therapeutics demand the continual investigation of COVID-19. Natural products containing active ingredients may be good therapeutic candidates. Here, we investigated the effectiveness of geraniin, the main ingredient in medical plants Elaeocarpus sylvestris var. ellipticus and Nephelium lappaceum, for treating COVID-19. The SARS-CoV-2 spike protein binds to the human angiotensin-converting enzyme 2 (hACE2) receptor to initiate virus entry into cells; viral entry may be an important target of COVID-19 therapeutics. Geraniin was found to effectively block the binding between the SARS-CoV-2 spike protein and hACE2 receptor in competitive enzyme-linked immunosorbent assay, suggesting that geraniin might inhibit the entry of SARS-CoV-2 into human epithelial cells. Geraniin also demonstrated a high affinity to both proteins despite a relatively lower equilibrium dissociation constant (KD) for the spike protein (0.63 µM) than hACE2 receptor (1.12 µM), according to biolayer interferometry-based analysis. In silico analysis indicated geraniin's interaction with the residues functionally important in the binding between the two proteins. Thus, geraniin is a promising therapeutic agent for COVID-19 by blocking SARS-CoV-2's entry into human cells.

Anticancer Effect of Salvia plebeia and Its Active Compound by Improving T-Cell Activity via Blockade of PD-1/PD-L1 Interaction in Humanized PD-1 Mouse Model.

Immune checkpoint inhibitors, increasingly used to treat malignant tumors, are revolutionizing cancer treatment by improving the patient survival expectations. Despite the high antitumor efficacy of antibody therapeutics that bind to PD-1/PD-L1, study on small molecule-based PD-1/PD-L1 inhibitors is required to overcome the side effects of antibody therapeutics caused by their size and affinity. Herein, we investigated antitumor potential of Salvia plebeia R. Br. extract (SPE), which has been used as a traditional oriental medicine and food in many countries, and its components by the blockade of PD-1/PD-L1 interaction. SPE and its component cosmosiin effectively blocked the molecular interaction between PD-1 and PD-L1. SPE also inhibited tumor growth by increasing CD8+ T-cells in the tumor through the activation of tumor-specific T-cells in a humanized PD-1 mouse model bearing hPD-L1 knock-in MC38 colon adenocarcinoma tumor. This finding presents a preclinical strategy to develop small molecule-based anticancer drugs targeting the PD-1/PD-L1 immune checkpoint pathway.

Anti-Influenza Activity of an Ethyl Acetate Fraction of a Rhus verniciflua Ethanol Extract by Neuraminidase Inhibition.

Antigenic mismatch can cause influenza vaccines to be ineffective, and influenza viruses resistant to antiviral drugs are rising. Thus, development of antiviral agents against these viruses is an immediate need. Rhus verniciflua (RVS) has long been used in herbal medicine and as a nutritional supplement. The effect of RVS and its components on influenza virus has not, however, been reported. We found that RVS treatment significantly reduced viral replication when evaluated with green fluorescent protein- (GFP-) tagged virus (influenza A virus, A/PR/8/34-GFP) in Madin-Darby canine kidney (MDCK) cells. RVS showed significant inhibition of neuraminidase from A/PR/8/34. Subsequently, three fractions were prepared from an ethanolic crude extract of RVS. In vitro assays indicated that an ethyl acetate fraction (RVSE) was more potent than H2O and CHCl3 fractions. RVSE significantly suppressed influenza virus infection in MDCK cells via neuraminidase inhibition. Additionally, RVSE treatment inhibited expression of several virus proteins and decreased mortality of mice exposed to influenza A/PR/8/34 by 50% and reduced weight loss by 11.5%. Active components in RVSE were isolated, and 5-deoxyluteolin (5) and sulfuretin (7) demonstrate the highest neuraminidase inhibitory activity against influenza A virus. RVS, RVSE, and their constituents may be useful for the development of anti-influenza agents.

Unripe Black Raspberry (Rubus coreanus Miquel) Extract and Its Constitute, Ellagic Acid Induces T Cell Activation and Antitumor Immunity by Blocking PD-1/PD-L1 Interaction.

Rubus coreanus Miquel (R. coreanus) is a unripen fruit of black raspberry native to eastern Asia. It is used as traditional oriental medicine and supplementary foods for centuries. Previous studies have shown that the R. coreanus extract (RCE) and its main constitute ellagic acid possess diverse biological activities. However, the effects of RCE on antitumor immunity and T cell function were not fully understood. The present study describes the anti-tumor effect of RCE in humanized PD-1 mice by blocking PD-1/PD-L1 interaction. Competitive enzyme-linked immunosorbent assay (ELISA) and pull down assay were performed to elucidate the binding properties of RCE in vitro. Cellular PD-1/PD-L1 blockade activities were measured by T cell receptor (TCR)-induced nuclear factor of activated T cells-luciferase activity in co-cultured cell models with PD-1/NFAT Jurkat and PD-L1/aAPC CHO-K1 cells. The in vivo efficacy of RCE was confirmed in humanized PD-1 mice bearing MC38 colorectal tumor. RCE and ellagic acid dose-dependently block the binding of PD-1 to PD-L1. Moreover, oral administration of RCE showed the potent anti-tumor activity similar to anti-PD-1 antibody. The present study suggests that RCE possesses potent anti-tumor effect via PD-1/PD-L1 blockade, and ellagic acid is the main compound in RCE. Thus, we provide new aspects of RCE as an immunotherapeutic agent.

Inhibition of Programmed Death Receptor-1/Programmed Death Ligand-1 Interactions by Ginsenoside Metabolites.

Evidence suggests that programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1) targeted inhibitors act as an immune checkpoint blockade, indicating that these compounds may be useful in cancer immunotherapy by inhibiting the immune response between T-cells and tumors. Previous studies have shown that ginsenosides can regulate the expression of PD-1 and PD-L1 in target diseases; however, it remains unknown whether ginsenosides act as a blockade of PD-1/PD-L1 interactions. In this study, we used competitive ELISA to investigate 12 ginsenosides for their ability to block PD-1/PD-L1 interactions. In addition, we performed a protein-ligand docking simulation and examined the hydrophobic interactions and hydrogen bonds formed at the interfaces between the ginsenosides and PD-L1/PD-1. Eight out of the 12 ginsenosides studied showed inhibition of PD-1/PD-L1 interactions at 35% at the maximum concentration (1 µM). Among them, Rg3 and Compound K (C-K) demonstrated the highest inhibitory effects. Rg3 and C-K were further identified for their interaction efficacy with PD-1/PD-L1, which supported our results demonstrating the blocking activity of these compounds against PD-1/PD-L1 binding interactions. Collectively, our findings suggest that some ginsenosides, including Rg3 and C-K, inhibit PD-1/PD-L1 binding interactions. Therefore, these compounds may prove useful as part of an overall immuno-oncological strategy.

Kaempferol and Its Glycoside, Kaempferol 7-O-Rhamnoside, Inhibit PD-1/PD-L1 Interaction In Vitro.

Kaempferol (KO) and kaempferol 7-O-rhamnoside (KR) are natural products from various oriental herbs such as Geranii Herba. Previous studies have reported some biological activities of KO and KR; however, their effects on PD-1/PD-L1 interaction have not been reported yet. To elucidate their inhibitory activities on PD-1/PD-L1 protein-protein interaction (PPI), biochemical assays including competitive ELISA and biolayer interferometry (BLI) systems were performed. Cellular PD-1/PD-L1 blocking activity was measured in a co-culture system with PD-1 Jurkat and PD-L1/aAPC CHO-K1 cells by T-cell receptor (TCR) activation-induced nuclear factor of activated T cells (NFAT)-luciferase reporter assay. The detailed binding mode of action was simulated by an in silico docking study and pharmacophore analysis. Competitive ELISA revealed that KO and its glycoside KR significantly inhibited PD-1/PD-L1 interaction. Cellular PD-1/PD-L1 blocking activity was monitored by KO and KR at non-cytotoxic concentration. Surface plasmon resonance (SPR) and biolayer interferometry (BLI) analysis suggested the binding affinity and direct inhibition of KR against PD-1/PD-L1. An in silico docking simulation determined the detailed mode of binding of KR to PD-1/PD-L1. Collectively, these results suggest that KR could be developed as a potent small molecule inhibitor for PD-1/PD-L1 blockade.

Immune Checkpoint PD-1/PD-L1 CTLA-4/CD80 are Blocked by Rhus verniciflua Stokes and its Active Compounds.

The bark of Rhus verniciflua Stokes (RVS) has been used to treat cancer in Korean herbal medicine. When we screened for PD-1 and CTLA-4 immune checkpoint inhibitors (PD-1/PD-L1 CTLA-4/CD80) from around 800 herbal extracts using competitive Enzyme-Linked Immunosorbent Assay (ELISA), we found that RVS blocked both the PD-1/PD-L1 and the CTLA-4/CD80 interactions. To identify the active compounds from RVS, we performed bioactivity-guided fractionation, and the ethyl acetate (EtOAc) fraction of RVS proved to be the most effective at blocking the PD-1/PD-L1 and CTLA-4/CD80 interactions. In addition, we isolated and identified 20 major compounds in the EtOAc fraction of RVS and then examined the blocking effects of these 20 compounds on PD-1/PD-L1 and CTLA-4/CD80. Among them, four compounds [eriodictyol (7) > fisetin (9) > quercetin (18) > liquiritigenin (13)] blocked the interaction of PD-1/PD-L1 on competitive ELISA. In addition, four different compounds [protocatechuic acid (2) > caffeic acid (19) > taxifolin (5) > butin (6)] blocked the interaction of CTLA-4/CD80. Our findings suggest that RVS and its components could be used as a potential immune checkpoint inhibitor blockade and could be developed for immuno-oncological therapeutics.

Antiviral activity of ethanol extract of Geranii Herba and its components against influenza viruses via neuraminidase inhibition.

Influenza viruses are a serious threat to human health, causing numerous deaths and pandemics worldwide. To date, neuraminidase (NA) inhibitors have primarily been used to treat influenza. However, there is a growing need for novel NA inhibitors owing to the emergence of resistant viruses. Geranii Herba (Geranium thunbergii Siebold et Zuccarini), which is edible, has long been used in a variety of disease treatments in Asia. Although recent studies have reported its various pharmacological activities, the effect of Geranii Herba and its components on influenza viruses has not yet been reported. In this study, Geranii Herba ethanol extract (GHE) and its component geraniin showed high antiviral activity against influenza A strain as well as influenza B strain, against which oseltamivir has less efficacy than influenza A strain, by inhibiting NA activity following viral infection in Madin-Darby canine kidney cells. Thus, GHE and its components may be useful for the development of anti-influenza drugs.