Targeting protein-protein interaction interfaces with antiviral N protein inhibitor in SARS-CoV-2.

Coronaviruses not only pose significant global public health threats but also cause extensive damage to livestock-based industries. Previous studies have shown that 5-benzyloxygramine (P3) targets the Middle East respiratory syndrome coronavirus (MERS-CoV) nucleocapsid (N) protein N-terminal domain (N-NTD), inducing non-native protein-protein interactions (PPIs) that impair N protein function. Moreover, P3 exhibits broad-spectrum antiviral activity against CoVs. The sequence similarity of N proteins is relatively low among CoVs, further exhibiting notable variations in the hydrophobic residue responsible for non-native PPIs in the N-NTD. Therefore, to ascertain the mechanism by which P3 demonstrates broad-spectrum anti-CoV activity, we determined the crystal structure of the SARS-CoV-2 N-NTD:P3 complex. We found that P3 was positioned in the dimeric N-NTD via hydrophobic contacts. Compared with the interfaces in MERS-CoV N-NTD, P3 had a reversed orientation in SARS-CoV-2 N-NTD. The Phe residue in the MERS-CoV N-NTD:P3 complex stabilized both P3 moieties. However, in the SARS-CoV-2 N-NTD:P3 complex, the Ile residue formed only one interaction with the P3 benzene ring. Moreover, the pocket in the SARS-CoV-2 N-NTD:P3 complex was more hydrophobic, favoring the insertion of the P3 benzene ring into the complex. Nevertheless, hydrophobic interactions remained the primary stabilizing force in both complexes. These findings suggested that despite the differences in the sequence, P3 can accommodate a hydrophobic pocket in N-NTD to mediate a non-native PPI, enabling its effectiveness against various CoVs.

Bioprospecting the American Alligator Peptidome for antiviral peptides against Venezuelan equine encephalitis virus.

The innate immune protection provided by cationic antimicrobial peptides (CAMPs) has been shown to extend to antiviral activity, with putative mechanisms of action including direct interaction with host cells or pathogen membranes. The lack of therapeutics available for the treatment of viruses such as Venezuelan equine encephalitis virus (VEEV) underscores the urgency of novel strategies for antiviral discovery. American alligator plasma has been shown to exhibit strong in vitro antibacterial activity, and functionalized hydrogel particles have been successfully employed for the identification of specific CAMPs from alligator plasma. Here, a novel bait strategy in which particles were encapsulated in membranes from either healthy or VEEV-infected cells was implemented to identify peptides preferentially targeting infected cells for subsequent evaluation of antiviral activity. Statistical analysis of peptide identification results was used to select five candidate peptides for testing, of which one exhibited a dose-dependent inhibition of VEEV and also significantly inhibited infectious titers. Results suggest our bioprospecting strategy provides a versatile platform that may be adapted for antiviral peptide identification from complex biological samples.

Echinocystic Acid Ameliorates Arthritis in SKG Mice by Suppressing Th17 Cell Differentiation and Human Rheumatoid Arthritis Fibroblast-Like Synoviocytes Inflammation.

Echinocystic acid (EA), a pentacyclic triterpene, exhibits anti-inflammatory, antioxidant, and analgesic activities to counteract pathological effects in various diseases. Here, we aimed to determine the immunomodulatory effect of EA on zymosan-induced arthritis in SKG mice and how it would influence Th17 differentiation and human rheumatoid arthritis fibroblast-like synoviocytes inflammation. Our results showed that EA (10 and 25 mg/kg) attenuated arthritis symptoms, including high arthritis scores, infiltrating inflammatory cells, synovial hyperplasia, bone erosion, and the high levels of proinflammatory cytokines, such as TNF-α, interleukin (IL)-6, and IL-1ß in paw tissues, and reduced the number of splenic Th17 cells. Mechanistically, we found that in vitro treatment of EA inhibited both IL-6- and transforming growth factor-ß (TGF-ß)-induced Th17 cell differentiation by suppressing the phosphorylation of signal transducers and transcriptional activators, especially STAT3. In line with the in vivo result, EA significantly reduced the protein and mRNA expression of IL-6 and IL-1ß in human RA-FLA cells, MH7A cells. Furthermore, the production of both cytokines was confirmed with the downregulation of mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB) signaling pathways under the stimulation of TNF-α. In conclusion, these findings revealed that EA was capable of amelioration of arthritic disorders in SKG mice through inhibiting Th17 cell differentiation and synovial fibroblast inflammation, supporting that EA is a promising therapeutic candidate for treating RA patients.

RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2.

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy toward them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3X (DDX3), a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented support the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.

Crassolide Induces G2/M Cell Cycle Arrest, Apoptosis, and Autophagy in Human Lung Cancer Cells via ROS-Mediated ER Stress Pathways.

Crassolide, a cembranoid diterpene extracted from the soft coral Lobophytum crissum, has been proven to possess antioxidant and immunomodulatory properties. In the present study, we assessed the anticancer effects of crassolide on human H460 non-small-cell lung cancer (NSCLC) cells. We found that crassolide exerted cytotoxic effects on H460 cancer cells in vitro, inducing G2/M phase arrest and apoptosis. In addition, in H460 cells exposed to crassolide, the expression of the autophagy-related proteins LC3-II and beclin was increased, while the expression of p62 was decreased. Moreover, inhibiting autophagy with chloroquine (CQ) suppressed the crassolide-induced G2/M arrest and apoptosis of H460 cells. Moreover, we also found that crassolide induced endoplasmic reticulum (ER) stress in lung cancer cells by increasing the expression of ER stress marker proteins and that the crassolide-induced G2/M arrest, apoptosis, and autophagy were markedly attenuated by the ER stress inhibitor 4-phenylbutyric acid (4-PBA). Furthermore, we found that crassolide promoted reactive oxygen species (ROS) production by H460 cells and that the ROS inhibitor N-acetylcysteine (NAC) decreased the crassolide-induced ER stress, G2/M arrest, apoptosis, and autophagy. In conclusion, our findings show that crassolide inhibits NSCLC cell malignant biological behaviors for the first time, suggesting that this effect may be mechanistically achieved by inducing G2/M arrest, apoptosis, and autophagy through ROS accumulation, which activates the ER stress pathway. As a result of our findings, we now have a better understanding of the molecular mechanism underlying the anticancer effect of crassolide, and we believe crassolide might be a candidate for targeted cancer therapy.

RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2.

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy towards them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3, a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented supports the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.

(-)-Agelasidine A Induces Endoplasmic Reticulum Stress-Dependent Apoptosis in Human Hepatocellular Carcinoma.

Liver cancers, such as hepatocellular carcinoma (HCC), are a highly prevalent cause of cancer-related deaths. Current treatments to combat liver cancer are limited. (-)-Agelasidine A, a compound isolated from the methanol extract of Agelasnakamurai, a sesquiterpene guanidine derived from sea sponge, has antibacterial activity. We demonstrated its anticancer capabilities by researching the associated mechanism of (-)-agelasidine A in human liver cancer cells. We found that (-)-agelasidine A significantly reduced viability in Hep3B and HepG2 cells, and we determined that apoptosis was involved in the (-)-agelasidine A-induced Hep3B cell deaths. (-)-Agelasidine A activated caspases 9, 8, and 3, as well as PARP. This effect was reversed by caspase inhibitors, suggesting caspase-mediated apoptosis in the (-)-agelasidine A-treated Hep3B cells. Moreover, the reduced mitochondrial membrane potential (MMP) and the release of cytochrome c indicated that the (-)-agelasidine A-mediated mitochondrial apoptosis was mechanistic. (-)-Agelasidine A also increased apoptosis-associated proteins (DR4, DR5, FAS), which are related to extrinsic pathways. These events were accompanied by an increase in Bim and Bax, proteins that promote apoptosis, and a decrease in the antiapoptotic protein, Bcl-2. Furthermore, our results presented that (-)-agelasidine A treatment bridged the intrinsic and extrinsic apoptotic pathways. Western blot analysis of Hep3B cells treated with (-)-agelasidine A showed that endoplasmic reticulum (ER) stress-related proteins (GRP78, phosphorylated PERK, phosphorylated eIF2α, ATF4, truncated ATF6, and CHOP) were upregulated. Moreover, 4-PBA, an ER stress inhibitor, could also abrogate (-)-agelasidine A-induced cell viability reduction, annexin V+ apoptosis, death receptor (DR4, DR5, FAS) expression, mitochondrial dysfunction, and cytochrome c release. In conclusion, by activating ER stress, (-)-agelasidine A induced the extrinsic and intrinsic apoptotic pathways of human HCC.

The Gastroprotective Effect of Naringenin against Ethanol-Induced Gastric Ulcers in Mice through Inhibiting Oxidative and Inflammatory Responses.

Naringenin is a major flavanone found in grapes, tangelos, blood oranges, lemons, pummelo, and tangerines. It is known to have anti-inflammatory, antioxidant, anticancer, antimutagenic, antifibrogenic, and antiatherogenic pharmacological properties. This study aims to investigate the anti-inflammatory effects of naringenin in ethanol-induced gastric damage in vivo and ethanol-stimulated KATO III cells in vitro. Our results showed that pretreatment with naringenin significantly protected mice from ethanol-induced hemorrhagic damage, epithelial cell loss, and edema with leucocytes. It reduced gastric ulcers (GU) by suppressing ethanol-induced nuclear factor-κB (NF-κB) activity and decreasing the levels of nitric oxide (NO), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and myeloperoxidase (MPO). In addition, pretreatment with naringenin might inhibit the secretion of TNF-α, IL-6, and IL-8, as well as the proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) via the suppression of NF-κB and mitogen-activated protein kinase (MAPK) signaling in ethanol-stimulated stomach epithelial KATO III cells. Together, the results of this study highlight the gastroprotective effect of naringenin in GU of mice by inhibiting gastric secretion and acidity, reducing inflammation and oxidative stress, suppressing NF-κB activity, and restoring the histological architecture. These findings suggested that naringenin has therapeutic potential in the alleviation of ethanol-induced GU.

Atractylodin Suppresses TGF-ß-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic change in alveolar epithelial cells and leads to the irreversible deterioration of pulmonary function. Transforming growth factor-beta 1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) in type 2 lung epithelial cells contributes to excessive collagen deposition and plays an important role in IPF. Atractylodin (ATL) is a kind of herbal medicine that has been proven to protect intestinal inflammation and attenuate acute lung injury. Our study aimed to determine whether EMT played a crucial role in the pathogenesis of pulmonary fibrosis and whether EMT can be utilized as a therapeutic target by ATL treatment to mitigate IPF. To address this topic, we took two steps to investigate: 1. Utilization of anin vitro EMT model by treating alveolar epithelial cells (A549 cells) with TGF-ß1 followed by ATL treatment for elucidating the underlying pathways, including Smad2/3 hyperphosphorylation, mitogen-activated protein kinase (MAPK) pathway overexpression, Snail and Slug upregulation, and loss of E-cadherin. Utilization of an in vivo lung injury model by treating bleomycin on mice followed by ATL treatment to demonstrate the therapeutic effectiveness, such as, less collagen deposition and lower E-cadherin expression. In conclusion, ATL attenuates TGF-ß1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in mice.

Microencapsulated Recombinant Human Epidermal Growth Factor Ameliorates Osteoarthritis in a Murine Model.

Osteoarthritis, a highly age-related and chronic inflammatory disorder with cartilage loss, causes patients difficultly in movement; there is no efficient and sustainable remedy for osteoarthritis currently. Although hyaluronic acid (HA) and platelet-rich plasma (PRP) have been used to alleviate osteoarthritis, the effects could be short and multiple injections might be required. To address this issue, we exploited the property of chitosan to encapsulate recombinant human epidermal growth factor and obtained microencapsulated rhEGF (Me-rhEGF). In the current study, we induced the osteoarthritis-like symptoms with monosodium iodoacetate (MIA) in rats and investigated the therapeutic effects of Me-rhEGF. Following administration of HA/Me-rhEGF in vivo, we observed that the total Mankin scores, cartilage oligomeric protein, C-telopeptide of type II collagen, IL-1ß, IL-6, IL-17A, and TNF-α cytokines, nitric oxide, and prostaglandin E2 expressions were significantly inhibited. Our results also strongly indicate that individual use of HA or rhEGF slightly decreased the inflammation and restored the destructive joint structure, but was not as drastic as seen in the HA/Me-rhEGF. Moreover, HA/Me-rhEGF profoundly reduced cartilage destruction and proteoglycan loss and downregulated matrix metalloproteinase expressions. These findings reveal that the treatment of HA/Me-rhEGF could be more beneficial than the use of single HA or rhEGF in reliving osteoarthritis and demonstrate the therapeutic application of microencapsulation technology in difficult joint disorders. In essence, we believe that the Me-rhEGF could be promising for further research and development as a clinical treatment against osteoarthritis.

Hesperidin and Chlorogenic Acid Synergistically Inhibit the Growth of Breast Cancer Cells via Estrogen Receptor/Mitochondrial Pathway.

Breast cancer is the most common cancer in women worldwide. Hesperidin (Hes) and chlorogenic acid (CA) are traditional medicinal molecules that abundantly exist in natural plants or foods. These compounds have been shown to prevent and suppress various cancers and therefore can be utilized as adjunctive therapies to aid cancer treatment. Here, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays show a greater synergistic inhibitory effect on the growth of breast cancer cells, MCF-7, but not normal breast cells, MCF-10A, than hesperidin or chlorogenic acid alone. We present the possible molecular signaling pathways in MCF-7 cells with or without herbal molecule treatments via proteomic approaches. The data were further analyzed by Ingenuity Pathway Analysis (IPA) and confirmed by quantifying mRNA associated with the estrogen-receptor signaling pathway and mitochondrial functions. We demonstrated that the expression of CYC1, TFAM, ATP5PB, mtATP6, mtDNA, and NRF-1 were decreased upon 12 h treatment, and subsequent ATP production was also significantly decreased at 24 h. These results identified a synergistic effect induced by combinational treatment with hesperidin and chlorogenic acid, which can regulate mitochondria and ATP production through the estrogen receptor pathway in MCF-7 cells. However, none of the treatments induced the generation of reactive oxygen species (ROS), suggesting that ROS likely plays no role in the observed pharmacological activities. Overall, our study sheds light on the adequacy of hesperidin and chlorogenic acid to serve as an adjunctive therapy when co-administrated with chemotherapy drugs in breast cancer patients.

Effect of the Chinese Herbal Medicine SS-1 on a Sjögren's Syndrome-Like Disease in Mice.

Sjögren's syndrome (SS) is an inflammatory autoimmune disease primarily affecting the exocrine glands; it has a major impact on patients' lives. The Chinese herbal formula SS-1 is composed of Gan Lu Yin, Sang Ju Yin, and Xuefu Zhuyu decoction, which exerts anti-inflammatory, immunomodulatory, and antifibrotic effects. Our previous study demonstrated that SS-1 alleviates clinical SS. This study aimed to evaluate the efficacy and mechanism of the Chinese herbal formula SS-1 for salivary gland protein-induced experimental Sjögren's syndrome (ESS). These results showed that ESS treatment with the Chinese herbal formula SS-1 (1500 mg/kg) significantly alleviated the severity of ESS. We found that SS-1 substantially improved saliva flow rates in SS mice and ameliorated lymphocytic infiltrations in submandibular glands. In addition, salivary gland protein-induced SS in mice treated with SS-1 significantly lowered proinflammatory cytokines (including IFN-γ, IL-6, and IL-17A) in mouse salivary glands and decreased serum anti-M3R autoantibody levels. In addition, we found that CD4+ T cells isolated from SS-1-treated SS mice significantly reduced the percentages of IFN-γ-producing CD4+ T cells (Th1) and IL-17A-producing CD4+ T cells (Th17). Our data show that SS-1 alleviates ESS through anti-inflammatory and immunomodulatory effects, which provides new insight into the clinical treatment of SS.

A Natural Botanical Product, Resveratrol, Effectively Suppresses Vesicular Stomatitis Virus Infection In Vitro.

Numerous natural phytochemicals such as resveratrol are acknowledged as potent botanical agents in regulating immune responses. However, it is less understood whether such immunomodulatory phytochemicals are appropriate for use as direct treatments in veterinary viral diseases. In the present study, we investigated the efficacy of resveratrol in suppressing vesicular stomatitis virus (VSV) infection. Outbreaks of VSV can cause massive economic loss in poultry and livestock husbandry farming, and VSV treatment is in need of therapeutic development. We utilized a recombinant VSV that expresses green fluorescent protein (GFP) to measure viral replication in cells treated with resveratrol. Our findings revealed that resveratrol treatment affords a protective effect, shown by increased viability and reduced viral replication, as indicated by a reduction in fluorescent signals. Additionally, we found that resveratrol inhibition of VSV infection occurs via suppression of the caspase cascade. Structural analysis also indicated that resveratrol potentially interacts with the active sites of caspase-3 and -7, facilitating antiviral activity. The potential effect of resveratrol on reducing VSV infection in vitro suggests that resveratrol should be further investigated as a potential veterinary therapeutic or prophylactic agent.

Use of magnetic nanotrap particles in capturing Yersinia pestis virulence factors, nucleic acids and bacteria.

BACKGROUND: Many pathogens, including Yersinia pestis, cannot be consistently and reliably cultured from blood. New approaches are needed to facilitate the detection of proteins, nucleic acid and microorganisms in whole blood samples to improve downstream assay performance. Detection of biomarkers in whole blood is difficult due to the presence of host proteins that obscure standard detection mechanisms. Nanotrap® particles are micron-sized hydrogel structures containing a dye molecule as the affinity bait and used to detect host biomarkers, viral nucleic acids and proteins as well as some bacterial markers. Nanotraps have been shown to bind and enrich a wide variety of biomarkers and viruses in clinically relevant matrices such as urine and plasma. Our objective was to characterize the binding ability of Nanotrap particle type CN3080 to Y. pestis bacteria, bacterial proteins and nucleic acids from whole human blood in order to potentially improve detection and diagnosis. RESULTS: CN3080 Nanotraps bind tightly to Yersinia bacteria, even after washing, and we were able to visualize the co-localized Nanotraps and bacteria by electron microscopy. These magnetic hydrogel Nanotraps were able to bind Yersinia DNA, supporting the utility of Nanotraps for enhancing nucleic acid-based detection methods. Nanotraps were capable of increasing Y. pestis nucleic acid yield by fourfold from whole human blood compared to standard nucleic acid extraction. Interestingly, we found CN3080 Nanotraps to have a high affinity for multiple components of the Yersinia type III secretion system (T3SS), including chaperone proteins, Yop effector proteins and virulence factor protein LcrV (V). Using Nanotraps as a rapid upstream sample-prep tool, we were able to detect LcrV in human blood by western blotting with minimal blood interference in contrast to direct western blotting of blood samples in which LcrV was obscured. We were able to computationally model the interaction of LcrV with the CN3080 Nanotrap dye and found that it had a low delta-G, suggesting high affinity. Importantly, Nanotraps were also able to enhance detection of secreted Yersinia proteins by mass spectrometry. CONCLUSION: Upstream use of magnetic CN3080 Nanotrap particles may improve the downstream workflow though binding and enrichment of biomarkers and speed of processing. Utilization of Nanotrap particles can improve detection of Yersinia pestis proteins and nucleic acid from whole human blood and contribute to downstream assays and diagnostics including molecular methods such as sequencing and PCR and protein-based methods.

Bavachinin Induces G2/M Cell Cycle Arrest and Apoptosis via the ATM/ATR Signaling Pathway in Human Small Cell Lung Cancer and Shows an Antitumor Effect in the Xenograft Model.

Lung cancer is grouped into small cell lung cancer (SCLC) and non-SCLC (NSCLC). SCLC exhibits a poor prognosis, and the current anticancer treatment remains unsatisfactory. Bavachinin, present in the seed of Psoralea corylifolia, shows anti-inflammatory effects, immune modulation, and anticancer potency. This study aims to investigate the antitumor effect of bavachinin on SCLC and its underlying mechanism. The SCLC cell line H1688 was treated with different concentrations of bavachinin and showed decreased viability with arrested G2/M and sub-G1 phase cell accumulation at a concentration as low as 25 µM. Expression levels of caspase-3, -8, and -9, as well as Fas, FasL, and Bax, increased with the concentration of bavachinin. The accumulated sub-G1 cells and annexin V confirmed increasing apoptotic cancer cells after treatment. The accumulated G2/M phase cells with increasing levels of phosphorylated CDC25C, CDC2, ATM/ATR, and CHK2/CHK1 confirmed the arrested cell cycle caused by bavachinin via a dose-dependent manner. This phenomenon can be reversed by an ATM/ATR inhibitor, caffeine. Following the administration of bavachinin to xenograft mice with SCLC, the tumor burden decreased without impairing hematologic or hepatorenal functions. Bavachinin induces SCLC apoptosis via intrinsic and extrinsic pathways and causes cancer cell cycle arrest via the ATM/ATR signaling pathway.

Targeting protein-protein interaction interfaces in COVID-19 drug discovery.

To date, the COVID-19 pandemic has claimed over 1 million human lives, infected another 50 million individuals and wreaked havoc on the global economy. The crisis has spurred the ongoing development of drugs targeting its etiological agent, the SARS-CoV-2. Targeting relevant protein-protein interaction interfaces (PPIIs) is a viable paradigm for the design of antiviral drugs and enriches the targetable chemical space by providing alternative targets for drug discovery. In this review, we will provide a comprehensive overview of the theory, methods and applications of PPII-targeted drug development towards COVID-19 based on recent literature. We will also highlight novel developments, such as the successful use of non-native protein-protein interactions as targets for antiviral drug screening. We hope that this review may serve as an entry point for those interested in applying PPIIs towards COVID-19 drug discovery and speed up drug development against the pandemic.

Homoseongomycin, a compound isolated from marine actinomycete bacteria K3-1, is a potent inhibitor of encephalitic alphaviruses.

Marine microorganisms have been a resource for novel therapeutic drugs for decades. In addition to anticancer drugs, the drug acyclovir, derived from a marine sponge, is FDA-approved for the treatment of human herpes simplex virus-1 infections. Most alphaviruses that are infectious to terrestrial animals and humans, such as Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV), lack efficient antiviral drugs and it is imperative to develop these remedies. To push the discovery and development of anti-alphavirus compounds forward, this study aimed to isolate and screen for potential antiviral compounds from cultured marine microbes originating from the marine environment. Compounds from marine microbes were of interest as they are prolific producers of bioactive compounds across the spectrum of human diseases and infections. Homoseongomycin, an actinobacteria isolated from a marine sponge displayed impressive activity against VEEV from a total of 76 marine bioactive products. The 50% effective concentration (EC50) for homoseongomycin was 8.6 µM for suppressing VEEV TC-83 luciferase reporter virus replication. Homoseongomycin was non-toxic up to 50 µM and partially rescued cells from VEEV induced cell death. Homoseongomycin exhibited highly efficient antiviral activity with a reduction of VEEV infectious titers by 8 log10 at 50 µM. It also inhibited EEEV replication with an EC50 of 1.2 µM. Mechanism of action studies suggest that homoseongomycin affects both early and late stages of the viral life cycle. Cells treated with 25 µM of homoseongomycin had a ~90% reduction in viral entry. In comparison, later stages showed a more robust reduction in infectious titers (6 log10) and VEEV extracellular viral RNA levels (4 log10), but a lesser impact on intracellular viral RNA levels (1.5 log10). In sum, this work demonstrates that homoseongomycin is a potential anti-VEEV and anti-EEEV compound due to its low cytotoxicity and potent antiviral activity.

Crassolide Suppresses Dendritic Cell Maturation and Attenuates Experimental Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the production of ß2-glycoprotein I (ß2GPI)-dependent autoantibodies, with vascular thrombosis or obstetrical complications. Around 20% of APS patients are refractory to current treatments. Crassolide, a cembranoid diterpene extracted from soft corals, is a potential therapeutic candidate. Here, to examine the anti-inflammatory properties of crassolide, we first determined its effects on bone marrow-derived and splenic dendritic cells (DC). Specifically, we applied lipopolysaccharide (LPS) or ß2GPI stimulation and measured the expressions of CD80 and CD86, and secretions of cytokines. We also determined in the OT-II mice, if bone marrow-derived DC was able to stimulate antigen-specific T cells. Moreover, we examined the therapeutic potential of crassolide postimmunization in a murine model of APS that depended on active immunization with ß2GPI. The vascular manifestations were evaluated in terms of fluorescein-induced thrombi in mesenteric microvessels, whereas the obstetric manifestations were evaluated based on the proportion of fetal loss after pregnancy. We also measured blood titers of anti-ß2GPI antibody, splenic cell proliferative responses and cytokine secretions after ß2GPI stimulation ex vivo. Finally, we determined in these mice, hematological, hepatic and renal toxicities of crassolide. Crassolide after LPS stimulation suppressed DC maturation and secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12 and IL-23, and downstream T cell activation. Crassolide could partially ameliorate both the vascular and obstetric manifestations of APS in BALB/c mice. Both blood titers of anti-ß2GPI antibody and splenic cell proliferation after ß2GPI stimulation were reduced. Splenic Th1 and Th17 responses were also lowered after ß2GPI stimulation. Finally, within therapeutic doses of crassolide, we found no evidence of its toxicity. In conclusion, we showed the ability of crassolide to suppress DC and downstream T cell responses. Crassolide is therefore a potential candidate for adjunctive therapy in APS.

Kurarinone Attenuates Collagen-Induced Arthritis in Mice by Inhibiting Th1/Th17 Cell Responses and Oxidative Stress.

Kurarinone is a flavanone, extracted from Sophora flavescens Aiton, with multiple biological effects. Here, we determine the therapeutic potential of kurarinone and elucidate the interplay between kurarinone and the autoimmune disease rheumatoid arthritis (RA). Arthritis was recapitulated by induction of bovine collagen II (CII) in DBA/1 mice as a collagen-induced arthritis (CIA) model. After the establishment of the CIA, kurarinone was given orally from day 21 to 42 (100 mg/kg/day) followed by determination of the severity based on a symptom scoring scale and with histopathology. Levels of cytokines, anti-CII antibodies, and the proliferation and lineages of T cells from the draining lymph nodes were measured using ELISA and flow cytometry, respectively. The expressional changes, including STAT1, STAT3, Nrf2, KEAP-1, and heme oxygenase-1 (HO-1) changes in the paw tissues, were evaluated by Western blot assay. Oxidative stress featured with malondiadehyde (MDA) and hydrogen peroxide (H2O2) activities in paw tissues were also evaluated. Results showed that kurarinone treatment reduced arthritis severity of CIA mice, as well as their levels of proinflammatory cytokines, TNF-α, IL-6, IFN-γ, and IL-17A, in the serum and paw tissues. T cell proliferation was also reduced by kurarinone even under the stimulation of CII and anti-CD3 antibody. In addition, kurarinone reduced STAT1 and STAT3 phosphorylation and the proportions of Th1 and Th17 cells in lymph nodes. Moreover, kurarinone suppressed the production of MDA and H2O2. All while promoting enzymatic activities of key antioxidant enzymes, SOD and GSH-Px. In the paw tissues, upregulation of Nrf-2 and HO-1, and downregulation of KEAP-1 were observed. Overall, kurarinone showed an anti-inflammatory effect by inhibiting Th1 and Th17 cell differentiation and an antioxidant effect exerted in part through activating the Nrf-2/KEAP-1 pathway. These beneficial effects in CIA mice contributed to the amelioration of their arthritis, indicating that kurarinone might be an adjunct treatment option for rheumatoid arthritis.

Galangin ameliorates experimental autoimmune encephalomyelitis in mice via modulation of cellular immunity.

Multiple sclerosis (MS) causes neurologic disabilities that effect musculature, sensory systems, and vision. This is largely due to demyelination of nerve fibers caused by chronic inflammation. Corticosteroid treatments ameliorate symptoms of MS, but do not successfully cure the disease itself. In the current study, the application of galangin, a phytochemical flavonoid extracted from the ginger family of Alpinis officinarum, on experimental autoimmune encephalomyelitis (EAE; mouse model for MS) was explored. This study investigated prophylactic and therapeutic activity of the drug and mechanisms by which it acts. The results revealed that galangin at 40 and 80 mg/kg could lower the incidence rate of MS, and alleviate clinical/pathological manifestations. Mice administered galangin presented with less limb paralysis, lower levels of inflammatory cell infiltrates, and decreased demyelination compared to vehicle controls. Levels of CD4+IFNγ+ (TH1) and CD4+IL-17A+ (TH17) cells in the spinal cords of EAE mice administered galangin were reduced and both cell types were not capable of expansion. More surprisingly, galangin inhibited antigen presentation and cytokine production by dendritic cells (DC). Formation of cytokines like IL-6, IL-12, and IL-23 were significantly decreased due to galangin in co-culture models of DC and T-cells. Taken together, the data lead one to conclude that galangin could potentially be used as a potent immunoregulatory agent to alleviate clinical symptoms and reduce the prevalence of MS.