Systematic Studies on the Anti-SARS-CoV-2 Mechanisms of Tea Polyphenol-Related Natural Products.

The causative pathogen of COVID-19, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), utilizes the receptor-binding domain (RBD) of the spike protein to bind to human receptor angiotensin-converting enzyme 2 (ACE2). Further cleavage of spike by human proteases furin, TMPRSS2, and/or cathepsin L facilitates viral entry into the host cells for replication, where the maturation of polyproteins by 3C-like protease (3CLpro) and papain-like protease (PLpro) yields functional nonstructural proteins (NSPs) such as RNA-dependent RNA polymerase (RdRp) to synthesize mRNA of structural proteins. By testing the tea polyphenol-related natural products through various assays, we found that the active antivirals prevented SARS-CoV-2 entry by blocking the RBD/ACE2 interaction and inhibiting the relevant human proteases, although some also inhibited the viral enzymes essential for replication. Due to their multitargeting properties, these compounds were often misinterpreted for their antiviral mechanisms. In this study, we provide a systematic protocol to check and clarify their anti-SARS-CoV-2 mechanisms, which should be applicable for all of the antivirals.

Plakoglobin and High-Mobility Group Box 1 Mediate Intestinal Epithelial Cell Apoptosis Induced by Clostridioides difficile TcdB.

Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated intestinal disease, resulting in severe diarrhea and fatal pseudomembranous colitis. TcdB, one of the essential virulence factors secreted by this bacterium, induces host cell apoptosis through a poorly understood mechanism. Here, we performed an RNA interference (RNAi) screen customized to Caco-2 cells, a cell line model of the intestinal epithelium, to discover host factors involved in TcdB-induced apoptosis. We identified plakoglobin, also known as junction plakoglobin (JUP) or γ-catenin, a member of the catenin family, as a novel host factor and a previously known cell death-related chromatin factor, high-mobility group box 1 (HMGB1). Disruption of those host factors by RNAi and CRISPR resulted in resistance of cells to TcdB-mediated and mitochondrion-dependent apoptosis. JUP was redistributed from adherens junctions to the mitochondria and colocalized with the antiapoptotic factor Bcl-XL. JUP proteins could permeabilize the mitochondrial membrane, resulting in the release of cytochrome c. Our results reveal a novel role of JUP in targeting the mitochondria to promote the mitochondrial apoptotic pathway. Treatment with glycyrrhizin, an HMGB1 inhibitor, resulted in significantly increased resistance to TcdB-induced epithelial damage in cultured cells and a mouse ligated colon loop model. These findings demonstrate the critical roles of JUP and HMGB1 in TcdB-induced epithelial cell apoptosis. IMPORTANCE Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea. Toxins, especially TcdB, cause epithelial cell apoptosis, but the underlying cell death mechanism is less clear. Through an apoptosis-focused RNAi screen using a bacterium-made small interfering (siRNA) library customized to a human colonic epithelial cell model, we found a novel host factor, plakoglobin (γ-catenin), as a key factor required for cell apoptosis induced by TcdB. Plakoglobin targets and permeabilizes mitochondria after stimulation by TcdB, demonstrating a hitherto underappreciated role of this catenin family member in the apoptosis of intestinal epithelial cells. We also found a previously known cell death-related chromatin factor, HMGB1, and explored the inhibition of HMGB1 for CDI therapy in vivo.

SARS-CoV-2 3CLpro displays faster self-maturation in vitro than SARS-CoV 3CLpro due to faster C-terminal cleavage.

The coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has become a worldwide pandemic. The 3C-like protease (3CLpro ), which cleaves 11 sites including its own N- and C-termini on the viral polyproteins, is essential for SARS-CoV-2 replication. In this study, we constructed the full-length inactive 3CLpro with N- and C-terminal extensions as substrates for monitoring self-cleavage by wild-type 3CLpro . We found that the rate-limiting C-terminal self-cleavage rate of SARS-CoV-2 3CLpro was 35-fold faster than that of SARS-CoV 3CLpro using the Trx/GST-tagged C145A 3CLpro substrates. Since self-cleavage of 3CLpro is the initial step for maturation of other viral proteins, our study suggests more facile SARS-CoV-2 replication than that of SARS-CoV.

Identification of non-covalent 3C-like protease inhibitors against severe acute respiratory syndrome coronavirus-2 via virtual screening of a Korean compound library.

The outbreak of coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has turned into a pandemic. The enzyme 3C-like protease (3CLpro) is essential for the maturation of viral polyproteins in SARS-CoV-2 and is therefore regarded as a key drug target for treating the disease. To identify 3CLpro inhibitors that can suppress SARS-CoV-2 replication, we performed a virtual screening of 500,282 compounds in a Korean compound bank. We then subjected the top computational hits to inhibitory assays against 3CLpro in vitro, leading to the identification of a class of non-covalent inhibitors. Among these inhibitors, compound 7 showed an EC50 of 39.89 µM against SARS-CoV-2 and CC50 of 453.5 µM. This study provides candidates for the optimization of potent 3CLpro inhibitors showing antiviral effects against SARS-CoV-2.

Kinetic Characterization and Inhibitor Screening for the Proteases Leading to Identification of Drugs against SARS-CoV-2.

Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CLpro) and papain-like protease (PLpro) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease. In this study, 3CLpro and PLpro assay platforms were established, and their substrate specificities were characterized. The assays were used to screen collections of 1,068 and 2,701 FDA-approved drugs. After excluding the externally used drugs which are too toxic, we totally identified 12 drugs as 3CLpro inhibitors and 36 drugs as PLpro inhibitors active at 10 µM. Among these inhibitors, six drugs were found to suppress SARS-CoV-2 with the half-maximal effective concentration (EC50) below or close to 10 µM. This study enhances our understanding on the proteases and provides FDA-approved drugs for prevention and/or treatment of COVID-19.

Overview of antiviral drug candidates targeting coronaviral 3C-like main proteases.

Coronaviruses (CoVs) are positive single-stranded RNA viruses that cause severe respiratory syndromes in humans, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). Coronavirus disease 2019 (COVID-19) caused by a novel severe acute respiratory syndrome CoV (SARS-CoV-2) at the end of 2019 became a global pandemic. The 3C-like cysteine protease (3CLpro) processes viral polyproteins to yield mature non-structural proteins, thus playing an important role in the CoV life cycle, and therefore is considered as a prominent target for antiviral drugs. To date, many 3CLpro inhibitors have been reported, and their molecular mechanisms have been illustrated. Here, we briefly introduce the structural features of 3CLpro of the human-related SARS-CoV, MERS-CoV and SARS-CoV-2, and explore the potency and mechanism of their cognate inhibitors. This information will shed light on the development and optimization of CoV 3CLpro inhibitors, which may benefit the further designation of therapeutic strategies for treating CoV diseases.

Structural-based virtual screening and in vitro assays for small molecules inhibiting the feline coronavirus 3CL protease as a surrogate platform for coronaviruses.

Feline infectious peritonitis (FIP) which is caused by feline infectious peritonitis virus (FIPV), a variant of feline coronavirus (FCoV), is a member of family Coronaviridae, together with severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. So far, neither effective vaccines nor approved antiviral therapeutics are currently available for the treatment of FIPV infection. Both human and animal CoVs shares similar functional proteins, particularly the 3CL protease (3CLpro), which plays the pivotal role on viral replication. We investigated the potential drug-liked compounds and their inhibitory interaction on the 3CLpro active sites of CoVs by the structural-bases virtual screening. Fluorescence resonance energy transfer (FRET) assay revealed that three out of twenty-eight compounds could hamper FIPV 3CLpro activities with IC50 of 3.57 ± 0.36 µM to 25.90 ± 1.40 µM, and Ki values of 2.04 ± 0.08 to 15.21 ± 1.76 µM, respectively. Evaluation of antiviral activity using cell-based assay showed that NSC629301 and NSC71097 could strongly inhibit the cytopathic effect and also reduced replication of FIPV in CRFK cells in all examined conditions with the low range of EC50 (6.11 ± 1.90 to 7.75 ± 0.48 µM and 1.99 ± 0.30 to 4.03 ± 0.60 µM, respectively), less than those of ribavirin and lopinavir. Analysis of FIPV 3CLpro-ligand interaction demonstrated that the selected compounds reacted to the crucial residues (His41 and Cys144) of catalytic dyad. Our investigations provide a fundamental knowledge for the further development of antiviral agents and increase the number of anti-CoV agent pools for feline coronavirus and other related CoVs.

High-level expression of ARID1A predicts a favourable outcome in triple-negative breast cancer patients receiving paclitaxel-based chemotherapy.

Paclitaxel-based chemotherapy is a common strategy to treat patients with triple-negative breast cancer (TNBC). As paclitaxel resistance is still a clinical issue in treating TNBCs, identifying molecular markers for predicting pathologic responses to paclitaxel treatment is thus urgently needed. Here, we report that an AT-rich interaction domain 1A (ARID1A) transcript is up-regulated in paclitaxel-sensitive TNBC cells but down-regulated in paclitaxel-resistant cells upon paclitaxel treatment. Moreover, ARID1A expression was negatively correlated with the IC50 concentration of paclitaxel in the tested TNBC cell lines. Kaplan-Meier analyses revealed that ARID1A down-regulation was related to a poorer response to paclitaxel-based chemotherapy in patients with TNBCs as measured by the recurrence-free survival probability. The pharmaceutical inhibition with p38MAPK-specific inhibitor SCIO-469 revealed that p38MAPK-related signalling axis regulates ARID1A expression and thereby modulates paclitaxel sensitivity in TNBC cells. These findings suggest that ARID1A could be used as a prognostic factor to estimate the pathological complete response for TNBC patients who decide to receive paclitaxel-based chemotherapy.

The Gαh-PLCδ1 signaling axis drives metastatic progression in triple-negative breast cancer.

BACKGROUND: Distant metastasis of triple-negative breast cancer (TNBC) to other organs, e.g., the lungs, has been correlated with poor survival rates among breast cancer patients. Therefore, the identification of useful therapeutic targets to prevent metastasis or even inhibit tumor growth of TNBC is urgently needed. Gαh is a novel GTP-binding protein and known as an inactive form of calcium-dependent tissue transglutaminase. However, the functional consequences of transamidating and G-protein activities of tissue transglutaminase in promoting cancer metastasis are still controversial. METHODS: Kaplan-Meier analyses were performed to estimate the prognostic values of Gαh and PLCδ1 by utilizing public databases and performing immunohistochemical staining experiments. Cell-based invasion assays and in vivo lung colony-forming and orthotropic lung metastasis models were established to evaluate the effectiveness of interrupting the protein-protein interaction (PPI) between Gαh and PLCδ1 in inhibiting the invasive ability and metastatic potential of TNBC cells. RESULTS: Here, we showed that the increased level of cytosolic, not extracellular, Gαh is a poor prognostic marker in breast cancer patients and correlates with the metastatic evolution of TNBC cells. Moreover, clinicopathological analyses revealed that the combined signature of high Gαh/PLCδ1 levels indicates worse prognosis in patients with breast cancer and correlates with lymph node metastasis of ER-negative breast cancer. Blocking the PPI of the Gαh/PLCδ1 complex by synthetically myristoylated PLCδ1 peptide corresponding to the Gαh-binding interface appeared to significantly suppress cellular invasiveness in vitro and inhibit lung metastatic colonies of TNBC cells in vivo. CONCLUSIONS: This study establishes Gαh/PLCδ1 as a poor prognostic factor for patients with estrogen receptor-negative breast cancers, including TNBCs, and provides therapeutic value by targeting the PPI of the Gαh/PLCδ1 complex to combat the metastatic progression of TNBCs.

Prevalence and molecular characterization of Clostridium difficile isolates from a pig slaughterhouse, pork, and humans in Taiwan.

Clostridium difficile causes antibiotic-associated diarrhea in both humans and animals. The ribotype 078, predominant in food animals, is associated with community-acquired C. difficile infection, and C. difficile is suggested to be a foodborne pathogen. Recently, the C. difficile ribotype 078 lineage emerged in patients and pigs in Taiwan. This study aimed to investigate the prevalence and molecular characterization of C. difficile isolated from a pig slaughterhouse, retail meat, ready-to-eat meals, and humans in Taiwan. We collected samples from one slaughterhouse (n=422), 29 retail markets (raw pork, n=62; ready-to-eat pork, n=65), and one hospital (non-diarrheal humans, stool, n=317) in 2015. The isolated C. difficile were subjected to ribotyping and multilocus variable-number tandem-repeat analysis (MLVA). In the slaughterhouse, the isolation rate from carcasses was high (23%, 21/92) and ribotype 126 dominated. Scalding water was found to have C. difficile contamination (44%, 4/9), and two of the seven isolates were ribotype 126. The isolation rates from raw pork and ready-to-eat pork were between 20% and 29%. Ribotypes 126, 127, and 014 were found in raw pork, whereas ribotype 078 was not identified in this study. Eight isolates-seven non-toxigenic isolates and one ribotype 017-were found in non-diarrheal human samples. Notably, MLVA showed that ribotype 126 isolates from the slaughterhouse, pig stool, colons, carcasses, and scalding water were closely genetically related, indicating serious risk for cross-contamination. However, the genetic evidence of foodborne transmission from carcasses to food and humans is still lacking.

Structural basis of inhibition specificities of 3C and 3C-like proteases by zinc-coordinating and peptidomimetic compounds.

Human coxsackievirus (CV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. In picornavirus, a chymotrypsin-like protease (3C(pro)) is required for viral replication by processing the polyproteins, and thus it is regarded as an antiviral drug target. A 3C-like protease (3CL(pro)) also exists in human coronaviruses (CoV) such as 229E and the one causing severe acute respiratory syndrome (SARS). To combat SARS, we previously had developed peptidomimetic and zinc-coordinating inhibitors of 3CL(pro). As shown in the present study, some of these compounds were also found to be active against 3C(pro) of CV strain B3 (CVB3). Several crystal structures of 3C(pro) from CVB3 and 3CL(pro) from CoV-229E and SARS-CoV in complex with the inhibitors were solved. The zinc-coordinating inhibitor is tetrahedrally coordinated to the His(40)-Cys(147) catalytic dyad of CVB3 3C(pro). The presence of specific binding pockets for the residues of peptidomimetic inhibitors explains the binding specificity. Our results provide a structural basis for inhibitor optimization and development of potential drugs for antiviral therapies.

Engineering a novel endopeptidase based on SARS 3CL(pro).

A 3C-like protease (3CLpro) from the severe acute respiratory syndrome-coronavirus (SARS-CoV) is required for viral replication, cleaving the replicase polyproteins at 11 sites with the conserved Gln [downward arrow](Ser, Ala, Gly) sequences. In this study, we developed a mutant 3CLpro (T25G) with an expanded S1' space that demonstrates 43.5-fold better k(cat)/K(m) compared with wild-type in cleaving substrates with a larger Met at P1' and is suitable for tag removal from recombinant fusion proteins. Two vectors for expressing fusion proteins with the T25G recognition site (Ala-Val-Leu-Gln [downward arrow]Met) in Escherichia coli and yeast were constructed. Identical cloning sites were used in these vectors for parallel cloning. PstI was chosen as a 5' cloning site because it overlapped the nucleotide sequence encoding the protease site and avoided addition of extra amino acids at the N terminus of recombinant proteins. 3CL(pro) (T25G) was found to have a 3-fold improvement over TEV(pro) in tag cleavage at each respective preferred cleavage site.

Design, synthesis, and evaluation of 3C protease inhibitors as anti-enterovirus 71 agents.

Human enterovirus (EV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. A peptidomimetic inhibitor AG7088 was developed to inhibit the 3C protease of rhinovirus (a member of the family), a chymotrypsin-like protease required for viral replication, by forming a covalent bond with the active site Cys residue. In this study, we have prepared the recombinant 3C protease from EV71 (TW/2231/98), a particular strain which causes severe outbreaks in Asia, and developed inhibitors against the protease and the viral replication. For inhibitor design, the P3 group of AG7088, which is not interacting with the rhinovirus protease, was replaced with a series of cinnamoyl derivatives directly linked to P2 group through an amide bond to simplify the synthesis. While the replacement caused decreased potency, the activity can be largely improved by substituting the alpha,beta-unsaturated ester with an aldehyde at the P1' position. The best inhibitor 10b showed EC(50) of 18 nM without apparent toxicity (CC(50)>25 microM). Our study provides potent inhibitors of the EV71 3C protease as anti-EV71 agents and facilitates the combinatorial synthesis of derivatives for further improving the inhibitory activity.

Structure-based inhibitors exhibit differential activities against Helicobacter pylori and Escherichia coli undecaprenyl pyrophosphate synthases.

Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase (UPPS), which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli.

Structural basis of mercury- and zinc-conjugated complexes as SARS-CoV 3C-like protease inhibitors.

Five active metal-conjugated inhibitors (PMA, TDT, EPDTC, JMF1586 and JMF1600) bound with the 3C-like protease of severe acute respiratory syndrome (SARS)-associated coronavirus were analyzed crystallographically. The complex structures reveal two major inhibition modes: Hg(2+)-PMA is coordinated to C(44), M(49) and Y(54) with a square planar geometry at the S3 pocket, whereas each Zn(2+) of the four zinc-inhibitors is tetrahedrally coordinated to the H(41)-C(145) catalytic dyad. For anti-SARS drug design, this Zn(2+)-centered coordination pattern would serve as a starting platform for inhibitor optimization.

Specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus.

In this study, 221 phytocompounds were evaluated for activity against anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV) activities using a cell-based assay measuring SARS-CoV-induced cytopathogenic effect on Vero E6 cells. Ten diterpenoids (1-10), two sesquiterpenoids (11 and 12), two triterpenoids (13 and 14), five lignoids (15-19), curcumin (20), and reference controls niclosamide (21) and valinomycin (22) were potent inhibitors at concentrations between 3.3 and 10 microM. The concentrations of the 22 compounds to inhibit 50% of Vero E6 cell proliferation (CC50) and viral replication (EC50) were measured. The selective index values (SI = CC50/EC50) of the most potent compounds 1, 5, 6, 8, 14, and 16 were 58, >510, 111, 193, 180, and >667, respectively. Betulinic acid (13) and savinin (16) were competitive inhibitors of SARS-CoV 3CL protease with Ki values = 8.2 +/- 0.7 and 9.1 +/- 2.4 microM, respectively. Our findings suggest that specific abietane-type diterpenoids and lignoids exhibit strong anti-SARS-CoV effects.

Structure-based drug design and structural biology study of novel nonpeptide inhibitors of severe acute respiratory syndrome coronavirus main protease.

Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)), a protein required for the maturation of SARS-CoV, is vital for its life cycle, making it an attractive target for structure-based drug design of anti-SARS drugs. The structure-based virtual screening of a chemical database containing 58,855 compounds followed by the testing of potential compounds for SARS-CoV M(pro) inhibition leads to two hit compounds. The core structures of these two hits, defined by the docking study, are used for further analogue search. Twenty-one analogues derived from these two hits exhibited IC50 values below 50 microM, with the most potent one showing 0.3 microM. Furthermore, the complex structures of two potent inhibitors with SARS-CoV M(pro) were solved by X-ray crystallography. They bind to the protein in a distinct manner compared to all published SARS-CoV M(pro) complex structures. They inhibit SARS-CoV M(pro) activity via intensive H-bond network and hydrophobic interactions, without the formation of a covalent bond. Interestingly, the most potent inhibitor induces protein conformational changes, and the inhibition mechanisms, particularly the disruption of catalytic dyad (His41 and Cys145), are elaborated.

Stable benzotriazole esters as mechanism-based inactivators of the severe acute respiratory syndrome 3CL protease.

Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus that infected more than 8000 individuals and resulted in more than 800 fatalities in 2003. Currently, there is no effective treatment for this epidemic. SARS-3CL(pro) has been shown to be essential for replication and is thus a target for drug discovery. Here, a class of stable benzotriazole esters was reported as mechanism-based inactivators of 3CL(pro), and the most potent inactivator exhibited a k(inact) of 0.0011 s(-1) and a K(i) of 7.5 nM. Mechanistic investigation with kinetic and mass spectrometry analyses indicates that the active site Cys145 is acylated, and that no irreversible inactivation was observed with the use of the C145A mutant. In addition, a noncovalent, competitive inhibition became apparent by using benzotriazole ester surrogates in which the bridged ester-oxygen group is replaced with carbon.

Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic alpha,beta-unsaturated esters.

The proteolytic processing of polyproteins by the 3CL protease of severe acute respiratory syndrome coronavirus is essential for the viral propagation. A series of tripeptide alpha,beta-unsaturated esters and ketomethylene isosteres, including AG7088, are synthesized and assayed to target the 3CL protease. Though AG7088 is inactive (IC50 > 100 microM), the ketomethylene isosteres and tripeptide alpha,beta-unsaturated esters containing both P1 and P2 phenylalanine residues show modest inhibitory activity (IC50 = 11-39 microM). The Phe-Phe dipeptide inhibitors 18a-e are designed on the basis of computer modeling of the enzyme-inhibitor complex. The most potent inhibitor 18c with an inhibition constant of 0.52 microM is obtained by condensation of the Phe-Phe dipeptide alpha,beta-unsaturated ester with 4-(dimethylamino)cinnamic acid. The cell-based assays also indicate that 18c is a nontoxic anti-SARS agent with an EC50 value of 0.18 microM.