Inhibition of dengue viruses by N-methylcytisine thio derivatives through targeting viral envelope protein and NS2B-NS3 protease.

Dengue virus (DENV) is a significant global health threat, causing millions of cases worldwide each year. Developing antiviral drugs for DENV has been a challenging endeavor. Our previous study identified anti-DENV properties of two (-)-cytisine derivatives contained substitutions within the 2-pyridone core from a pool of 19 (-)-cytisine derivatives. This study aimed to expand on the previous research by investigating the antiviral potential of N-methylcytisine thio (mCy thio) derivatives against DENV, understanding the molecular mechanisms of antiviral activity for the active thio derivatives. The inhibitory assays on DENV-2-induced cytopathic effect and infectivity revealed that mCy thio derivatives 3 ((1R,5S)-3-methyl-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyrido[1,2-a][1,5]diazocine-8-thione) and 6 ((1S,5R)-3-methyl-2-thioxo-1,2,3,4,5,6-hexahydro-8H-1,5-methanopyrido[1,2-a][1,5]diazocin-8-one) were identified as the active compounds against both DENV-1 and DENV-2. Derivative 6 displayed robust antiviral activity against DENV-2, with EC50 values ranging from 0.002 to 0.005 µM in different cell lines. Derivative 3 also exhibited significant antiviral activity against DENV-2. The study found that these compounds are effective at inhibiting DENV-2 at both the entry stage (including virus attachment) and post-entry stages of the viral life cycle. The study also investigated the inhibition of the DENV-2 NS2B-NS3 protease activity by these compounds. Derivative 6 demonstrated notably stronger inhibition compared to mCy thio 3, revealing its dual antiviral action at both the entry and post-entry stages. Molecular docking simulations indicated that mCy thio derivatives 3 and 6 bind to the domain I and III of the DENV E protein, as well as the active of NS2B-NS3 protease, suggesting their molecular interactions with the virus. The study demonstrates the antiviral efficacy of N-methylcytisine thio derivatives against DENV. It provides valuable insights into the potential interactions between these compounds and viral target proteins, which could be useful in the development of antiviral drugs for DENV.

Tafenoquine and its derivatives as inhibitors for the severe acute respiratory syndrome coronavirus 2.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely affected human lives around the world as well as the global economy. Therefore, effective treatments against COVID-19 are urgently needed. Here, we screened a library containing Food and Drug Administration (FDA)-approved compounds to identify drugs that could target the SARS-CoV-2 main protease (Mpro), which is indispensable for viral protein maturation and regard as an important therapeutic target. We identified antimalarial drug tafenoquine (TFQ), which is approved for radical cure of Plasmodium vivax and malaria prophylaxis, as a top candidate to inhibit Mpro protease activity. The crystal structure of SARS-CoV-2 Mpro in complex with TFQ revealed that TFQ noncovalently bound to and reshaped the substrate-binding pocket of Mpro by altering the loop region (residues 139-144) near the catalytic Cys145, which could block the catalysis of its peptide substrates. We also found that TFQ inhibited human transmembrane protease serine 2 (TMPRSS2). Furthermore, one TFQ derivative, compound 7, showed a better therapeutic index than TFQ on TMPRSS2 and may therefore inhibit the infectibility of SARS-CoV-2, including that of several mutant variants. These results suggest new potential strategies to block infection of SARS-CoV-2 and rising variants.

Your height affects your health: genetic determinants and health-related outcomes in Taiwan.

BACKGROUND: Height is an important anthropometric measurement and is associated with many health-related outcomes. Genome-wide association studies (GWASs) have identified hundreds of genetic loci associated with height, mainly in individuals of European ancestry. METHODS: We performed genome-wide association analyses and replicated previously reported GWAS-determined single nucleotide polymorphisms (SNPs) in the Taiwanese Han population (Taiwan Biobank; n = 67,452). A genetic instrument composed of 251 SNPs was selected from our GWAS, based on height and replication results as the best-fit polygenic risk score (PRS), in accordance with the clumping and p-value threshold method. We also examined the association between genetically determined height (PRS251) and measured height (phenotype). We performed observational (phenotype) and genetic PRS251 association analyses of height and health-related outcomes. RESULTS: GWAS identified 6843 SNPs in 89 genomic regions with genome-wide significance, including 18 novel loci. These were the most strongly associated genetic loci (EFEMP1, DIS3L2, ZBTB38, LCORL, HMGA1, CS, and GDF5) previously reported to play a role in height. There was a positive association between PRS251 and measured height (p < 0.001). Of the 14 traits and 49 diseases analyzed, we observed significant associations of measured and genetically determined height with only eight traits (p < 0.05/[14 + 49]). Height was positively associated with body weight, waist circumference, and hip circumference but negatively associated with body mass index, waist-hip ratio, body fat, total cholesterol, and low-density lipoprotein cholesterol (p < 0.05/[14 + 49]). CONCLUSIONS: This study contributes to the understanding of the genetic features of height and health-related outcomes in individuals of Han Chinese ancestry in Taiwan.

In vivo measurement of trabecular meshwork stiffness in a corticosteroid-induced ocular hypertensive mouse model.

Ocular corticosteroids are commonly used clinically. Unfortunately, their administration frequently leads to ocular hypertension, i.e., elevated intraocular pressure (IOP), which, in turn, can progress to a form of glaucoma known as steroid-induced glaucoma. The pathophysiology of this condition is poorly understood yet shares similarities with the most common form of glaucoma. Using nanotechnology, we created a mouse model of corticosteroid-induced ocular hypertension. This model functionally and morphologically resembles human ocular hypertension, having titratable, robust, and sustained IOPs caused by increased resistance to aqueous humor outflow. Using this model, we then interrogated the biomechanical properties of the trabecular meshwork (TM), including the inner wall of Schlemm's canal (SC), tissues known to strongly influence IOP and to be altered in other forms of glaucoma. Specifically, using spectral domain optical coherence tomography, we observed that SC in corticosteroid-treated mice was more resistant to collapse at elevated IOPs, reflecting increased TM stiffness determined by inverse finite element modeling. Our noninvasive approach to monitoring TM stiffness in vivo is applicable to other forms of glaucoma and has significant potential to monitor TM function and thus positively affect the clinical care of glaucoma, the leading cause of irreversible blindness worldwide.

Glycyrrhizic Acid Derivatives Bearing Amino Acid Residues in the Carbohydrate Part as Dengue Virus E Protein Inhibitors: Synthesis and Antiviral Activity.

Dengue virus (DENV) is one of the most geographically distributed mosquito-borne flaviviruses, like Japanese encephalitis virus (JEV), and Zika virus (ZIKV). In this study, a library of the known and novel Glycyrrhizic acid (GL) derivatives bearing amino acid residues or their methyl/ethyl esters in the carbohydrate part were synthesized and studied as DENV inhibitors in vitro using the cytopathic effect (CPE), viral infectivity and virus yield assays with DENV1 and DENV-2 in Vero E6 and A549 cells. Among the GL conjugates tested, compound hits GL-D-ValOMe 3, GL-TyrOMe 6, GL-PheOEt 11, and GL-LysOMe 21 were discovered to have better antiviral activity than GL, with IC50 values ranging from <0.1 to 5.98 µM on the in vitro infectivity of DENV1 and DENV2 in Vero E6 and A549 cells. Compound hits 3, 6, 11, and 21 had a concentration-dependent inhibition on the virus yield in Vero E6, in which GL-D-ValOMe 3 and GL-PheOEt 11 were the most active inhibitors of DENV2 yield. Meanwhile, the time-of-addition assay indicated that conjugates GL-D-ValOMe 3 and GL-PheOEt 11 exhibited a substantial decrease in the DENV2 attachment stage. Subsequently, chimeric single-round infectious particles (SRIPs) of DENV2 C-prM-E protein/JEV replicon and DENV2 prM-E/ZIKV replicon were utilized for the DENV envelope I protein-mediated attachment assay. GL conjugates 3 and 11 significantly reduced the attachment of chimeric DENV2 C-prM-E/JEV and DENV2 prM-E/ZIKV SRIPs onto Vero E6 cells in a concentration-dependent manner but did not impede the attachment of wild-type JEV CprME/JEV and ZIKV prM-E/ZIKV SRIPs, indicating the inhibition of Compounds 3 and 11 on DENV2 E-mediated attachment. Molecular docking data revealed that Compounds 3 and 11 have hydrophobic interactions within a hydrophobic pocket among the interfaces of Domains I, II, and the stem region of the DENV2 envelope (E) protein. These results displayed that Compounds 3 and 11 were the lead compounds targeting the DENV E protein. Altogether, our findings provide new insights into the structure−activity relationship of GL derivatives conjugated with amino acid residues and can be the new fundamental basis for the search and development of novel flavivirus inhibitors based on natural compounds.

Synthesis and antiviral evaluation of cytisine derivatives against dengue virus types 1 and 2.

Dengue virus (DENV) causes about 50-100 million cases per year worldwide. However, there is still a big challenge in developing antiviral drugs against DENV infection. Some derivatives of alkaloid (-)-cytisine, like other alkaloid analogs, have been proposed for their antiviral potential. This study investigated antiviral activity and mechanisms of the cytisine derivatives, and discovered the structure-activity relationship against DENV. The antiviral assays were performed using one strain of DENV1 and DENV2, and two cell lines Vero E6 and A549. The structure-activity relationship of the effective compounds was also evaluated using combination of time-of-addition/removal assay and molecular docking. Compounds 3, 4, 12 (N-allylcytisine-3-thiocarbamide), 16, and 20 exhibited the high antiviral activity with IC50 values of lower than 3 µM against DENV1 and DENV2. Of them, the derivative 12 showed the highest antiviral activities against DENV1 (IC50 = 0.14 µM) and DENV-2 (IC50 = <0.1 µM), exhibiting the potent inhibition on virus attachment and entry stages. Meanwhile, the compounds 4 and 20 had a strong inhibition at the post-entry stage (IC50 = <0.1 µM). A correlation between the experimental pIC50 values and predicted pKi calculated by docking of compounds into DENV E protein was significant, correlating with the impact of compound 12 on the attachment stage, but compounds 4, and 20 on post-entry stage. The results provided the insight into the directions of synthetic modifications of starting (-)-cytisine as the inhibitors of DENV E protein at attachment and entry stages of DENV life cycle.

Glycyrrhetinic acid derivatives as Zika virus inhibitors: Synthesis and antiviral activity in vitro.

Zika virus (ZIKV) is an arbovirus of the Flaviviridae family (Flavivirus genus), causing serious neurological complications, such as Guillain-Barre Syndrome (GBS) in adults and fetal microcephaly. Licensed vaccines or specific antiviral agents against ZIKV do not currently exist. Therefore, the search and development of anti-ZIKV agents are particularly relevant and necessary. Glycyrrhetinic (3ß-hydroxy-11-oxo-18ßH-Olean-12-en-30-oic acid) (GA) 1 is one of the well-known pentacyclic triterpenoids isolated from licorice root (Glycyrrhiza glabra L., Gl. uralensis Fisher) (Leguminosae) possessing many biological features, including antiviral activity. This paper is devoted to the synthesis and studies of a number of nitrogen and sulfur-containing GA derivatives as ZIKV inhibitors. Sixteen GA and related triterpenoids (3ß-hydroxy-18ßH-Olean-12-en-30-oic acid and 3ß-hydroxy-11-oxo-18ßH-Olean-12(13),18(19)-dien-30-oic acid) derivatives were synthesized (amides, semi- and thiosemicarbazones, and 1,2,3-thiadiazoles) and antiviral activity against ZIKV was studied in vitro, including the inhibitory assays on cytopathic effect (CPE), viral protein synthesis, and replication stages. Four active compounds were found among GA derivatives tested, 13 (3-O-acetyl-30-aminopyridine GA), 16 (3-semicarbazone-30-butyl GA), 18 (1,2,3-thiadiazole-30-methyl GA), and 19 (1,2,3-thiadiazole-30-butyl GA) with IC50 < 1 µM against ZIKV replication. These compounds had a stronger inhibitory activity on ZIKV-induced CPE and viral protein translation in infected cells as compared to derivatives of 11-desoxo-GA. The most active compound was amide 13 (IC50 0.13 µM, TI ˃ 384). Time-of-addition assays indicated that 1,2,3-thiadiazole ring is important for inhibiting viral entry stage (compounds 18 and 19), while the 30-butyl ester group influenced on post-entry stage (compound 19). The molecular docking analysis demonstrated that lead compounds 13 and 19 forms a hydrogen-bond interaction with the catalytic triad (His51-Asp75-Ser135) of ZIKV NS2B-NS3 protease. Therefore, the active GA derivatives are promising for developing new antiviral agents against ZIKV infection.

SARS Unique Domain (SUD) of Severe Acute Respiratory Syndrome Coronavirus Induces NLRP3 Inflammasome-Dependent CXCL10-Mediated Pulmonary Inflammation.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) initiates the cytokine/chemokine storm-mediated lung injury. The SARS-CoV unique domain (SUD) with three macrodomains (N, M, and C), showing the G-quadruplex binding activity, was examined the possible role in SARS pathogenesis in this study. The chemokine profile analysis indicated that SARS-CoV SUD significantly up-regulated the expression of CXCL10, CCL5 and interleukin (IL)-1ß in human lung epithelial cells and in the lung tissues of the mice intratracheally instilled with the recombinant plasmids. Among the SUD subdomains, SUD-MC substantially activated AP-1-mediated CXCL10 expression in vitro. In the wild type mice, SARS-CoV SUD-MC triggered the pulmonary infiltration of macrophages and monocytes, inducing CXCL10-mediated inflammatory responses and severe diffuse alveolar damage symptoms. Moreover, SUD-MC actuated NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome-dependent pulmonary inflammation, as confirmed by the NLRP3 inflammasome inhibitor and the NLRP3-/- mouse model. This study demonstrated that SARS-CoV SUD modulated NLRP3 inflammasome-dependent CXCL10-mediated pulmonary inflammation, providing the potential therapeutic targets for developing the antiviral agents.

Antiviral efficacy of bromo-anilino substituents of 4,5-dihydrofuran-3-carboxylate compound CW-33 against Japanese encephalitis virus.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, occasionally causes severe central nervous system disorders in the risk zone where more than 3 billion people reside. Our prior studies demonstrated antiviral potential of 4,5-dihydrofuran-3-carboxylate compound CW-33 (ethyl 2-(3',5'-dimethylanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) and its derivative CW-33A ((ethyl 2-(2-fluoroanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) against JEV infection ((Int. J. Mol. Sci. 2016, 17: E1386; Sci. Rep. 2018, 8: 16595). This study synthesized six new CW-33 derivatives containing chloro, or bromo groups at the C-2, C-3, or C-4 of anilino ring of CW-33, and assessed the antiviral activity and mechanisms of these chloro- and bromo-anilino substitutedderivatives. CW-33K, CW-33L and CW-33M had the bromo-substituents at the C-2, C-3, or C-4 of anilino ring of CW-33, respectively, showing the higher anti-JEV activity than CW-33 and other derivatives. CW-33K (ethyl 2-(2-bromoanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) exhibited the highest antiviral efficacy and therapeutic index. The IC50 value of CW-33K was less than 5 µM for reducing JEV-induced cytopathic effect, virus infectivity and virus yield. CW-33K significantly inhibited the JEV replication at the early and late stages, suppressing viral RNA synthesis and intracellular JEV particle production. The study demonstrated that the CW-33 derivative with a bromosubstitutionat the C-2 anilino ring improved the antiviral activity JEV, providing the structure-antiviral activity relationship for the development of anti-JEV agents.

Glycyrrhizic acid derivatives as Dengue virus inhibitors.

Dengue virus (DENV) is one of the most geographically distributed pathogenic flaviviruses transmitted by mosquitoes Aedes sps. In this study, the structure-antiviral activity relationships of Glycyrrhizic acid (GL) derivatives was evaluated by the inhibitory assays on the cytopathic effect (CPE) and viral infectivity of DENV type 2 (DENV2) in Vero E6 cells. GL (96% purity) had a low cytotoxicity to Vero E6 cells, inhibited DENV2-induced CPE, and reduced the DENV-2 infectivity with the IC50 of 8.1 µM. Conjugation of GL with amino acids or their methyl esters and the introduction of aromatic acylhydrazide residues into the carbohydrate part strongly influenced on the antiviral activity. Among compounds tested GL conjugates with isoleucine 13 and 11-aminoundecanoic acid 17 were found as potent anti-DENV2 inhibitors (IC50 1.2-1.3 µM). Therefore, modification of GL is a perspective way in the search of new antivirals against DENV2 infection.

Ursolic acid induces apoptosis and autophagy in oral cancer cells.

Oral squamous cell carcinoma (OSCC) is the fifth common cause of cancer mortality in Taiwan with high incidence and recurrence and needs new therapeutic strategies. In this study, ursolic acid (UA), a triterpenoid, was examined the antitumor potency in OSCC cells. Our results showed that UA inhibited the proliferation of OSCC cells in a dose- and time-dependent manner in both Ca922 and SCC2095 oral cancer cells. UA induced caspase-dependent apoptosis accompanied with the modulation of various biological biomarkers including downregulating Akt/mTOR/NF-κB signaling, ERK, and p38. In addition, UA inhibited angiogenesis as evidenced by abrogation of migration/invasion and blocking MMP-2 secretion in Ca922 cells. Interestingly, UA induced autophagy in OSCC cells, as manifested by LC3B-II conversion and increased p62 expression and accumulation of autophagosomes. Inhibition by autophagy inhibitor enhanced UA-mediated apoptosis in Ca922 cells. The experiment provides a rationale for using triterpenoid in the treatment of OSCC.

KCNQ1 variants associate with hypertension in type 2 diabetes and affect smooth muscle contractility in vitro.

KCNQ1 encodes a potassium voltage-gated channel and represents a susceptibility locus for type 2 diabetes mellitus (T2DM). Here, we explored the association between KCNQ1 polymorphisms and hypertension risk in individuals with T2DM, as well as the role of KCNQ1 in vascular smooth muscle cell contraction in vitro. To investigate the relationship between KCNQ1 and the risk of developing hypertension in patients with T2DM, we divided the T2DM cohort into hypertension (n = 452) and non-hypertension (n = 541) groups. The Mann-Whitney U test, chi-square test, and multivariate regression analyses were used to assess the clinical characteristics and genotypic frequencies. In vitro studies utilized the rat aortic smooth muscle A10 cell line. Patients in the hypertension group were significantly older at the time of enrollment and had higher levels of body mass index, waist-to-hip ratio, and triglyceride than those in the non-hypertension group. The KCNQ1 rs3864884 and rs12576239 genetic variants were associated with hypertension in T2DM. KCNQ1 expression was lower in the individuals with the CC versus the CT and TT genotypes. Smooth muscle cell contractility was inhibited by treatment with a KCNQ1 inhibitor. These results suggest that KCNQ1 might be associated with hypertension in individuals with T2DM.

Overexpression of SmeDEF Efflux Pump Decreases Aminoglycoside Resistance in Stenotrophomonas maltophilia.

The SmeDEF pump of Stenotrophomonas maltophilia is negatively regulated by SmeT. In this study, strains KJΔT (smeT deletion mutant) and KJT-Dm (mutant with a defective SmeT-binding site) showed increased resistance to chloramphenicol/nalidixic acid/macrolides and susceptibility to aminoglycoside. Overexpression of the SmeDEF pump, in either KJΔT or KJT-Dm, downregulated smeYZ expression, which is responsible for the reduced aminoglycoside resistance. Furthermore, the SmeRySy two-component regulatory system was downregulated in response to SmeDEF overexpression, which supports its involvement in the regulatory circuit.

Development of a fluorescence resonance energy transfer-based intracellular assay to identify novel enterovirus 71 antivirals.

Enterovirus 71 (EV71) is considered one of the most virulent pathogens in the family Picornaviridae. However, there have been no effective treatments for the severe complications caused by EV71. Development of new drugs against targets that are essential for viral replication often requires screening large collections of compounds, for which a high-throughput screening platform is needed. In this study, a drug-screening platform was developed based on a genetically engineered cell line that displays fluorescence resonance energy transfer (FRET) and shows a real-time and quantifiable impairment of FRET upon EV71 infection. A library of small molecules consisting of 1280 compounds with defined bioactivities was used for screening drugs with anti-EV71 activity; accurate, rapid, and robust results were obtained from this screening procedure. Ten drugs were identified in the primary screening, and their antiviral activities were indicated by dose-dependent elevation of FRET. Among these, AC-93253, mitoxantrone and N-bromoacetamide had not been reported as enterovirus inhibitors, and it was confirmed that they were able to suppress viral yields in a dose-dependent manner. Taken together, these studies demonstrate the feasibility of this FRET-based platform for efficient screening and identification of novel compounds with activity against EV71 infection.

Tubacin, an HDAC6 Selective Inhibitor, Reduces the Replication of the Japanese Encephalitis Virus via the Decrease of Viral RNA Synthesis.

Japanese encephalitis virus (JEV), a neurotropic flavivirus, annually causes over 30,000 Japanese Encephalitis (JE) cases in East and Southeast Asia. Histone deacetylases (HDACs) modulate lysine acetylation of histones and non-histone proteins, regulating many processes including inflammation and antiviral immune response. This study investigated antiviral activity of pan- and selective-HDAC inhibitors as host-targeting agents against JEV. Among HDAC inhibitors, selective HDAC6 inhibitors (tubastatin-A (TBSA) and tubacin) concentration-dependently inhibited JEV-induced cytopathic effect and apoptosis, as well as reduced virus yield in human cerebellar medulloblastoma cells. The 50% inhibitory concentration (IC50) values of virus yield was 0.26 µM for tubacin and 1.75 µM for TBSA, respectively. Tubacin (IC50 of 1.52 µM), but not TBSA, meaningfully blocked the production of intracellular infectious virus particles. In time-of-addition assays, the greatest potency of antiviral activity was observed in the mode of pre-treatment with tubacin (IC50 of 1.89 µM) compared to simultaneous (IC50 of 4.88 µM) and post-treatment (IC50 of 2.05 µM) modes. Interestingly, tubacin induced the hyperacetylation of a HDAC6 substrate Hsp90 and reduced the interaction of Hsp90 with JEV NS5 protein. Novobiocin, an Hsp90 inhibitor, diminished the NS5 protein amount and virus replication in JEV-infected cells. Meantime, tubacin suppressed the NS5 expression and antisense RNA genome synthesis in infected cells. Tubacin-induced Hsp90 hyperacetylation was suggested to influence the NS5 activity in JEV replication. Therefore, tubacin had a high potential of a host-targeting agent against JEV, exhibiting preventive and therapeutic activities against JEV infection.

Idarubicin is a broad-spectrum enterovirus replication inhibitor that selectively targets the virus internal ribosomal entry site.

Enterovirus 71 (EV71) causes life-threatening diseases with neurological manifestations in young children. However, the treatment of EV71 infections remains an unmet medical need. Idarubicin (IDR) is an anthracycline compound that is used therapeutically for certain types of tumour. In this study, we identified IDR as an EV71 inhibitor, which displayed antiviral potency in the submicromolar range and substantially protected cells from the cytopathic effects and cell death caused by EV71 infections. The antiviral effects extended to several other enterovirus (EV) species, and these effects were independent of cytotoxicity or topoisomerase inhibition. Structure-activity relationship studies indicated the importance of the anthracycline scaffold for anti-EV potency. IDR effectively blocked the synthesis of viral protein and RNA, but not the viral proteolysis processes. Moreover, anthracyclines were demonstrated to suppress EV internal ribosomal entry site (IRES)-mediated translation; conversely, the cellular p53 IRES activity was not sensitive to IDR action. Inhibition of IRES-mediated translation by IDR correlated with the affinity of binding between IDR and the particular IRES. Moreover, IDR impaired binding between the EV71 IRES RNA and hnRNP A1, a known host IRES trans-acting factor. In sum, we have identified a USA FDA-approved anticancer drug with the new indication as a selective EV IRES binder and inhibitor. The finding may also provide leads for the development of novel antiviral therapies directed at the EV IRES RNA.

Discovery of the ROCK inhibitor netarsudil for the treatment of open-angle glaucoma.

Inhibition of Rho kinase (ROCK) to improve fluid outflow through the trabecular meshwork and lower intraocular pressure is a strategy for the development of new anti-glaucoma agents. Alpha-aryl-beta-amino isoquinoline analogs were identified as potent ROCK inhibitors. Compounds that provided a longer duration of intraocular pressure reduction in Dutch Belted rabbits also inhibited norepinephrine transporter. Ester 60 improved bioavailability of its parent ROCK inhibitor, 29 (Ki=0.2nM) and demonstrated an effective and sustained IOP reduction for 24h after dosing. From these studies, netarsudil (a.k.a. AR-13324) was discovered and is currently in clinical trials for the treatment of glaucoma and ocular hypertension.

SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6.

Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLPro) reportedly inhibits the production of type I interferons (IFNs) and pro-inflammatory cytokines in Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I) pathways. The study investigated the inhibitory effect and its antagonistic mechanism of SARS-CoV PLPro on TLR7-mediated cytokine production. TLR7 agonist (imiquimod (IMQ)) concentration-dependently induced activation of ISRE-, NF-κB- and AP-1-luciferase reporters, as well as the production of IFN-α, IFN-ß, TNF-α, IL-6 and IL-8 in human promonocyte cells. However, SARS-CoV PLPro significantly inhibited IMQ-induced cytokine production through suppressing the activation of transcription factors IRF-3, NF-κB and AP-1. Western blot analysis with anti-Lys48 and anti-Lys63 ubiquitin antibodies indicated the SARS-CoV PLPro removed Lys63-linked ubiquitin chains of TRAF3 and TRAF6, but not Lys48-linked ubiquitin chains in un-treated and treated cells. The decrease in the activated state of TRAF3 and TRAF6 correlated with the inactivation of TBK1 in response to IMQ by PLPro. The results revealed that the antagonism of SARS-CoV PLPro on TLR7-mediated innate immunity was associated with the negative regulation of TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals.

Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 µM. Time-of-addition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 µM, respectively. CW-33 significantly moderated JEV-triggered Ca(2+) overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents.

Anticancer Activity of γ-Bisabolene in Human Neuroblastoma Cells via Induction of p53-Mediated Mitochondrial Apoptosis.

γ-Bisabolene has demonstrated antiproliferative activities against several human cancer cell lines. This study first discloses the antiproliferative and apoptosis induction activities of γ-bisabolene to human neuroblastoma TE671 cells. A CC50 value of γ-bisabolene was 8.2 µM to TE671 cells. Cell cycle analysis with PI staining showed γ-bisabolene elevating the sub-G1 fractions in a time-dependent manner. In addition, annexin V-FITC/PI staining showed γ-bisabolene significantly triggering early (annexin-V positive/PI negative) and late (annexin-V positive/PI positive) apoptosis in dose-dependent manners. γ-Bisabolene induced caspase 3/8/9 activation, intracellular ROS increase, and mitochondrial membrane potential decrease in apoptosis of human neuro-blastoma cells. Moreover, γ-bisabolene increased p53 phosphorylation and up-regulated p53-mediated apoptotic genes Bim and PUMA, as well as decreased the mRNA and protein levels of CK2α. Notably, the results indicated the involvement of CK2α-p53 pathways in mitochondria-mediated apoptosis of human neuroblastoma cells treated with γ-bisabolene. This study elucidated the apoptosis induction pathways of γ-bisabolene-treated neuroblastoma cells, in which could be useful for developing anti-neuroblastoma drugs.