Inhibition of NF-{kappa}B improves left ventricular remodeling and cardiac dysfunction after myocardial infarction

Several studies have demonstrated that NF-kappaB is substantially involved in the progression of cardiac remodeling; however, it remains uncertain whether the continuous inhibition of NF-kappaB is effective for the prevention of myocardial remodeling. Myocardial infarction (MI) was produced by ligation of the left anterior coronary artery of rats. IMD-0354 (10 mg/kg per day), a novel phosphorylation inhibitor of IkappaB that acts via inhibition of IKK-beta, was injected intraperitoneally starting 24 h after induction of MI for 28 days. After 28 days, the IMD-0354-treated group showed significantly improved survival rate compared with that of the vehicle-treated group (P < 0.05). Although infarct size was similar in both groups, improved left ventricular (LV) remodeling and diastolic dysfunction, as indicated by smaller LV cavity (LV end-diastolic area: vehicle, 74.13 +/- 3.57 mm(2); IMD-0354, 55.00 +/- 3.73 mm(2); P < 0.05), smaller peak velocity of early-to-late filling wave (E/A) ratio (vehicle, 3.87 +/- 0.26; IMD-0354, 2.61 +/- 0.24; P < 0.05), and lower plasma brain natriuretic peptide level (vehicle, 167.63 +/- 14.87 pg/ml; IMD-0354, 110.75 +/- 6.41 pg/ml; P < 0.05), were observed in the IMD-0354-treated group. Moreover, fibrosis, accumulation of macrophages, and expression of several factors (transforming growth factor-beta1, monocyte chemoattractant protein-1, matrix metalloproteinase-9 and -2) in the noninfarcted myocardium was remarkably inhibited by IMD-0354. In conclusion, inhibition of NF-kappaB activation may reduce the proinflammatory reactions and modulate the extracellular matrix and provide an effective approach to prevent adverse cardiac remodeling after MI.     

IMD-0354 Targets Breast Cancer Stem Cells: A Novel Approach for an Adjuvant to Chemotherapy to Prevent Multidrug Resistance in a Murine Model

Although early detection of breast cancer improved in recent years, prognosis of patients with late stage breast cancer remains poor, mostly due to development of multidrug resistance (MDR) followed by tumor recurrence. Cancer stem cells (CSCs), with higher drug efflux capability and other stem cell-like properties, are concentrated in a side population (SP) of cells, which were proposed to be responsible for MDR and tumor repopulation that cause patients to succumb to breast cancer. Therefore, targeting of CSCs as an adjuvant to chemotherapy should be able to provide a more effective treatment of this disease. Here, we used IMD-0354, an inhibitor of NF-κB, identified for targeting CSCs, in a combination therapy with doxorubicin encapsulated in targeted nanoparticles. IMD-0354 did target CSCs, evidenced by a decrease in the SP, demonstrated by the inhibition of the following: dye/drug efflux, reduction in ABC transporters as well as in colony formation in soft agar and low attachment plates. Decrease of stem-like gene expression of Oct4, Nanog and Sox2, and apoptosis resistance related to the Survivin gene also was observed after treatment with this compound. In addition, IMD-0354 targeted non-CSCs as indicated by reducing viability and increasing apoptosis. Targeted drug delivery, achieved with a legumain inhibitor, proved to enhance drug delivery under hypoxia, a hallmark of the tumor microenvironment, but not under normoxia. Together, this allowed a safe, non-toxic delivery of both anticancer agents to the tumor microenvironment of mice bearing syngeneic metastatic breast cancer. Targeting both bulk tumor cells with a chemotherapeutic agent and CSCs with IMD-0354 should be able to reduce MDR. This could eventually result in decreasing tumor recurrences and/or improve the outcome of metastatic disease.     

Pentoxifylline Attenuates Proteinuria in Anti-Thy1 Glomerulonephritis via Downregulation of Nuclear Factor-κB and Smad2/3 Signaling

Anti-Thy1 glomerulonephritis is a rat nephritis model closely simulating human mesangial proliferative glomerulonephritis. It affects primarily the mesangium, yet displays substantial proteinuria during the course. This study investigated the molecular signals underlying proteinuria in this disease and the modulation of which by the known antiproteinuric agent, pentoxifylline. Male Wistar rats were randomly divided into a control group and nephritic groups with or without treatment with IMD-0354 (an IκB kinase inhibitor), SB431542 (an activin receptor–like kinase inhibitor) or pentoxifylline. Kidney sections were prepared for histological examinations. Glomeruli were isolated for mRNA and protein analysis. Urine samples were collected for protein and nephrin quantitation. One day after nephritis induction, proteinuria developed together with ultrastructural changes of the podocyte and downregulation of podocyte mRNA and protein expression. These were associated with upregulation of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β/activins mRNAs and activation of nuclear factor (NF)-κB p65 and Smad2/3. IMD-0354 attenuated proteinuria on d 1, whereas SB431542 decreased proteinuria on d 3 and 5, in association with partial restoration of downregulated podocyte mRNA and protein expression. Pentoxifylline attenuated proteinuria and nephrinuria through the course, plus inhibition of p-NF-κB p65 (d 1) and p-Smad2/3 (d 5) and partial reversal of downregulated podocyte mRNA and protein. Our data show that the pathogenesis of proteinuria in anti-Thy1 glomerulonephritis involves TNF-α and TGF-β/activin pathways, and the evolution of this process can be attenuated by pentoxifylline via downregulation of NF-κB and Smad signals and restoration of the podocyte component of the glomerular filtration barrier.     

Synthesis and evaluation of diterpenic Mannich bases as antiviral agents against influenza A and SARS-CoV-2

A chemical library was constructed based on the resin acids (abietic, dehydroabietic, and 12-formylabietic) and its diene adducts (maleopimaric and quinopimaric acid derivatives). The one-pot three-component CuCl-catalyzed aminomethylation of the abietane diterpenoid propargyl derivatives was carried out by formaldehyde and secondary amines (diethylamine, pyrrolidine, morpholine, and homopiperazine). All compounds were tested for cytotoxicity and antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells and SARS-CoV-2 pseudovirus in BHK-21-hACE2 cells. Among 21 tested compounds, six derivatives demonstrated a selectivity index (SI) higher than 10, and their IC₅₀ values ranged from 0.19 to 5.0 μM. Moreover, two derivatives exhibited potent anti-SARS-CoV-2 infection activity. The antiviral activity and toxicity strongly depended on the nature of the diterpene core and heterocyclic substituent. Compounds 12 and 21 bearing pyrrolidine moieties demonstrated the highest virus-inhibiting activity with SIs of 128.6 and 146.8, respectively, and appeared to be most effective when added at the time points 0–10 and 1–10 h of the viral life cycle. Molecular docking and dynamics modeling were adopted to investigate the binding mode of compound 12 into the binding pocket of influenza A virus M2 protein. Compound 9 with a pyrrolidine group at C20 of 17-formylabietic acid was a promising anti-SARS-CoV-2 agent with an EC₅₀ of 10.97 µM and a good SI value > 18.2. Collectively, our data suggested the potency of diterpenic Mannich bases as effective anti-influenza and anti-COVID-19 compounds.     

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro

COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CL^pro) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CL^pro activity in vitro with IC₅₀’s of 8.52 µg/ml and 0.39 µM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC₅₀’s of 0.74 µg/ml and 2.9 µM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CL^pro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CL^pro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.     

Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes – NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.     

3-O-sulfated glucuronide derivative as a potential anti-dengue virus agent

A series of 12 carbohydrate compounds were synthesized by introduction of a sulfated group at specific positions and evaluated for their activities against dengue virus (DENV) infection as well as binding to BHK-21 cells. 3-O-sulfated GlcA was active against DENV infection, whereas 2-O-sulfated GlcA and 3,6-di-O-sulfated Glc showed negligible activity. Persulfated compounds did not inhibit DENV infection. These results provided a rationale for designing sulfated carbohydrate compounds with low molecular mass as anti-DENV agents targeting E protein functions. 3-O-Sulfated GlcA showed no significant cytotoxicity at 1mM. The EC(50) value (120μM) was lower than that of sucrose octasulfate (SOS), a small molecular weight inhibitor of DENV infection. Two negatively charged groups, 3-O-sulfate and 6-C-carboxylic acid, appear to be essential for anti-DENV activity. We performed docking study to investigate the binding potential of 3-O-sulfated GlcA with respect to DENV E protein. The docking study showed that distance and conformation of these negative charges on the carbohydrate may be suitable for association with three amino acid residues of E protein critically involved in virus adsorption (Lys295, Ser145, and Gly159). This interaction may competitively prevent functional DENV binding to receptor(s) on host cells. In conclusion, 3-O-sulfated GlcA is a chemical probe that may facilitate exploration of the molecular mechanisms underlying manifestations of dengue diseases.     

新型冠状病毒的糖基化、糖受体识别及糖链抑制剂的发现北大核心CSCD

新型冠状病毒疫情(COVID-19)是21世纪截至目前人类面对的最为严重的公共卫生事件。疫苗、中和抗体以及小分子化合药物的出现有效预防和阻止了COVID-19的快速传播,而不断出现的病毒突变体却使这些疫苗及药物的效价降低,这对COVID-19的预防及治疗提出了新的挑战。新型冠状病毒(SARS-CoV-2)通常会先黏附于呼吸道表面的大分子糖链——硫酸乙酰肝素,进而与特异性受体人血管紧张素转化酶2(human angiotensin-converting enzyme 2,hACE2)结合,从而实现对人体的侵入。SARS-CoV-2的刺突(spike,S)蛋白是高度糖基化的,而糖基化对于hACE2与S蛋白的结合也有着重要影响,S蛋白在宿主体内还会被一系列凝集素受体所结合,这意味着糖链在SARS-CoV-2的入侵及感染过程中有着重要的作用。基于SARS-CoV-2的糖基化及糖受体识别机制开发糖链抑制剂可能是预防或治疗新型冠状病毒感染的有效手段,相关研究发现海洋来源的硫酸化多糖、肝素分子及其他的一些糖类具有抗SARS-CoV-2的活性。本文系统阐述了新型冠状病毒的糖基化及其糖链在入侵、感染中的作用,并对抗SARS-CoV-2糖链抑制剂的发现和机制研究现状进行了总结,在此基础上还对糖类抗病毒药物的机遇与挑战进行了展望。     

Repurposing of phytomedicine-derived bioactive compounds with promising anti-SARS-CoV-2 potential: Molecular docking,MD simulation and drug-likeness/ADMET studies

In view of the potential of traditional plant-based remedies (or phytomedicines) in the management of COVID-19, the present investigation was aimed at finding novel anti-SARS-CoV-2 molecules by in silico screening of bioactive phytochemicals (database) using computational methods and drug repurposing approach. A total of 160 compounds belonging to various phytochemical classes (flavonoids, limonoids, saponins, triterpenoids, steroids etc.) were selected (as initial hits) and screened against three specific therapeutic targets (Mpro/3CLpro, PLpro and RdRp) of SARS-CoV-2 by docking, molecular dynamics simulation and drug-likeness/ADMET studies. From our studies, six phytochemicals were identified as notable ant-SARS-CoV-2 agents (best hit molecules) with promising inhibitory effects effective against protease (Mpro and PLpro) and polymerase (RdRp) enzymes. These compounds are namely, ginsenoside Rg2, saikosaponin A, somniferine, betulinic acid, soyasapogenol C and azadirachtin A. On the basis of binding modes and dynamics studies of protein–ligand intercations, ginsenoside Rg2, saikosaponin A, somniferine were found to be the most potent (in silico) inhibitors potentially active against Mpro, PLpro and RdRp, respectively. The present investigation can be directed towards further experimental studies in order to confirm the anti-SARS-CoV-2 efficacy along with toxicities of identified phytomolecules.     

Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron-sulphur cluster metabolism

COG0354 proteins have been implicated in synthesis or repair of iron/sulfur (Fe/S) clusters in all domains of life, and those of bacteria, animals, and protists have been shown to require a tetrahydrofolate to function. Two COG0354 proteins were identified in Arabidopsis and many other plants, one (At4g12130) related to those of α-proteobacteria and predicted to be mitochondrial, the other (At1g60990) related to those of cyanobacteria and predicted to be plastidial. Grasses and poplar appear to lack the latter. The predicted subcellular locations of the Arabidopsis proteins were validated by in vitro import assays with purified pea organelles and by targeting assays in Arabidopsis and tobacco protoplasts using green fluorescent protein fusions. The At4g12130 protein was shown to be expressed mainly in flowers, siliques, and seeds, whereas the At1g60990 protein was expressed mainly in young leaves. The folate dependence of both Arabidopsis proteins was established by functional complementation of an Escherichia coli COG0354 (ygfZ) deletant; both plant genes restored in vivo activity of the Fe/S enzyme MiaB but restoration was abrogated when folates were eliminated by deleting folP. Insertional inactivation of At4g12130 was embryo lethal; this phenotype was reversed by genetic complementation of the mutant. These data establish that COG0354 proteins have a folate-dependent function in mitochondria and plastids, and that the mitochondrial protein is essential. That plants retain mitochondrial and plastidial COG0354 proteins with distinct phylogenetic origins emphasizes how deeply the extant Fe/S cluster assembly machinery still reflects the ancient endosymbioses that gave rise to plants.     

Differentially Expressed in Chondrocytes 2 (DEC2) Increases the Expression of IL-1β and Is Abundantly Present in Synovial Membrane in Rheumatoid Arthritis

Objective

Patients with rheumatoid arthritis (RA) have altered circadian rhythm of circulating serum cortisol, melatonin and IL-6, as well as disturbance in the expression of clock genes ARNTL2 and NPAS2. In humans, TNFα increases the expression ARNTL2 and NPAS2 but paradoxically suppresses clock output genes DPB and PER3. Our objective was to investigate the expression of direct clock suppressors DEC1 and DEC2 (BHLHE 40 and 41 proteins) in response to TNFα and investigate their role during inflammation.

Methods

Cultured primary fibroblasts were stimulated with TNFα. Effects on DEC2 were studied using RT-qPCR and immunofluorescence staining. The role of NF-κB in DEC2 increase was analyzed using IKK-2 specific inhibitor IMD-0354. Cloned DEC2 was transfected into HEK293 cells to study its effects on gene expression. Transfections into primary human fibroblasts were used to confirm the results. The presence of DEC2 was analyzed in (RA) and osteoarthritis (OA) synovial membranes by immunohistochemistry.

Results

TNFα increased DEC2 mRNA and DEC2 was mainly detected at nuclei after the stimulus. The effects of TNFα on DEC2 expression were mediated via NF-κB. Overexpression, siRNA and promoter activity studies disclosed that DEC2 directly regulates IL-1β, in both HEK293 cells and primary human fibroblasts. DEC2 was increased in synovial membrane in RA compared to OA.

Conclusion

Not only ARNTL2 and NPAS2 but also DEC2 is regulated by TNFα in human fibroblasts. NF-κB mediates the effect on DEC2, which upregulates IL-1β. Circadian clock has a direct effect on inflammation in human fibroblasts.     

The design, synthesis and in vitro immunosuppressive evaluation of novel isobenzofuran derivatives

The synthesis and biological evaluation of a series of novel isobenzofuran-based compounds are described. The compounds were evaluated for their immunosuppressive effects of T-cell proliferation and IMPDH type II inhibitor activity in vitro, as well as their structure-activity relationships were assessed. Several compounds demonstrated highly efficacious immunosuppressive properties, especially compounds 2d, 2e, 2h and 2j, which were superior to MPA, while compounds 2k, 2m, 2n, 4c and 5d exhibited an equipotent inhibitory activity compared to MPA. Generally, it was obviously demonstrated that α,β-unsaturated amides proved more potent than the diamide and urea series. The present study provides a guide for further research on development of safe and effective immunosuppressive agents.     

Comparison of five commercial anti-SARS-CoV-2 total antibodies and IgG immunoassays after vaccination with BNT162b2 mRNA

BackgroundSince universal vaccinations represents the most effective strategy to mitigate coronavirus disease 2019 (COVID-19), baseline assessment and post-vaccine monitoring of anti-SARS-CoV-2 neutralizing antibodies are essential to vaccination programs. Therefore, this study aimed to compare data of five commercial anti-SARS-CoV2 immunoassays after administration of an mRNA vaccine.MethodsVenous blood was collected from three healthcare workers, receiving a double (30 g) dose of BNT162b2 mRNA Covid-19 vaccine (Comirnaty, Pfizer), on the day of the first vaccine dose and then at fixed intervals for the following 2 months. Anti-SARS-CoV-2 neutralizing antibody response was assayed with Roche Total Ig anti-RBD (receptor binding domain), DiaSorin TrimericS IgG (spike trimer), Beckman Coulter IgG anti-RBD, SNIBE IgG anti-RBD and Technogenetics IgG anti-N/S1.ResultsA total number of 45 samples were drawn at the end of the 2-month study period. The Spearman''s correlations of absolute anti-SARS-CoV-2 antibodies were always excellent (all p<0.001), comprised between 0.967-0.994. Satisfactory results were also observed when absolute antiSARS-CoV-2 antibodies values of the five methods were compared with the mean consensus value, with correlations always higher than 0.979 (all p<0.001). The agreement of anti-SARS-CoV-2 antibodies positivity versus the consensus median positivity ranged between 0.764 and 1.000 (always p<0.001), but become always >0.900 after readjustment of one assay cutoff.ConclusionsAll the immunoassays evaluated in this study appear suitable for monitoring anti-SARS-CoV-2 neutralizing antibodies response in subjects undergoing mRNA COVID-19 vaccination.     

Newer human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2) inhibitors as potential anticancer agents

Human inosine 5′-monophosphate dehydrogenase 2 (hIMPDH2), being an age-old target, has attracted attention recently for anticancer drug development. Mycophenolic acid (MPA), a well-known immunosuppressant drug, was used a lead structure to design and develop modestly potent and selective analogues. The steep structure–activity relationship (SAR) requirements of the lead molecule left little scope to synthesise newer analogues. Here, newer MPA amides were designed, synthesised and evaluated for hIMPDH2 inhibition and cellular efficacy in breast, prostate and glioblastoma cell lines. Few title compounds exhibited cellular activity profile better than MPA itself. The observed differences in the overall biological profile could be attributed to improved structural and physicochemical properties of the analogues over MPA. This is the first report of the activity of MPA derivatives in glioblastoma, the most aggressive brain cancer.     

Establishment of a stable cell line coexpressing dengue virus-2 and green fluorescent protein for screening of antiviral compounds

This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A-derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5'- and 3'-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds.     

Autoantibodies to heat shock protein 60, 70, and 90 are not altered in the anti-SARS-CoV-2 IgG-seropositive humans without or with mild symptoms

Highly conserved heat shock proteins (Hsps) are localized in the cytoplasm and cellular organelles, and act as molecular chaperones or proteases. Members of Hsp families are released into the extracellular milieu under both normal and stress conditions. It is hypothesized that the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) has the potential to elicit autoimmunity due to molecular mimicry between human extracellular Hsps and immunogenic proteins of the virus. To confirm the above hypothesis, levels of circulating autoantibodies directed to the key human chaperones i.e., Hsp60, Hsp70, and Hsp90 in the anti-SARS-CoV-2 IgG-seropositive participants have been evaluated. Twenty-six healthy volunteers who got two doses of the mRNA vaccine encoding the viral spike protein, anti-SARS-CoV-2 IgG-positive participants (n = 15), and healthy naïve (anti-SARS-CoV-2 IgG-negative) volunteers (n = 51) have been included in this study. We found that the serum levels of anti-Hsp60, anti-Hsp70, and anti-Hsp90 autoantibodies of the IgG, IgM, or IgA isotype remained unchanged in either the anti-COVID-19-immunized humans or the anti-SARS-CoV-2 IgG-positive participants when compared to healthy naïve volunteers, as measured by enzyme-linked immunosorbent assay. Our results showing that the humoral immune response to SARS-CoV-2 did not include the production of anti-SARS-CoV-2 antibodies that also recognized extracellular heat shock protein 60, 70, and 90 represent a partial evaluation of the autoimmunity hypothesis stated above. Further testing for cell-based immunity will be necessary to fully evaluate this hypothesis.     

Selective inhibition of prostacyclin synthase activity by rofecoxib

The development of cyclooxygenase-2 (COX-2) selective inhibitors prompted studies aimed at treating chronic inflammatory diseases and cancer by using this new generation of drugs.Yet, several recent reports pointed out that long-term treatment of patients with COX-2 selective inhibitors (especially rofecoxib) caused severe cardiovascular complicances. The aim of this study was to ascertain whether, in addition to inhibiting COX-2, rofecoxib may also affect prostacyclin (PGI2) level by inhibiting PGI2 forming enzyme (prostacyclin synthase, PGIS). In order to evaluate if selective (celecoxib, rofecoxib) and non-selective (aspirin, naproxen) anti-inflammatory compounds could decrease PGI2 production in endothelial cells by inhibiting PGIS, we analyzed the effect of anti-inflammatory compounds on the enzyme activity by ELISA assay after addition of exogenous substrate, on PGIS protein levels by Western blotting and on its subcellular distribution by confocal microscopy. We also analyzed the effect of rofecoxib on PGIS activity in bovine aortic microsomal fractions enriched in PGIS. This study demonstrates an inhibitory effect of rofecoxib on PGIS activity in human umbilical vein endothelial (HUVE) cells and in PGIS-enriched bovine aortic microsomal fractions, which is not observed by using other anti-inflammatory compounds. The inhibitory effect of rofecoxib is associated neither to a decrease of PGIS protein levels nor to an impairment of the enzyme intracellular localization. The results of this study may explain the absence of a clear relationship between COX-2 selectivity and cardiovascular side effects. Moreover, in the light of these results we propose that novel selective COX-2 inhibitors should be tested on PGI2 synthase activity inhibition.     

Binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with SARS-CoV 3CL(pro): structure-activity relationship studies reveal salient pharmacophore features

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for discovery of drugs against SARS, because of its critical role in the viral life cycle. In this study, a natural compound called quercetin-3-β-galactoside was identified as an inhibitor of the protease by molecular docking, SPR/FRET-based bioassays, and mutagenesis studies. Both molecular modeling and Q189A mutation revealed that Gln189 plays a key role in the binding. Furthermore, experimental evidence showed that the secondary structure and enzymatic activity of SARS-CoV 3CL^pro were not affected by the Q189A mutation. With the help of molecular modeling, eight new derivatives of the natural product were designed and synthesized. Bioassay results reveal salient features of the structure–activity relationship of the new compounds: (1) removal of the 7-hydroxy group of the quercetin moiety decreases the bioactivity of the derivatives; (2) acetoxylation of the sugar moiety abolishes inhibitor action; (3) introduction of a large sugar substituent on 7-hydroxy of quercetin can be tolerated; (4) replacement of the galactose moiety with other sugars does not affect inhibitor potency. This study not only reveals a new class of compounds as potential drug leads against the SARS virus, but also provides a solid understanding of the mechanism of inhibition against the target enzyme.     

IMD-4690, a Novel Specific Inhibitor for Plasminogen Activator Inhibitor-1, Reduces Allergic Airway Remodeling in a Mouse Model of Chronic Asthma via Regulating Angiogenesis and Remodeling-Related Mediators

Plasminogen activator inhibitor (PAI)-1 is the principal inhibitor of plasminogen activators, and is responsible for the degradation of fibrin and extracellular matrix. IMD-4690 is a newly synthesized inhibitor for PAI-1, whereas the effect on allergic airway inflammation and remodeling is still unclear. We examined the in vivo effects by using a chronic allergen exposure model of bronchial asthma in mice. The model was generated by an immune challenge for 8 weeks with house dust mite antigen, Dermatophagoides pteronyssinus (Dp). IMD-4690 was intraperitoneally administered during the challenge. Lung histopathology, hyperresponsiveness and the concentrations of mediators in lung homogenates were analyzed. The amount of active PAI-1 in the lungs was increased in mice treated with Dp. Administration with IMD-4690 reduced an active/total PAI-1 ratio. IMD-4690 also reduced the number of bronchial eosinophils in accordance with the decreased expressions of Th2 cytokines in the lung homogenates. Airway remodeling was inhibited by reducing subepithelial collagen deposition, smooth muscle hypertrophy, and angiogenesis. The effects of IMD-4690 were partly mediated by the regulation of TGF-β, HGF and matrix metalloproteinase. These results suggest that PAI-1 plays crucial roles in airway inflammation and remodeling, and IMD-4690, a specific PAI-1 inhibitor, may have therapeutic potential for patients with refractory asthma due to airway remodeling.