Project description:Inflammation and oxidative stress are hallmarks and mediators of the progression of CKD. Bardoxolone methyl, a potent activator of the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant and anti-inflammatory response, increases estimated GFR and decreases BUN, serum phosphorus, and uric acid concentrations in patients with moderate to severe CKD. However, it also increases albuminuria, which is associated with inflammation and disease progression. Therefore, we investigated whether this bardoxolone methyl-induced albuminuria may result from the downregulation of megalin, a protein involved in the tubular reabsorption of albumin and lipid-bound proteins. Administration of bardoxolone methyl to cynomolgus monkeys significantly decreased the protein expression of renal tubular megalin, which inversely correlated with the urine albumin-to-creatinine ratio. Moreover, daily oral administration of bardoxolone methyl to monkeys for 1 year did not lead to any adverse effects on renal histopathologic findings but did reduce serum creatinine and BUN, as observed in patients with CKD. Finally, the bardoxolone methyl-induced decrease in megalin corresponded with pharmacologic induction of renal Nrf2 targets, including NAD(P)H:quinone oxidoreductase 1 enzyme activity and glutathione content. This result indicates that Nrf2 may have a role in megalin regulation. In conclusion, these data suggest that the increase in albuminuria that accompanies bardoxolone methyl administration may result, at least in part, from reduced expression of megalin, which seems to occur without adverse effects and with strong induction of Nrf2 targets.
Project description:The SARS coronavirus 2 (SARS-CoV-2) pandemic remains a major problem in many parts of the world and infection rates remain at extremely high levels. This high prevalence drives the continued emergence of new variants, and possibly ones that are more vaccine-resistant and that can drive infections even in highly vaccinated populations. The high rate of variant evolution makes clear the need for new therapeutics that can be clinically applied to minimize or eliminate the effects of COVID-19. With a hurdle of 10 years, on average, for first in class small molecule therapeutics to achieve FDA approval, the fastest way to identify therapeutics is by drug repurposing. To this end, we developed a high throughput cell-based screen that incorporates the essential viral 3C-like protease and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or FDA-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Medicinal chemistry efforts to optimize the hits identified Tranilast as a potential lead. Here, we report the rapid screening and identification of potentially relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral 3C-like protease.
Project description: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 (3CLpro) 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 3CLpro 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 3CLpro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CLpro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.
Project description:Foot-and-mouth disease (FMD) is a highly contagious disease in cloven-hoofed animals, caused by the foot-and-mouth disease virus (FMDV). It is endemic in Asia and Africa but spreads sporadically throughout the world, resulting in significant losses in the livestock industry. Effective anti-FMDV therapeutics could be a supportive control strategy. Herein, we utilized computer-aided, structure-based virtual screening to filter lead compounds from the National Cancer Institute (NCI) diversity and mechanical libraries using FMDV 3C protease (3Cpro) as the target. Seven hit compounds were further examined via cell-based antiviral and intracellular protease assays, in which two compounds (NSC116640 and NSC332670) strongly inhibited FMDV, with EC50 values at the micromolar level of 2.88 µM (SI = 73.15) and 5.92 µM (SI = 11.11), respectively. These compounds could inactivate extracellular virus directly in a virucidal assay by reducing 1.00 to 2.27 log TCID50 of the viral titers in 0-60 min. In addition, the time-of-addition assay revealed that NSC116640 inhibited FMDV at the early stage of infection (0-8 h), while NSC332670 diminished virus titers when added simultaneously at infection (0 h). Both compounds showed good FMDV 3Cpro inhibition with IC50 values of 10.85 µM (NSC116640) and 4.21 µM (NSC332670). The molecular docking of the compounds on FMDV 3Cpro showed their specific interactions with amino acids in the catalytic triad of FMDV 3Cpro. Both preferentially reacted with enzymes and proteases in physicochemical and ADME analysis studies. The results revealed two novel small molecules with antiviral activities against FMDV and probably related picornaviruses.
Project description:Oxidative stress plays an important role in the pathogenesis of chronic heart failure (CHF) in many tissues. Increasing evidence suggests that systemic activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling can protect against postinfarct cardiac remodeling by reducing oxidative stress. However, it remains to be elucidated if Nrf2 activation exerts therapeutic effects in the CHF state. Here, we investigated the beneficial hemodynamic effects of bardoxolone methyl (2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester, CDDO-Me), a pharmacological activator of Nrf2, in a rodent model of CHF. Based on echocardiographic analysis, rats at 12 weeks post-myocardial infarction (MI) were randomly split into four groups. CDDO-Me (5 mg/kg, i.p.) was administered daily for another 2 weeks in sham and CHF rats and compared with vehicle treatment. Echocardiographic and hemodynamic analysis suggest that short-term CDDO-Me administration increased stroke volume and cardiac output in CHF rats and decreased left ventricle end-diastolic pressure. Molecular studies revealed that CDDO-Me-induced cardiac functional improvement was attributed to an increase of both Nrf2 transcription and translation, and a decrease of oxidative stress in the noninfarcted areas of the heart. Furthermore, CDDO-Me reduced NF-κB binding and increased Nrf2 binding to the CREB-binding protein, which may contribute to the selective increase of Nrf2 downstream targets, including NADPH Oxidase Quinone 1, Heme Oxygenase 1, Catalase, and Glutamate-Cysteine Ligase Catalytic Subunit, and the attenuation of myocardial inflammation in CHF rats. Our findings suggest that Nrf2 activation may provide beneficial cardiac effects in MI-mediated CHF. SIGNIFICANCE STATEMENT: Chronic heart failure (CHF) is the leading cause of death among the aged worldwide. The imbalance between pro- and antioxidant pathways is a determinant in the pathogenesis of CHF. Systemic activation of Nrf2 and antioxidant protein signaling by bardoxolone methyl may have beneficial effects on cardiac function and result in improvements by enhancing antioxidant enzyme expression and attenuating myocardial inflammation.
Project description:In addition to antioxidative and anti-inflammatory properties, activators of the cytoprotective nuclear factor erythroid-2-like-2 (NRF2) signaling pathway have antiviral effects, but the underlying antiviral mechanisms are incompletely understood. We evaluated the ability of the NRF2 activators 4-octyl itaconate (4OI), bardoxolone methyl (BARD), sulforaphane (SFN), and the inhibitor of exportin-1 (XPO1)-mediated nuclear export selinexor (SEL) to interfere with influenza virus A/Puerto Rico/8/1934 (H1N1) infection of human cells. All compounds reduced viral titers in supernatants from A549 cells and vascular endothelial cells in the order of efficacy SEL>4OI>BARD = SFN, which correlated with their ability to prevent nucleo-cytoplasmic export of viral nucleoprotein and the host cell protein p53. In contrast, intracellular levels of viral HA mRNA and nucleocapsid protein (NP) were unaffected. Knocking down mRNA encoding KEAP1 (the main inhibitor of NRF2) or inactivating the NFE2L2 gene (which encodes NRF2) revealed that physiologic NRF2 signaling restricts IAV replication. However, the antiviral effect of all compounds was NRF2-independent. Instead, XPO1 knock-down greatly reduced viral titers, and incubation of Calu3 cells with an alkynated 4OI probe demonstrated formation of a covalent complex with XPO1. Ligand-target modelling predicted covalent binding of all three NRF2 activators and SEL to the active site of XPO1 involving the critical Cys528. SEL and 4OI manifested the highest binding energies, whereby the 4-octyl tail of 4OI interacted extensively with the hydrophobic groove of XPO1, which binds nuclear export sequences on cargo proteins. Conversely, SEL as well as the three NRF2 activators were predicted to covalently bind the functionally critical Cys151 in KEAP1. Blocking XPO1-mediated nuclear export may, thus, constitute a "noncanonical" mechanism of anti-influenza activity of electrophilic NRF2 activators that can interact with similar cysteine environments at the active sites of XPO1 and KEAP1. Considering the importance of XPO1 function to a variety of pathogenic viruses, compounds that are optimized to inhibit both targets may constitute an important class of broadly active host-directed treatments that embody anti-inflammatory, cytoprotective, and antiviral properties.
Project description:Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses.
Project description:The targeted activation of nuclear factor erythroid-derived-2-like 2 (Nrf2) to alleviate symptoms of chronic kidney disease has recently garnered much attention. Unfortunately, the greatest clinical success to date, bardoxolone, failed in phase III clinical trial for unspecified safety reasons. The present letter to the editor discusses the clinical development of bardoxolone and explores potential reasons for the ultimate withdrawal from clinical trials. In particular, was the correct clinical indication pursued and would improved specificity have mitigated the safety concerns? Ultimately, it is concluded that the right clinical indication and heightened specificity will lead to successful Nrf2-based therapies. Therefore, the bardoxolone clinical results do not dampen enthusiasm for Nrf2-based therapies; rather it illuminates the clinical potential of the Nrf2 pathway as a drug target.
Project description:BACKGROUND:Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), which is sometimes associated with severe central nervous system disease in children. There is currently no specific medication for EV71 infection. Quercetin, one of the most widely distributed flavonoids in plants, has been demonstrated to inhibit various viral infections. However, investigation of the anti-EV71 mechanism has not been reported to date. METHODS:The anti-EV71 activity of quercetin was evaluated by phenotype screening, determining the cytopathic effect (CPE) and EV71-induced cells apoptosis. The effects on EV71 replication were evaluated further by determining virus yield, viral RNA synthesis and protein expression, respectively. The mechanism of action against EV71 was determined from the effective stage and time-of-addition assays. The possible inhibitory functions of quercetin via viral 2Apro, 3Cpro or 3Dpol were tested. The interaction between EV71 3Cpro and quercetin was predicted and calculated by molecular docking. RESULTS:Quercetin inhibited EV71-mediated cytopathogenic effects, reduced EV71 progeny yields, and prevented EV71-induced apoptosis with low cytotoxicity. Investigation of the underlying mechanism of action revealed that quercetin exhibited a preventive effect against EV71 infection and inhibited viral adsorption. Moreover, quercetin mediated its powerful therapeutic effects primarily by blocking the early post-attachment stage of viral infection. Further experiments demonstrated that quercetin potently inhibited the activity of the EV71 protease, 3Cpro, blocking viral replication, but not the activity of the protease, 2Apro, or the RNA polymerase, 3Dpol. Modeling of the molecular binding of the 3Cpro-quercetin complex revealed that quercetin was predicted to insert into the substrate-binding pocket of EV71 3Cpro, blocking substrate recognition and thereby inhibiting EV71 3Cpro activity. CONCLUSIONS:Quercetin can effectively prevent EV71-induced cell injury with low toxicity to host cells. Quercetin may act in more than one way to deter viral infection, exhibiting some preventive and a powerful therapeutic effect against EV71. Further, quercetin potently inhibits EV71 3Cpro activity, thereby blocking EV71 replication.
Project description:Ischemic acute kidney injury (AKI) triggers expression of adaptive (protective) and maladaptive genes. Agents that increase expression of protective genes should provide a therapeutic benefit. We now report that bardoxolone methyl (BARD) ameliorates ischemic murine AKI as assessed by both renal function and pathology. BARD may exert its beneficial effect by increasing expression of genes previously shown to protect against ischemic AKI, NF-E2-related factor 2 (Nrf2), peroxisome proliferator-activated receptor-? (PPAR?), and heme oxygenase 1 (HO-1). Although we found that BARD alone or ischemia-reperfusion alone increased expression of these genes, the greatest increase occurred after the combination of both ischemia-reperfusion and BARD. BARD had a different mode of action than other agents that regulate PPAR? and Nrf2. Thus we report that BARD regulates PPAR?, not by acting as a ligand but by increasing the amount of PPAR? mRNA and protein. This should increase ligand-independent effects of PPAR?. Similarly, BARD increased Nrf2 mRNA; this increased Nrf2 protein by mechanisms in addition to the prolongation of Nrf2 protein half-life previously reported. Finally, we localized expression of these protective genes after ischemia and BARD treatment. Using double-immunofluorescence staining for CD31 and Nrf2 or PPAR?, we found increased Nrf2 and PPAR? on glomerular endothelia in the cortex; Nrf2 was also present on cortical peritubular capillaries. In contrast, HO-1 was localized to different cells, i.e., tubules and interstitial leukocytes. Although Nrf2-dependent increases in HO-1 have been described, our data suggest that BARD's effects on tubular and leukocyte HO-1 during ischemic AKI may be Nrf2 independent. We also found that BARD ameliorated cisplatin nephrotoxicity.