Crystal structure of Staphylococcus aureus lipase complex with unsaturated petroselinic acid.

Staphylococcus aureus produces large amounts of toxins and virulence factors. In patients with underlying diseases or compromised immune systems, this bacterium can lead to severe infections and potentially death. In this study, the crystal structure of the complex of S. aureus lipase (SAL), which is involved in the growth of this bacterium, with petroselinic acid (PSA), an inhibitor of unsaturated fatty acids, was determined by X-ray crystallography. Recently, PSA was shown to inhibit S. aureus biofilm formation and the enzymatic activity of SAL. To further characterize the inhibitory mechanism, we determined the half-inhibitory concentration of SAL by PSA and the crystal structure of the complex. The IC50 of the inhibitory effect of PSA on SAL was 3.4 µm. SAL and PSA inhibitors were co-crystallized, and diffraction data sets were collected to 2.19 Å resolution at SPring-8 to determine the crystal structure and elucidate the detailed structural interactions. The results show that the fatty acid moiety of PSA is tightly bound to a hydrophobic pocket extending in two directions around the catalytic residue Ser116. Ser116 was also covalently bonded to the carbon of the unsaturated fatty acid moiety, and an oxyanion hole in SAL stabilized the electrons of the double bond. The difference in inhibitory activity between PSA and ester compounds revealed a structure-activity relationship between SAL and PSA. Additional research is required to further characterize the clinical potential of PSA.

Investigation of a practical assessment index to capture the clinical presentation of cachexia in patients with lung cancer.

OBJECTIVE: Cancer cachexia constitutes a poor prognostic factor in patients with lung cancer. However, the factors associated with cancer cachexia remain unclear. This study aimed to identify factors that influence cancer cachexia in patients with lung cancer. METHODS: In this retrospective observational study conducted at the Kansai Medical University, 76 patients with lung cancer were evaluated for physical function, nutritional status (Mini Nutritional Assessment-Short Form) and physical activity (International Physical Activity Questionnaire-Short Form) at the first visit to the rehabilitation outpatient clinic. The patients were divided into cachexia and noncachexia groups. The log-rank tests and Cox proportional hazards model were used to investigate the relationship between cachexia and prognosis. To examine the factors that influence cachexia, multivariate regression analysis with significant (P < 0.05) variables in the univariate logistic regression analysis was performed. Spearman's correlation analysis was performed to investigate the association between International Physical Activity Questionnaire-Short Form and performance status. RESULTS: Overall, 42 patients (55.2%) had cachexia associated with survival time since their first visit to the outpatient rehabilitation clinic, even after confounders adjustment (hazard ratio: 3.24, 95% confidence interval: 1.12-9.45, P = 0.031). In the multivariate analysis, Mini Nutritional Assessment-Short Form (odds ratio: 20.34, 95% confidence interval: 4.18-99.02, P < 0.001) and International Physical Activity Questionnaire-Short Form (odds ratio: 4.63, 95% confidence interval: 1.20-17.89, P = 0.026) were identified as independent factors for cachexia. There was no correlation between International Physical Activity Questionnaire-Short Form and performance status (r = 0.155, P = 0.181). CONCLUSION: Malnutrition and low physical activity were associated with cachexia in patients with lung cancer. The International Physical Activity Questionnaire-Short Form may be a useful indicator of physical activity in cachexia. Regularly assessing these factors and identifying suitable interventions for cachexia remain challenges to be addressed in the future.

Newly developed preclinical models reveal broad-spectrum CDK inhibitors as potent drugs for CRPC exhibiting primary resistance to enzalutamide.

Androgen-deprivation therapy is a standard treatment for advanced prostate cancer. However, most patients eventually acquire resistance and progress to castration-resistant prostate cancer (CRPC). In this study, we established new CRPC cell lines, AILNCaP14 and AILNCaP15, from LNCaP cells under androgen-deprived conditions. Unlike most pre-existing CRPC cell lines, both cell lines expressed higher levels of androgen receptor (AR) and prostate-specific antigen (PSA) than parental LNCaP cells. Moreover, these cells exhibited primary resistance to enzalutamide. Since AR signaling plays a significant role in the development of CRPC, PSA promoter sequences fused with GFP were introduced into AILNCaP14 cells to conduct GFP fluorescence-based chemical screening. We identified flavopiridol, a broad-spectrum CDK inhibitor, as a candidate drug that could repress AR transactivation of CRPC cells, presumably through the inhibition of phosphorylation of AR on the serine 81 residue (pARSer81 ). Importantly, this broad-spectrum CDK inhibitor inhibited the proliferation of AILNCaP14 cells both in vitro and in vivo. Moreover, a newly developed liver metastatic model using AILNCaP15 cells revealed that the compound attenuated tumor growth of CRPC harboring highly metastatic properties. Finally, we developed a patient-derived xenograft (PDX) model of CRPC and DCaP CR from a patient presenting therapeutic resistance to enzalutamide, abiraterone, and docetaxel. Flavopiridol successfully suppressed the tumor growth of CRPC in this PDX model. Since ARSer81 was found to be phosphorylated in clinical CRPC samples, our data suggested that broad-spectrum CDK inhibitors might be a potent candidate drug for the treatment of CRPC, including those exhibiting primary resistance to enzalutamide.

Cryptotanshinone is a candidate therapeutic agent for interstitial lung disease associated with a BRICHOS-domain mutation of SFTPC.

Interstitial lung disease (ILD) represents a large group of diseases characterized by chronic inflammation and fibrosis of the lungs, for which therapeutic options are limited. Among several causative genes of familial ILD with autosomal dominant inheritance, the mutations in the BRICHOS domain of SFTPC cause protein accumulation and endoplasmic reticulum stress by misfolding its proprotein. Through a screening system using these two phenotypes in HEK293 cells and evaluation using alveolar epithelial type 2 (AT2) cells differentiated from patient-derived induced pluripotent stem cells (iPSCs), we identified Cryptotanshinone (CPT) as a potential therapeutic agent for ILD. CPT decreased cell death induced by mutant SFTPC overexpression in A549 and HEK293 cells and ameliorated the bleomycin-induced contraction of the matrix in fibroblast-dependent alveolar organoids derived from iPSCs with SFTPC mutation. CPT and this screening strategy can apply to abnormal protein-folding-associated ILD and other protein-misfolding diseases.

A novel cell-based assay for the high-throughput screening of epithelial-mesenchymal transition inhibitors: Identification of approved and investigational drugs that inhibit epithelial-mesenchymal transition.

OBJECTIVES: Lung cancer with distant metastases is associated with a very poor prognosis, and epithelial-mesenchymal transition (EMT) contributes to cancer metastasis. Therefore, elucidation and inhibition of EMT signaling in lung cancer may be a new therapeutic strategy for improving the prognosis of patients. We constructed a high-throughput screening system for EMT inhibitors. Using this system, we aimed to identify compounds that indeed inhibit EMT. MATERIALS AND METHODS: We generated a luciferase reporter cell line using A549 human lung cancer cells and E-cadherin or vimentin as EMT markers. EMT was induced by transforming growth factor ß1 (TGF-ß1), and candidate EMT inhibitors were screened from a library of 2,350 compounds. The selected compounds were further tested using secondary assays to verify the inhibition of EMT and invasive capacity of cells. RESULTS: Values obtained by the assay were adjusted for the number of viable cells and scored by determining the difference between mean values of the positive and negative control groups. Four compounds were identified as novel candidate drugs. Among those, one (avagacestat) and two compounds (GDC-0879 and levothyroxine) improved the expression of E-cadherin and vimentin, respectively, in epithelial cells. GDC-0879 and levothyroxine also significantly inhibited the invasive capacity of cells. CONCLUSION: We systematically screened approved, investigational, and druggable compounds with inhibitory effects using a reporter assay, and identified candidate drugs for EMT inhibition.

High-throughput Screening in Combination With a Cohort Study for Iodothyronine Deiodinases.

Regulatory mechanisms of iodothyronine deiodinases (DIOs) require further elucidation, and conventional methods for evaluating DIOs are unsuitable for high-throughput screening (HTS). Here we explored factors of transcriptional regulation of 3 types of DIOs (DIO1, DIO2, and DIO3) from a chemical library using our designed HTS. We constructed HTS based on a promoter assay and performed a screen of 2480 bioactive compounds. For compounds that were clinically approved, we validated hit compounds through a retrospective cohort study in our department that evaluated changes in thyroid function in patients using the compounds as drug therapy. Furthermore, we verified the involvement of DIOs using mice treated with the compounds. Of the hit compounds, 6 and 7 compounds transcriptionally up- and downregulated DIO1, respectively; 34 transcriptionally upregulated DIO2; and 5 and 2 compounds transcriptionally up- and downregulated DIO3, respectively. The cohort study clarified the clinical effects of some hit compounds: ritodrine increased free triiodothyronine (fT3)/free thyroxine (fT4) ratio and decreased serum thyroid-stimulating hormone (TSH) levels, tadalafil increased serum fT3 levels, and tyrosine kinase inhibitors (TKIs) decreased serum fT3 and fT4 levels and increased serum TSH levels. Following in vivo experiments using treated mice, consistent results were observed in ritodrine, which upregulated DIO2 in the thyroid gland. In conclusion, we completed HTS for DIOs and obtained attractive hit compounds. Our cohort study revealed the clinical significance of ritodrine, sildenafil, and TKIs. We hope our unique method will contribute to analyzing various targets and lists of hit compounds will promote understanding of DIOs.

Albendazole induces the terminal differentiation of acute myeloid leukaemia cells to monocytes by stimulating the Krüppel-like factor 4-dihydropyrimidinase-like 2A (KLF4-DPYSL2A) axis.

Differentiation therapy is a less toxic but still a very effective treatment for a subset of acute myeloid leukaemia (AML) cases. With the goal to identify novel compounds that can effectively and safely induce the terminal differentiation of non-acute promyelocytic leukaemia (APL) AML cells, we performed a chemical screening and identified albendazole (ABZ), a widely used anti-helminthic drug, as a promising lead compound that can differentiate non-APL AML cells by stimulating the Krüppel-like factor 4-dihydropyrimidinase-like 2A (KLF4-DPYSL2A) differentiation axis to the monocytes. Our in vitro and in vivo findings demonstrate that ABZ is an attractive candidate drug as a novel differentiation chemotherapy for patients with non-APL AML.

Crystal structure of pathogenic Staphylococcus aureus lipase complex with the anti-obesity drug orlistat.

Staphylococcus aureus lipase (SAL), a triacylglycerol esterase, is an important virulence factor and may be a therapeutic target for infectious diseases. Herein, we determined the 3D structure of native SAL, the mutated S116A inactive form, and the inhibitor complex using the anti-obesity drug orlistat to aid in drug development. The determined crystal structures showed a typical α/ß hydrolase motif with a dimeric form. Fatty acids bound near the active site in native SAL and inactive S116A mutant structures. We found that orlistat potently inhibits SAL activity, and it covalently bound to the catalytic Ser116 residue. This is the first report detailing orlistat-lipase binding. It provides structure-based information on the production of potent anti-SAL drugs and lipase inhibitors. These results also indicated that orlistat can be repositioned to treat bacterial diseases.

Prenatal neurogenesis induction therapy normalizes brain structure and function in Down syndrome mice.

Down syndrome (DS) caused by trisomy of chromosome 21 is the most common genetic cause of intellectual disability. Although the prenatal diagnosis of DS has become feasible, there are no therapies available for the rescue of DS-related neurocognitive impairment. A growth inducer newly identified in our screen of neural stem cells (NSCs) has potent inhibitory activity against dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) and was found to rescue proliferative deficits in Ts65Dn-derived neurospheres and human NSCs derived from individuals with DS. The oral administration of this compound, named ALGERNON (altered generation of neurons), restored NSC proliferation in murine models of DS and increased the number of newborn neurons. Moreover, administration of ALGERNON to pregnant dams rescued aberrant cortical formation in DS mouse embryos and prevented the development of abnormal behaviors in DS offspring. These data suggest that the neurogenic phenotype of DS can be prevented by ALGERNON prenatal therapy.

RBM24 promotes U1 snRNP recognition of the mutated 5' splice site in the IKBKAP gene of familial dysautonomia.

The 5' splice site mutation (IVS20+6T>C) of the inhibitor of κ light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene in familial dysautonomia (FD) is at the sixth intronic nucleotide of the 5' splice site. It is known to weaken U1 snRNP recognition and result in an aberrantly spliced mRNA product in neuronal tissue, but normally spliced mRNA in other tissues. Aberrantly spliced IKBKAP mRNA abrogates IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1) expression and results in a defect of neuronal cell development in FD. To elucidate the tissue-dependent regulatory mechanism, we screened an expression library of major RNA-binding proteins (RBPs) with our mammalian dual-color splicing reporter system and identified RBM24 as a regulator. RBM24 functioned as a cryptic intronic splicing enhancer binding to an element (IVS20+13-29) downstream from the intronic 5' splice site mutation in the IKBKAP gene and promoted U1 snRNP recognition only to the mutated 5' splice site (and not the wild-type 5' splice site). Our results show that tissue-specific expression of RBM24 can explain the neuron-specific aberrant splicing of IKBKAP exon 20 in familial dysautonomia, and that ectopic expression of RBM24 in neuronal tissue could be a novel therapeutic target of the disease.

In vitro expansion of mouse primordial germ cell-like cells recapitulates an epigenetic blank slate.

The expansion of primordial germ cells (PGCs), the precursors for the oocytes and spermatozoa, is a key challenge in reproductive biology/medicine. Using a chemical screening exploiting PGC-like cells (PGCLCs) induced from mouse embryonic stem cells (ESCs), we here identify key signaling pathways critical for PGCLC proliferation. We show that the combinatorial application of Forskolin and Rolipram, which stimulate cAMP signaling via different mechanisms, expands PGCLCs up to ~50-fold in culture. The expanded PGCLCs maintain robust capacity for spermatogenesis, rescuing the fertility of infertile mice. Strikingly, during expansion, PGCLCs comprehensively erase their DNA methylome, including parental imprints, in a manner that precisely recapitulates genome-wide DNA demethylation in gonadal germ cells, while essentially maintaining their identity as sexually uncommitted PGCs, apparently through appropriate histone modifications. By establishing a paradigm for PGCLC expansion, our system reconstitutes the epigenetic "blank slate" of the germ line, an immediate precursory state for sexually dimorphic differentiation.

Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease. Various attempts are underway to convert severe DMD to a milder phenotype by modulating the splicing of the dystrophin gene and restoring its expression. In our previous study, we reported TG003, an inhibitor of CDC2-like kinase 1 (CLK1), as a splice-modifying compound for exon-skipping therapy; however, its metabolically unstable feature hinders clinical application. Here, we show an orally available inhibitor of CLK1, named TG693, which promoted the skipping of the endogenous mutated exon 31 in DMD patient-derived cells and increased the production of the functional exon 31-skipped dystrophin protein. Oral administration of TG693 to mice inhibited the phosphorylation of serine/arginine-rich proteins, which are the substrates of CLK1, and modulated pre-mRNA splicing in the skeletal muscle. Thus, TG693 is a splicing modulator for the mutated exon 31 of the dystrophin gene in vivo, possibly possessing therapeutic potential for DMD patients.

Retinoid derivative Tp80 exhibits anti-hepatitis C virus activity through restoration of GI-GPx expression.

Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus with an estimated infection in ∼180 million people worldwide, and its chronic infection leads to development of cirrhosis and hepatocellular carcinoma. Although recent development of direct acting antiviral (DAA) compounds improved anti-HCV regimens, alternative therapeutic compounds are still demanded due to an expected emergence of escape mutants for those DAAs. In order to identify novel anti-HCV agents, we conducted chemical library screening for 2086 compounds using HCV Rep-Feo reporter replicon in Huh7 hepatoma cells. Our screening identified retinoid derivative Tp80, which inhibits replication of HCV Rep-Feo (genotype 1b) and JFH1 HCV (genotype 2a) with 0.62 µM and 1.0 µM, respectively, of 50% effective concentration (EC50 ), at which cytotoxicity is not evident for host hepatocytes. Subsequent transcriptome profiling revealed Tp80 exhibits anti-HCV activity through restoration of gastrointestinal glutathione peroxidase (GI-GPx), suppression of which is responsible for HCV-induced oxidative stress to facilitate HCV replication. Furthermore, comparison of Tp80 with other retinoid derivatives revealed Tp80 shows best potency in both GI-GPx restoration and anti-HCV activity among compounds we examined. In conclusion, our current study provides Tp80 as a promising candidate of anti-HCV compound, suppressing host cellular oxidative stress through a restoration of GI-GPx.

Establishment of Nephrin Reporter Mice and Use for Chemical Screening.

Nephrin is a critical component of glomerular filtration barrier, which is important to maintain glomerular structure and avoid proteinuria. Downregulation of nephrin expression is commonly observed at early stage of glomerular disorders, suggesting that methods to increase nephrin expression in podocytes may have therapeutic utility. Here, we generated a knockin mouse line carrying single copy of 5.5 kb nephrin promoter controlling expression of enhanced green fluorescent protein (EGFP) at Rosa26 genomic locus (Nephrin-EGFP mouse). In these mice, EGFP was specifically expressed in podocytes. Next, we isolated and cultivated glomeruli from these mice, and developed a protocol to automatically quantitate EGFP expression in cultured glomeruli. EGFP signal was markedly reduced after 5 days of culture but reduction was inhibited by vitamin D treatment. We confirmed that vitamin D increased mRNA and protein expression of endogenous nephrin in cultivated glomeruli. Thus, we generated a mouse line converting nephrin promoter activity into fluorescence, which can be used to screen compounds having activity to enhance nephrin gene expression.

Selective inhibition of the kinase DYRK1A by targeting its folding process.

Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors.

Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ.

The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimer's disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases.

Identification of a Dual Inhibitor of SRPK1 and CK2 That Attenuates Pathological Angiogenesis of Macular Degeneration in Mice.

Excessive angiogenesis contributes to numerous diseases, including cancer and blinding retinopathy. Antibodies against vascular endothelial growth factor (VEGF) have been approved and are widely used in clinical treatment. Our previous studies using SRPIN340, a small molecule inhibitor of SRPK1 (serine-arginine protein kinase 1), demonstrated that SRPK1 is a potential target for the development of antiangiogenic drugs. In this study, we solved the structure of SRPK1 bound to SRPIN340 by X-ray crystallography. Using pharmacophore docking models followed by in vitro kinase assays, we screened a large-scale chemical library, and thus identified a new inhibitor of SRPK1. This inhibitor, SRPIN803, prevented VEGF production more effectively than SRPIN340 owing to the dual inhibition of SRPK1 and CK2 (casein kinase 2). In a mouse model of age-related macular degeneration, topical administration of eye ointment containing SRPIN803 significantly inhibited choroidal neovascularization, suggesting a clinical potential of SRPIN803 as a topical ointment for ocular neovascularization. Thus SRPIN803 merits further investigation as a novel inhibitor of VEGF.

Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia.

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by missplicing of exon 20, resulting from an intronic mutation in the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase complex-associated protein (IKBKAP) gene encoding IKK complex-associated protein (IKAP)/elongator protein 1 (ELP1). A newly established splicing reporter assay allowed us to visualize pathogenic splicing in cells and to screen small chemicals for the ability to correct the aberrant splicing of IKBKAP. Using this splicing reporter, we screened our chemical libraries and identified a compound, rectifier of aberrant splicing (RECTAS), that rectifies the aberrant IKBKAP splicing in cells from patients with FD. Here, we found that the levels of modified uridine at the wobble position in cytoplasmic tRNAs are reduced in cells from patients with FD and that treatment with RECTAS increases the expression of IKAP and recovers the tRNA modifications. These findings suggest that the missplicing of IKBKAP results in reduced tRNA modifications in patients with FD and that RECTAS is a promising therapeutic drug candidate for FD.

Discovery of protein disulfide isomerase P5 inhibitors that reduce the secretion of MICA from cancer cells.

In order to regulate the activity of P5, which is a member of the protein disulfide isomerase family, we screened a chemical compound library for P5-specific inhibitors, and identified two candidate compounds (anacardic acid and NSC74859). Interestingly, anacardic acid inhibited the reductase activity of P5, but did not inhibit the activity of protein disulfide isomerase (PDI), thiol-disulfide oxidoreductase ERp57, or thioredoxin. NSC74859 inhibited all these enzymes. When we examined the effects of these compounds on the secretion of soluble major histocompatibility complex class-I-related gene A (MICA) from cancer cells, anacardic acid was found to decrease secretion. In addition, anacardic acid was found to reduce the concentration of glutathione up-regulated by the anticancer drug 17-demethoxygeldanamycin in cancer cells. These results suggest that anacardic acid can both inhibit P5 reductase activity and decrease the secretion of soluble MICA from cancer cells. It might be a novel and potent anticancer treatment by targeting P5 on the surface of cancer cells.

Identification of novel N-(morpholine-4-carbonyloxy) amidine compounds as potent inhibitors against hepatitis C virus replication.

To identify novel compounds that possess antiviral activity against hepatitis C virus (HCV), we screened a library of small molecules with various amounts of structural diversity using an HCV replicon-expressing cell line and performed additional validations using the HCV-JFH1 infectious-virus cell culture. Of 4,004 chemical compounds, we identified 4 novel compounds that suppressed HCV replication with 50% effective concentrations of ranging from 0.36 to 4.81 µM. N'-(Morpholine-4-carbonyloxy)-2-(naphthalen-1-yl) acetimidamide (MCNA) was the most potent and also produced a small synergistic effect when used in combination with alpha interferon. Structure-activity relationship (SAR) analyses revealed 4 derivative compounds with antiviral activity. Further SAR analyses revealed that the N-(morpholine-4-carbonyloxy) amidine moiety was a key structural element for antiviral activity. Treatment of cells with MCNA activated nuclear factor κB and downstream gene expression. In conclusion, N-(morpholine-4-carbonyloxy) amidine and other related morpholine compounds specifically suppressed HCV replication and may have potential as novel chemotherapeutic agents.