Novel Neplanocin A Derivatives as Selective Inhibitors of Hepatitis B Virus with a Unique Mechanism of Action.

Novel neplanocin A derivatives have been identified as potent and selective inhibitors of hepatitis B virus (HBV) replication in vitro. These include (1S,2R,5R)-5-(5-bromo-4-methyl-7H-pyrrolo[2,3-d]-pyrimidin-7-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (AR-II-04-26) and (1S,2R,5R)-5-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3-(hydroxylmethyl)cyclopent-3-ene-1,2-diol (MK-III-02-03). The 50% effective concentrations of AR-II-04-26 and MK-III-02-03 were 0.77 ± 0.23 and 0.83 ± 0.36 µM in HepG2.2.15.7 cells, respectively. These compounds reduced intracellular HBV RNA levels in HepG2.2.15.7 cells and infected primary human hepatocytes. Accordingly, they could reduce HBs and HBe antigen production in the culture supernatants, which was not observed with clinically approved anti-HBV nucleosides and nucleotides (reverse transcriptase inhibitors). The neplanocin A derivatives also inhibited HBV RNA derived from cccDNA. In addition, unlike neplanocin A itself, the compounds did not inhibit S-adenosyl-l-homocysteine hydrolase activity. Thus, it appears that the mechanism of action of AR-II-04-26 and MK-III-02-03 differs from that of the clinically approved anti-HBV agents. Although their exact mechanism (target molecule) remains to be elucidated, the novel neplanocin A derivatives are considered promising candidate drugs for inhibition of HBV replication.

Induction of E. coli-derived endonuclease MazF suppresses HIV-1 production and causes apoptosis in latently infected cells.

The current antiretroviral therapy cannot cure the patients infected with human immunodeficiency virus type 1 (HIV-1) due to the existence of latently infected cells capable of virus production from harboring proviral DNA. MazF is an ACA nucleotide sequence-specific endoribonuclease derived from Escherichia coli. The conditional expression of MazF by binding of HIV-1 Tat to the promoter region of a MazF-expression vector has previously been shown to selectively inhibit HIV-1 replication in acutely infected cells. The expression of MazF significantly suppressed tumor necrosis factor (TNF)-α-induced HIV-1 production and viral RNA expression in the HIV-1 latently infected cell line OM-10.1 transduced with the MazF-expression vector (OM-10.1/MFR). Moreover, the viability of OM-10.1/MFR cells decreased with increasing concentrations of TNF-α, whereas such decrease was not observed for HL-60 cells transduced with the MazF-expression vector (HL-60/MFR), the uninfected parental cell line of OM-10.1. TNF-α increased the expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase in OM-10.1/MFR cells, indicating that the cell death was caused by the induction of apoptosis. TNF-α-induced expression of MazF mRNA was detected in OM-10.1/MFR but not HL-60/MFR cells, suggesting that TNF-α-induced apoptosis of latently infected cells was due to the expression of MazF. Thus, the anti-HIV-1 gene therapy using the MazF-expression vector may have potential for the cure of HIV-1 infection in combination with suitable latency reversing agents through reducing the size of latently infected cells without viral reactivation.

Successful treatment of an AIDS patient with prolonged Mycobacterium avium bacteremia, high HIV RNA, HBV infection, Kaposi's sarcoma and cytomegalovirus retinitis.

We report an AIDS patient with a high HIV RNA copy number in the plasma who was successfully treated for prolonged Mycobacterium avium bacteremia and other complications. An HIV-infected patient with high fever, anemia, high alkaline phosphatase, cystic lung lesions, hepatitis B virus infection and Kaposi's sarcoma was referred to our hospital. PCR of the blood revealed Mycobacterium avium bacteremia and the time to blood culture positivity was 8 days. The HIV-1 RNA copy number in the plasma was more than ten million copies/ml and the CD4-positive T cell count was 21 cells/µL. Although the high fever resolved five days after therapy for Mycobacterium avium was started, the fever recurred just before starting anti-retroviral therapy (ART) including dolutegravir. The patient experienced repeated but self-limiting bouts of severe inflammation. Mycobacteremia was intermittently detected up to 79 days, suggesting that the recurrent episodes of inflammation were due to the intermittent dissemination of mycobacteria, and that persistent treatment is needed. Five months after the beginning of ART, the HIV-1 RNA copy number in the plasma was still 28,000 copies/ml. An HIV drug-resistance test revealed sensitivity to all anti-retroviral drugs. Eleven months after the initiation of ART, the HIV RNA copy number in the plasma decreased to 45 copies/mL and the CD4-positive T cell count recovered to 205 cells/µL. Our case also suggests that dolutegravir can be effective in cases with prolonged high levels of HIV RNA. Our findings emphasize that prompt diagnosis and persistent therapy for mycobacterial infection are important for successful treatment.

Genome-wide association study for age-related hearing loss in CFW mice.

Age-related hearing impairment is the most common cause of hearing loss and is one of the most prevalent conditions affecting the elderly globally. It is influenced by a combination of environmental and genetic factors. The mouse and human inner ears are functionally and genetically homologous. Investigating the genetic basis of age-related hearing loss (ARHL) in an outbred mouse model may lead to a better understanding of the molecular mechanisms of this condition. We used Carworth Farms White (CFW) outbred mice, because they are genetically diverse and exhibit variation in the onset and severity of ARHL. The goal of this study was to identify genetic loci involved in regulating ARHL. Hearing at a range of frequencies was measured using Auditory Brainstem Response (ABR) thresholds in 946 male and female CFW mice at the age of 1, 6, and 10 months. We obtained genotypes at 4.18 million single nucleotide polymorphisms (SNP) using low-coverage (mean coverage 0.27x) whole-genome sequencing followed by imputation using STITCH. To determine the accuracy of the genotypes we sequenced 8 samples at >30x coverage and used calls from those samples to estimate the discordance rate, which was 0.45%. We performed genetic analysis for the ABR thresholds for each frequency at each age, and for the time of onset of deafness for each frequency. The SNP heritability ranged from 0 to 42% for different traits. Genome-wide association analysis identified several regions associated with ARHL that contained potential candidate genes, including Dnah11, Rapgef5, Cpne4, Prkag2, and Nek11. We confirmed, using functional study, that Prkag2 deficiency causes age-related hearing loss at high frequency in mice; this makes Prkag2 a candidate gene for further studies. This work helps to identify genetic risk factors for ARHL and to define novel therapeutic targets for the treatment and prevention of ARHL.

Identification of novel inhibitors of human immunodeficiency virus type 1 replication by in silico screening targeting cyclin T1/Tat interaction.

Human immunodeficiency virus type 1 (HIV-1) transcription is essential for viral replication and the only step for viral genome amplification. Cyclin T1 (CycT1) interacts with HIV-1 Tat and transactivation-responsive (TAR) RNA, leading to the activation of viral transcription through the hyperphosphorylation of RNA polymerase II (RNAPII). Thus, the CycT1/Tat/TAR RNA interaction represents a novel target for inhibition of HIV-1 replication. In this study, we conducted in silico screening of compounds targeting the CycT1/Tat/TAR RNA complex and found that two structurally related compounds (C1 and C2) had high docking scores for a model of the complex. These compounds proved inhibitory to HIV-1 replication in tumor necrosis factor alpha-stimulated chronically infected cells. In addition, C3, a derivative of C1 and C2, was found to be a more potent inhibitor of HIV-1 replication in chronically infected cells. C3 also inhibited HIV-1 replication in acutely infected cells. The compound could suppress Tat-mediated HIV-1 long terminal repeat-driven gene expression and phosphorylation of RNAPII through inhibition of Tat binding to CycT1. Furthermore, the docking pose of C3 was defined by analyses for its in silico docking energy and in vitro antiviral activity, which indicates that C3 interacts with Tat-binding amino acids of CycT1. Thus, a series of compounds described herein are novel inhibitors of HIV-1 transcription through inhibition of CycT1/Tat interaction.

Amodiaquine derivatives as inhibitors of severe fever with thrombocytopenia syndrome virus (SFTSV) replication.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral infection caused by a bandavirus in the family of Phenuiviridae, commonly known as SFTS virus (SFTSV). We have previously isolated SFTSV from blood samples of SFTS patients and established an antiviral assay system to identify selective inhibitors of SFTSV in vitro. Using the assay system, the antimalarial agent amodiaquine was identified as a selective inhibitor of SFTSV replication. However, due to its insufficient antiviral activity, 98 amodiaquine derivatives were newly synthesized and examined for their anti-SFTSV activity. Among the derivatives, some compounds showed selective inhibitory effect on SFTSV replication in vitro. The 50% effective concentration (EC50) and cytotoxic concentration (CC50) of the most active compound (C-90) were 2.6 ± 0.6 and >50 µM, respectively. This EC50 value was comparable to or slightly better than that of favipiravir (4.1 ± 0.6 µM). On the other hand, pharmacokinetic studies in vivo revealed that C-90 was poor in its oral bioavailability in mice. Therefore, we further designed and synthesized derivatives and obtained 2 compounds with selective anti-SFTSV activity in vitro and improved pharmacokinetics in vivo.

Exploring Novel Vitamin K Derivatives with Anti-SARS-CoV-2 Activity.

From our compound library of vitamin K derivatives, we found that some compounds exhibited anti-SARS-CoV-2 activity in VeroE6/TMPRSS2 cells. The common structure of these compounds was menaquinone-2 (MK-2) with either the m-methylphenyl or the 1-naphthyl group introduced at the end of the side chain. Therefore, new vitamin K derivatives having more potent anti-SARS-CoV-2 activity were explored by introducing various functional groups at the ω-position of the side chain. MK-2 derivatives with a purine moiety showed the most potent antiviral activity among the derivatives. We also found that their mechanism of action was the inhibition of RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. The chemical structures of our compounds were completely different from those of nucleic acid derivatives such as remdesivir and molnupiravir, clinically approved RdRp inhibitors for COVID-19 treatment, suggesting that our compounds may be effective against viruses resistant to these nucleic acid derivatives.

Novel RT-PCR Using Sugar Chain-Immobilized Gold-Nanoparticles Correlates Patients' Symptoms: The Follow-Up Study of COVID-19 Hospitalized Patients.

Background: The transmissible capacity and toxicity of SARS-CoV-2 variants are continually changing. We report here the follow-up study of hospitalized COVID-19 patients from 2020 to 2022. It is known that the PCR diagnosis for hospitalized patients sometimes causes confusion because of the incompatibility between their diagnosis and symptoms. We applied our sugar chain-immobilized gold-nanoparticles for the extraction and partial purification of RNA from specimens for quantitative RT-PCR assay and evaluated whether the results correlate with patients' symptoms. Methods and Results: Saliva specimens were taken from hospitalized patients with mild or moderate symptoms every early morning. At the time of RT-PCR diagnosis, two methods for the extraction and partial purification of RNA from the specimen were performed: a commonly used Boom (Qiagen) method and our original sugar chain-immobilized gold nanoparticle (SGNP) method. For symptoms, body temperature and oxygen saturation (SpO2) of patients were monitored every 4 h. Conclusions: It was clear that patients infected with the Delta variant needed more time to recover than those with the Omicron variant, and that the SGNP method showed more realistic correlation with the symptoms of patients compared with the common Qiagen method.

Potent and selective inhibition of hepatitis C virus replication by novel phenanthridinone derivatives.

A number of novel phenanthridinone derivatives were examined for their inhibitory effect on hepatitis C virus (HCV) replication in Huh-7 cells harboring self-replicating subgenomic viral RNA replicons with a luciferase reporter (LucNeo#2). The activity of compounds was further confirmed by inhibition of viral RNA copy number in different subgenomic and full-genomic replicon cells using real-time reverse transcription polymerase chain reaction. Among the compounds, 4-butyl-11-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-7-methoxy-[1,3]dioxolo[4,5-c]phenanthridin-5(4H)-one (HA-719) was found to be the most active with a 50% effective concentration of 0.063 ± 0.010 µM in LucNeo#2 cells. The compound did not show apparent cytotoxicity to the host cells at concentrations up to 40 µM. Western blot analysis demonstrated that HA-719 reduced the levels of NS3 and NS5A proteins in a dose-dependent fashion in the replicon cells. Interestingly, the phenanthridinone derivatives including HA-719 were less potent inhibitors of JFH1 strain (genobtype 2a HCV) in cell-free virus infection assay. Although biochemical assays revealed that HA-719 proved not to inhibit NS3 protease or NS5B RNA polymerase activity at the concentrations capable of inhibiting viral replication, their molecular target (mechanism of inhibition) remains unknown. Considering the fact that most of the anti-HCV agents currently approved or under clinical trials are protease and polymerase inhibitors, the phenanthridinone derivatives are worth pursuing for their mechanism of action and potential as novel anti-HCV agents.

Fused heterocyclic amido compounds as anti-hepatitis C virus agents.

We identified a fused heteroaromatic amido structure based on the phenanthridine skeleton as a superior scaffold for candidate drugs with potent anti-HCV activity. Among the compounds synthesized, a phenanthridine analogue with a 1,3-dioxolyl group (24) possessed the most potent anti-HCV activity (EC(50) value: 50 nM), with acceptable cytotoxicity. The structural development and structure-activity relationships of these compounds are described.

Structural development studies of anti-hepatitis C virus agents with a phenanthridinone skeleton.

A phenanthridinone skeleton was derived from our previous researches on thalidomide and retinoids as a multi-template for generation of anti-viral lead compounds. Structural development studies focusing on anti-hepatitis C virus activity afforded 5-butyl-2-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenanthridin-6(5H)-one (10) and 5-butylbenzo[b]phenanthridin-6(5H)-one (39), which showed EC(50) values of approximately 3.7 and 3.2microM, respectively.

The chemokine receptor antagonist cenicriviroc inhibits the replication of SARS-CoV-2 in vitro.

Cenicriviroc (CVC) is a small-molecule chemokine receptor antagonist with highly potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity through antagonizing C-C chemokine receptor type 5 (CCR5) as a coreceptor of HIV-1. CVC also strongly antagonizes C-C chemokine receptor type 2b (CCR2b), thereby it has potent anti-inflammatory and immunomodulatory effects. CVC is currently under clinical trials in the patients for treatment of nonalcoholic steatohepatitis, in which immune cell activation and dysregulation of proinflammatory cytokines play an important role in its pathogenesis. In this study, CVC was examined for its inhibitory effect on the replication of SARS-CoV-2, the causative agent of COVID-19, in cell cultures and found to be a selective inhibitor of the virus. The 50% effective concentrations of CVC were 19.0 and 2.9 µM in the assays based on the inhibition of virus-induced cell destruction and viral RNA levels in culture supernatants of the infected cells, respectively. Interestingly, the CCR5-specific antagonist maraviroc did not show any anti-SARS-CoV-2 activity. Although the mechanism of SARS-CoV-2 inhibition by CVC remains to be elucidated, CCR2b does not seem to be its target molecule. Considering the fact that the regulation of excessive immune activation is required to treat COVID-19 patients at the late stage of the disease, CVC should be further pursued for its potential in the treatment of SARS-CoV-2 infection.

Novel Anti-CD70 Antibody Drug Conjugate for the Treatment of Adult T-Cell Leukemia (ATL).

BACKGROUND/AIM: Adult T-cell leukemia (ATL) is a hematological malignancy caused by infection with human T-cell leukemia virus type 1 (HTLV-1). Chemotherapy, antibody therapy, and bone marrow transplantation are used to treat this disease, however, median survival time has not been significantly improved. Our aim was to develop and evaluate a novel antibody-drug conjugate (ADC) with regards to cell cytotoxicity and target specificity. MATERIALS AND METHODS: In this study, we have constructed a novel ADC, which is composed of an anti-CD70 single chain Fv-Fc antibody conjugated with the anticancer agent emtansine using a novel antibody modification method. Cell cytotoxicity and target specificity were assessed using a cell proliferation assay. RESULTS: The anti-CD70 ADC selectively killed HTLV-1-infected cells and ATL cells without affecting other cells. CONCLUSION: The anti-CD70 ADC offers some chemotherapeutic potential for the treatment of ATL.

Highly enhanced expression of CD70 on human T-lymphotropic virus type 1-carrying T-cell lines and adult T-cell leukemia cells.

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). In Japan, the number of HTLV-1 carriers is estimated to be 1.2 million and more than 700 cases of ATL have been diagnosed every year. Considering the poor prognosis and lack of curative therapy of ATL, it seems mandatory to establish an effective strategy for the treatment of ATL. In this study, we attempted to identify the cell surface molecules that will become suitable targets of antibodies for anti-ATL therapy. The expression levels of approximately 40,000 host genes of three human T-cell lines carrying HTLV-1 genomes were analyzed by oligonucleotide microarray and compared with the expression levels of the genes in an HTLV-1-negative T-cell line. The HTLV-1-carrying T-cell lines used for experiments had totally different expression patterns of viral genome. Among the genes evaluated, the expression levels of 108 genes were found to be enhanced more than 10-fold in all of the T-cell lines examined and 11 of the 108 genes were considered to generate the proteins expressed on the cell surface. In particular, the CD70 gene was upregulated more than 1,000-fold and the enhanced expression of the CD70 molecule was confirmed by laser flow cytometry for various HTLV-1-carrying T-cell lines and primary CD4(+) T cells isolated from acute-type ATL patients. Such expression was not observed for primary CD4(+) T cells isolated from healthy donors. Since CD70 expression is strictly restricted in normal tissues, such as highly activated T and B cells, CD70 appears to be a potential target for effective antibody therapy against ATL.

Discovery of diphenylmethane analogs as anti-bovine diarrhea viral agents.

Based on antiviral screening of our diphenylmethane derivatives prepared as steroid substitutes, we identified a 1,1-diphenylcyclobutane analog (9) and two diethyldiphenylsilane analogs (12 and 13) as superior lead compounds with potent anti-bovine viral diarrhea virus (BVDV) activity, having 50% effective concentration (EC(50): based on reduction of BVDV replication-induced cell destruction) and 50% cytotoxic concentration (CC(50): based on reduction of viable cell number) values of 6.2-8.4 microM and >100 microM, respectively, in Madin-Darby bovine kidney (MDBK) cells infected with BVDV.

Galectin-3 is involved in HIV-1 expression through NF-κB activation and associated with Tat in latently infected cells.

Galectin-3 (Gal-3) is involved in many biological processes and pathogenesis of diseases in part through nuclear factor (NF)-κB activation. We demonstrated that Gal-3 expression was significantly induced by tumor necrosis factor (TNF)-α or phorbol 12-myristate 13-acetate in OM-10.1 and ACH-2 cells, which are considered as a model of HIV-1 latently infected cells. The expression of Gal-3 was also associated with their viral production. However, the induction of Gal-3 by TNF-α was not observed in their uninfected parental cells. Knockdown of Gal-3 resulted in the suppression of NF-κB activation and HIV-1 replication in the latently infected cells. The expression level of Gal-3 was highly correlated with that of HIV-1 Tat in the latently infected cells stimulated with TNF-α. Furthermore, colocalization and possible interaction of Gal-3 and Tat were observed in the stimulated cells. These results suggent that Gal-3 expression is closely correlated with HIV-1 expression in latently infected cells through NF-κB activation and the interaction with Tat.

Pyrimidotriazine derivatives as selective inhibitors of HBV capsid assembly.

Chronic hepatitis B virus (HBV) infection is currently treated with nucleoside/nucleotides analogs. They are potent inhibitors of HBV DNA polymerase, which also functions as reverse transcriptase. Although nucleoside/nucleotide analogs efficiently suppress HBV replication in liver cells, they cannot eradicate HBV DNA from liver cells and cure the disease. Therefore, it is still mandatory to identify and develop effective inhibitors that target a step other than reverse transcription in the viral replication cycle. HBV capsid assembly is a critical step for viral replication and an attractive target for inhibition of HBV replication. We conducted in silico screening of compounds expected to bind to the HBV capsid dimer-dimer interaction site. The selected compounds were further examined for their anti-HBV activity in vitro. Among the test compounds, novel pyrimidotriazine derivatives were found to be selective inhibitors of HBV replication in HepG2.2.15.7 cells. Among the compounds, 2-[(2,3-dichlorophenyl)amino]-4-(4-tert-butylphenyl)-8-methyl-4H,9H-pyrimido[1,2-a][1,3,5]triazin-6-one was the most active against HBV replication. Studies on its mechanism of action revealed that the compound interfered with HBV capsid assembly determined by a cell-free capsid assembly system. Thus, the pyrimidotriazine derivatives are considered to be potential leads for novel HBV capsid assembly inhibitors.

Potent and selective inhibition of Tat-dependent HIV-1 replication in chronically infected cells by a novel naphthalene derivative JTK-101.

In search for effective human immunodeficiency virus type 1 (HIV-1) transcription inhibitors, we have evaluated more than 100,000 compounds for their inhibitory effects on HIV-1 long terminal repeat (LTR)-driven reporter gene expression, and identified a novel naphthalene derivative, JTK-101. This compound could suppress tumour necrosis factor (TNF)-alpha-induced HIV-1 production in latently infected OM-10.1 cells at nanomolar concentrations. JTK-101 could also potently inhibit constitutive HIV-1 production in MOTL-4/IIIB. However, the antiviral activity of JTK-101 was found to be much weaker in acutely infected cells and the chronically infected cells U937/IIIB cells than in OM-10.1 and MOLT-4/IIIB cells. JTK-101 selectively suppressed TNF-alpha-induced HIV-1 mRNA synthesis in OM-10.1 cells in a dose-dependent fashion. JTK-101 modestly inhibited TNF-alpha-induced HIV-1 LTR-driven reporter gene expression, but potently inhibited Tat-induced gene expression. Immunoblot analysis revealed that low-level expression of the Tat cofactors CDK9 and cyclin T1 might contribute to the diminished antiviral activity in U937/IIIB cells. Furthermore, JTK-101 could not inhibit HIV-1 replication in chronically infected monocytes/macrophages, in which CDK9 and cyclin T1 were undetectable. These results suggest that JTK-101 exerts its anti-HIV-1 activity through the inhibition of known or unknown Tat cofactors, presumably CDK9/cyclin T1.

Establishment of an antiviral assay system and identification of severe fever with thrombocytopenia syndrome virus inhibitors.

Aims Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease. SFTS is epidemic in Asia, and its fatality rate is around 30% in Japan. The causative virus severe fever with thrombocytopenia syndrome virus (SFTSV) is a phlebovirus of the family Phenuiviridae (the order Bunyavirales). Although effective treatments are required, there are no antiviral agents currently approved for clinical use. Ribavirin and favipiravir were examined for their anti-SFTSV activity and found to be selective inhibitors of SFTSV replication in vitro. However, their activity was not sufficient. Therefore, it is mandatory to identify novel compounds active against SFTSV. To this end, we have established a safe and rapid assay system for screening selective inhibitors of SFTSV. Methods The virus was isolated from SFTS patients treated in Kagoshima University Hospital. Vero cells were infected with SFTSV and incubated in the presence of various concentrations of test compounds. After three days, the cells were examined for their intracellular viral RNA levels by real-time reverse transcription-PCR without extracting viral RNA. The cytotoxicity of test compounds was determined by a tetrazolium dye method. Results Among the test compounds, the antimalarial agent amodiaquine was identified as a selective inhibitor of SFTSV replication. Its 50% effective concentration (EC50) and cytotoxic concentration (CC50) were 19.1 ± 5.1 and >100 µM, respectively. The EC50 value of amodiaquine was comparable to those of ribavirin and favipiravir. Conclusion Amodiaquine is considered to be a promising lead of novel anti-SFTSV agents, and evaluating the anti-SFTSV activity of its derivatives is in progress.

SELECTIVE INHIBITION OF HEPATITIS C VIRUS REPLICATION BY ALPHA-ZAM, A NIGELLA SATIVA SEED FORMULATION.

BACKGROUND: Hepatitis C virus (HCV) infection became curable because of the development of direct acting antivirals (DAAs). However, the high cost of DAAs has greatly impeded their potential impact on the treatment of HCV infection. As a result, hepatitis C will continue to cause substantial morbidity, and mortality among chronically infected individuals in low and middle income countries. Thus, urgent need exists for developing cheaper drugs available to hepatitis C patients in these countries. MATERIALS AND METHODS: Alpha-zam, an indigenous herbal formulation from Nigella sativa seed, was examined for its anti-HCV activity and cytotoxicity in genotype 1b HCV replicon cells. The antiviral activity was determined by luciferase expression and viral RNA synthesis, while the cytotoxicity was assessed by viable cell number and glyceraldehyde-3-phosphate dehydrogenase RNA synthesis in the replicon cells. RESULTS: Alpha-zam was found to be a selective inhibitor of HCV replication. The 50% effective dilution and 50% cytotoxic dilution of Alpha-zam were 761- and < 100-fold, respectively, in the subgenomic replicon cells LucNeo#2. Its selective inhibition of HCV was also confirmed by HCV RNA levels in LucNeo#2 and in the full-genome HCV replicon cells NNC#2 using real-time reverse transcriptase polymerase chain reaction. Furthermore, the anti-HCV activity of Alpha-zam was not due to the induction of interferon. CONCLUSION: Alpha-zam selectively inhibits HCV replication and therefore has potential for a novel antiviral agent against HCV infection.