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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.

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.

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.

Background The novel phenanthridinone derivative HA-719 has recently been identified as a highly potent and selective inhibitor of hepatitis C virus replication. To elucidate its mechanism of inhibition, we have isolated and analyzed a clone of hepatitis C virus replicon cells resistant to HA-719. Methods To isolate HA-719-resistant replicon cells, Huh-7 cells containing subgenomic hepatitis C virus replicons (genotype 1b) with a luciferase reporter (LucNeo#2) were cultured in the presence of G418 and escalating concentrations of HA-719. After several passages, total RNA was extracted from the growing cells, and Huh-7 cells were transfected with the extracted RNA. Limiting dilution of the transfected cells was performed to obtain an HA-719-resistant clone. Results The 50% effective concentration (EC50) of HA-719 for hepatitis C virus replication was 0.058 ± 0.012 µM in LucNeo#2 cells. The replicon cells capable of growing in the presence of G418 and 3 µM HA-719 were obtained after 18 passages (72 days). The HA-719-resistant clone LucNeo719R showed 98.3-fold resistant to the compound (EC50 = 5.66 ± 0.92 µM), but the clone had no cross-resistance to telaprevir (NS3 inhibitor), daclatasvir (NS5A inhibitor), and VX-222 (NS5B inhibitor). The sequence analysis for the wild-type and LucNeo719R identified 3, 2 and 7 mutations in NS3/4 A, NS4B, and NS5A, respectively, but no mutations in NS5B. Conclusion None of the amino acid mutations in the resistant clone corresponds to those reported to confer drug-resistance to current anti-hepatitis C virus agents, suggesting that the target of HA-719 for hepatitis C virus inhibition differs from those of the existing agents.

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.

Background: A number of compounds were examined for their inhibitory effects on bovine viral diarrhoea virus (BVDV), a surrogate model of hepatitis C virus, in cell cultures. Among them, some diphenylmethane derivatives were found to be selective inhibitors of BVDV. Methods: Determination of compounds for their anti-BVDV activity was based on the inhibition of virus-induced cytopathic effect in Madin–Darby bovine kidney cells and reduction of infectious virus particles in culture supernatants. To gain insight into the mechanism of action, the inhibition of viral entry and RNA synthesis in the host cells was also determined by real-time reverse transcription-PCR. Results: Among the test compounds, four diphenylmethane derivatives significantly inhibited BVDV replication with a 50% effective concentration ranging between 6.3 and 10.8 μM. They were not cytotoxic at concentrations up to 100 μM. The representative compound, SH-595A, reduced the virus titre of culture supernatants in a dose-dependent manner. In addition, the compound appeared to somewhat affect viral entry to the host cells. Although SH-595A was inhibitory to viral RNA synthesis, the inhibition was achieved only at high concentrations and was not comparable to its antiviral activity. Conclusions: The novel diphenylmethane derivatives are effective against BVDV replication and might have a unique mechanism of action.

Background: A number of compounds were examined for their inhibitory effect on bovine viral diarrhoea virus (BVDV) replication in cell cultures and found that some cyclooxygenase (COX) inhibitors had antiviral activity against the virus. Methods: Determination of compounds for their anti-BVDV activity was on the basis of the inhibition of virus-induced cytopathogenicity in Mardin–Darby bovine kidney (MDBK) cells. Anti-hepatitis C virus (HCV) activity was assessed by the inhibition of viral RNA synthesis in the subgenomic HCV RNA replicon cells. Results: Among the test compounds, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1 H-pyrazole (SC-560) was the most active against BVDV, and its 50% effective and cytotoxic concentrations were 10.9 ±2.8 and 93.9 ±24.5 μM in virus and mock-infected MDBK cells, respectively. The compound also suppressed BVDV RNA synthesis in a dose-dependent fashion. Studies on the mechanism of action revealed that SC-560 did not interfere with viral entry to the host cells. Furthermore, it was assumed that the antiviral activity of SC-560 was not associated with its inhibitory effect on COX. The combination of SC-560 and interferon-α was additive to synergistic in inhibiting BVDV replication. More importantly, the compound proved to be a selective inhibitor of HCV replication. Conclusions: SC-560 and its derivative might have potential as novel antiviral agents against HCV.

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.

TAK-652, a novel small-molecule chemokine receptor antagonist, is a highly potent and selective inhibitor of CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) replication in vitro. Since TAK-652 is orally bioavailable and has favorable pharmacokinetic profiles in humans, it is considered a promising candidate for an entry inhibitor of HIV-1. To investigate the resistance to TAK-652, peripheral blood mononuclear cells were infected with the R5 HIV-1 primary isolate KK and passaged in the presence of escalating concentrations of the compound for more than 1 year. After 67 weeks of cultivation, the escape virus emerged even in the presence of a high concentration of TAK-652. This virus displayed more than 200,000-fold resistance to TAK-652 compared with the wild type. The escape virus appeared to have cross-resistance to the structurally related compound TAK-779 but retained full susceptibility to TAK-220, which is from a different class of CCR5 antagonists. Furthermore, the escape virus was unable to use CXCR4 as a coreceptor. Analysis for Env amino acid sequences of escape viruses at certain points of passage revealed that amino acid changes accumulated with an increasing number of passages. Several amino acid changes not only in the V3 region but also in other Env regions seemed to be required for R5 HIV-1 to acquire complete resistance to TAK-652.

Background A new series of 1-aromatic methyl-substituted 3-(3,5-dimethylbenzyl)uracil and N-3,5-dimethylbenzyl-substituted urea derivatives were synthesized and evaluated as non-nucleoside HIV-1 reverse transcriptase inhibitors. Methods A series of new 6-azido and 6-amino derivatives of 1-substituted-3-(3,5-dimethylbenzyl)uracils were synthesized using our previously reported method, and three acyclic derivatives were synthesized from urea. The anti-HIV-1 activities of these compounds were determined based on the inhibition of virus-induced cytopathogenicity in MT-4 cells. The cytotoxicities of the compounds were evaluated using the viability of mock-infected cells. Results Some of these compounds showed good-to-moderate activities against HIV-1 with half maximal effective concentration (EC50) values in the submicromolar or subnanomolar range. Compared with emivirine, compound 6-amino-3-(3,5-dimethylbenzyl)-1-(4-aminobenzyl)uracil showed significant anti-HIV-1 activity with an EC50 value of 10 nM and a high selectivity index of 1923. Preliminary structure–activity relationship studies and molecular modeling analyses were carried out to explore the major interactions between HIV-1 reverse transcriptase and the potent inhibitor 6-amino-3-(3,5-dimethylbenzyl)-1-(4-aminobenzyl)uracil; these results may be important for further development of this class of compounds as anti-HIV-1 agents. Conclusion The excellent activity of 6-amino-3-(3,5-dimethylbenzyl)-1-(4-aminobenzyl)uracil (EC50: 0.010 ± 0.006 µM, SI: >1923) may serve as the basis for conducting further investigations on the behavior of this class of compounds against drug-resistant mutants.

Background The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is an attractive target for the development of drugs used in the treatment of HIV-1 infection and acquired immune deficiency syndrome (AIDS). We have continued the search for novel anti-HIV-1 agents using the structure–activity relationships of the successful 1,3-disubstituted and 1,3,6-trisubstituted uracil-type HIV-1 RT inhibitors. Methods A series of new triazine analogs were synthesized using an established method. The anti-HIV-1 activities of these compounds were determined based on the inhibition of virus-induced cytopathogenicity in MT-4 cells. The cytotoxicity of the compounds was evaluated by assessing the viability of mock-infected cells. Results Some of the compounds showed good-to-moderate activities against HIV-1, with half-maximal effective concentrations (EC50) in the submicromolar range. In particular, a dihydro-1-(4-aminobenzyl)triazine analog showed satisfactory anti-HIV-1 activity with an EC50 of 0.110 µM and a selectivity index (SI) of 909. Furthermore, molecular modeling analyses were performed to explore the major interactions between HIV-1 RT and potent inhibitors. These results may be important for further development of this class of compounds as anti-HIV-1 agents. Conclusion The satisfactory anti-HIV-1 activity of triazine analogs may serve as the basis for further investigations of the behavior of this class of compounds against drug-resistant mutants.

EM2487, a Streptomyces-derived substance, has previously been shown to inhibit HIV-1 replication in both acutely and chronically infected cells. In this study, we found that EM2487 was also a selective inhibitor of human T-lymphotropic virus type I (HTLV-I) replication in persistently infected cells. Its 50% effective concentrations for HTLV-I p19 antigen production were 3.6 and 1.2 μM in MT-2 and MT-4 cells, respectively. However, the compound did not reduce cell proliferation and viability at these concentrations. The 50% cytotoxic concentrations of EM2487 were 30.6 and 5.7 μM in MT-2 and MT-4 cells, respectively. The compound also displayed selective inhibition of HTLV-I production in peripheral blood mononuclear cells obtained from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis. Quantitative reverse transcription PCR analysis revealed that EM2487 selectively suppressed HTLV-I mRNA synthesis in MT-2 cells in a dose-dependent fashion. However, the compound did not inhibit endogenous Tax-induced HTLV-I long terminal repeat-driven reporter gene expression. Furthermore, intracellular Tax accumulation was not suppressed in MT-2 cells exposed to EM2487. These results suggest that the inhibition occurred at the viral transcription level, but it cannot be attributed to the inhibition of the Tax function.

Adult T-cell leukemia (ATL) is caused by infection with human T-cell leukemia virus type-1 (HTLV-1). The tetrahydrotetramethylnaphthalene derivative TMNAA has recently been identified as a selective inhibitor of HTLV-1-infected T-cell lines and adult T-cell leukemia (ATL) cells but not of uninfected T-cell lines and peripheral blood mononuclear cells (PBMCs). In the present study, more than 100 derivatives of TMNAA were synthesized and examined for their inhibitory effects on the proliferation of various T-cell lines and PBMCs. Among the compounds, MN417 is a more potent inhibitor of ATL cells than TMNAA. This compound is a novel phenanthridinone derivative with the tetrahydrotetramethylnaphthalene structure. Interestingly, PN-H and MN314-B, which are also phenanthridinone derivatives but do not have the tetrahydrotetramethylnaphthalene structure, could not distinguish between HTLV-1-infected and uninfected T-cell lines in terms of their anti-proliferative activity. These results suggest that the tetrahydrotetramethylnaphthalene structure is required for the selective inhibition of HTLV-1-infected cells.

Cepharanthine (12- O-methyl cepharanoline) is a plant alkaloid and has been shown to inhibit tumour necrosis factor-α- or phorbol 12-myristate 13-acetate-induced HIV-1 replication in the chronically infected promonocytic cell line, U1. Its mechanism of action is considered to be the inhibition of nuclear factor κB, a potent inducer of HIV-1 gene expression. In this study, we have synthesized 96 derivatives of cepharanoline, including cepharanthine, and examined their inhibitory effects on HIV-1 replication in U1 cells. Among the 12- O-alkyl derivatives, cepharanthine proved to be the most active, and the activity decreased as the length of the alkyl chain increased. All of the 12- O-acyl derivatives were totally inactive, while a few 12- O-carbamoyl derivatives displayed modest activity. Since 12- O-ethyl derivatives were found to be as active as cepharanthine against HIV-1 replication, we further synthesized various 12- O-ethyl derivatives of cepharanoline. Among the derivatives, five proved to be more active inhibitors than cepharanthine, and the most active compound was 12- O-ethylpiperazinyl cepharanoline. The 50% effective concentrations of this compound and cepharanthine were 0.0041 and 0.028 μg/ml (0.0060 and 0.046 μM), respectively.

Human immunodeficiency virus type 1 (HIV-1) is unique in that it encodes its own transcriptional activator Tat, which specifically binds to the viral mRNA sequence TAR (transactivation response) element and activates viral transcription at the step of elongation as well as initiation. We recently reported that fluoroquinoline derivatives inhibited HIV-1 replication most likely by blocking viral transcription. In this report, we investigated the mechanism of action of one such compound 7-(3,4-dehydro-4-phenyl-1-piperidinyl)-1,4-dihydro-6-fluoro-1-methyl-8-trifluoromethyl-4-oxoquinoline-3-carboxylic acid (K-37). We demonstrated that K-37 inhibited not only Tat but also other RNA-dependent transactivators. No effect was observed with DNA-dependent transactivators such as p65 (NF-κB) and Gal4VP16. Moreover, K-37 did not inhibit carboxyl-terminal domain (CTD)-kinase activities of CDK-activating kinase (CAK) and positive transcription elongation factor b (P-TEFb), which are known to be involved in Tat-mediated transactivation at the step of transcriptional elongation. It is suggested that RNA-mediated transactivation may involve a common unknown factor to which K-37 directly interacts. Since K-37 did not appear to block DNA-mediated transactivation and thus did not show strong nonspecific cytotoxicity as reported previously, K-37 and its derivative compounds are considered to be feasible candidates for a novel AIDS therapy.

In a search for effective HIV-1 transcription inhibitors, we have evaluated more than 75,000 compounds for their inhibitory effects on Tat-induced human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-driven reporter gene expression and found that EM2487, a novel small-molecule substance produced by a Streptomyces species, is a potent and selective inhibitor of HIV-1 replication in both acutely and chronically infected cells. Its 50% effective concentration for acute HIV-1 infection was 0.27 μM in peripheral blood mononuclear cells (PBMCs), while the 50% cytotoxic concentration for mock-infected PBMCs was 13.3 μM. EM2487 proved inhibitory to a variety of HIV-1 strains and HIV-2 in acutely infected T-cell lines (MOLT-4 and MT-4). The compound could suppress tumor necrosis factor alpha (TNF-α)-induced HIV-1 production in latently infected cells (OM-10.1 and ACH-2) as well as constitutive viral production in chronically infected cells (MOLT-4/III B and U937/III B ) without showing any cytotoxicity. EM2487 did not affect early events of the HIV-1 replication cycle, as determined by proviral DNA synthesis in acutely infected MOLT-4 cells. In contrast, the compound selectively prevented viral mRNA synthesis in OM-10.1 cells, suggesting that HIV-1 inhibition occurs at the transcriptional level. Furthermore, EM2487 did not inhibit TNF-α-induced HIV-1 LTR-driven reporter gene expression but did inhibit that induced by Tat, irrespective of the presence or absence of the nuclear factor κB binding sites in the LTR. These results suggest that the mechanism of action is attributable in part to the inhibition of Tat function.

Interleukin 6 (IL-6) has been reported to upregulate the expression of human immuno-deficiency virus type 1 (HIV-1) in infected monocyte/macrophages. Since IL-6 signal is transduced through the membrane glycoprotein gp130, we investigated the inhibitory effect of anti-gp130 monoclonal antibody (mAb) on IL-6-induced viral expression in monocytic cell lines latently infected with HIV-I. IL-6 significantly induced the expression of HIV-1 in U1 cells, whereas it had no effect in OM-10.1 cells. Flow cytometric analysis revealed that U1 but not OM-10.1 cells expressed gp130 on their surface. The anti-gp130 mAb could inhibit IL-6-induced HIV-1 expression in U1 cells in a dose dependent fashion. These results suggest that blocking the gp130 signal transduction may have therapeutic potential for the treatment of HIV-1 infection.

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.

The tetrahydrotetramethylnaphthalene derivative TMNAA has recently been identified as a selective inhibitor of human T-lymphotropic virus type 1 (HTLV-1)-infected T-cell lines and adult T-cell leukemia (ATL) cells but not of uninfected T-cell lines and peripheral blood mononuclear cells (PBMCs). Although the target molecule of TMNAA is still unknown, it does not inhibit nuclear factor-κB (NF-κB) activity. Therefore, TMNAA was examined for its inhibitory effect on the cell proliferation in combination with the NF-κB inhibitor cepharanthine. Synergism was observed for the combination, in inhibiting the proliferation of HTLV-1-infected T-cell lines. Although TMNAA alone did not induce the apoptosis of HTLV-1-infected T-cell lines, it strongly enhanced their apoptosis induced by cepharanthine. Thus, TMNAA may have potential as a therapeutic agent against ATL either alone or in combination with cepharanthine, which is clinically used as an anti-inflammatory drug in Japan.

Adult T-cell leukemia (ATL) is caused by infection with human T-lymphotropic virus type 1 (HTLV-1). A novel tetramethylnaphthalene derivative, TMNAA, selectively inhibited the proliferation of various HTLV-1-infected cells, including ATL cell lines and peripheral blood mononuclear cells (PBMCs) from ATL patients. In contrast, the proliferation of uninfected cell lines and PBMCs from healthy donors was hardly affected by the compound. Cell-cycle analysis revealed that TMNAA increased the population of the G0/G1 phase and reduced that of the S phase in HTLV-1-infected cells. TMNAA was found to suppress the phosphorylation of retinoblastoma protein and the expression of cyclin-dependent kinase 4 in HTLV-1-infected cells. Furthermore, the inhibition of cell proliferation was partially annihilated by removing the compound. These results indicate that TMNAA exerts selective inhibition of HTLV-1-infected cells through a novel mechanism, presumably modulating cell cycle regulatory proteins associated with the G0/G1 phase.

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)-α-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-α-induced HIV-1 mRNA synthesis in OM-10.1 cells in a dose-dependent fashion. JTK-101 modestly inhibited TNF-α-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.

RNA helicase A (RHA) has two double-stranded (ds) RNA-binding domains (dsRBD1 and dsRBD2). These domains are conserved with the cis-acting transactivation response element (TAR)-binding protein (TRBP) and dsRNA-activated protein kinase (PKR). TRBP and PKR are involved in the regulation of HIV-1 gene expression through their binding to TAR RNA. This study shows that RHA also plays an important role in TAR-mediated HIV-1 gene expression. Wild-type RHA preferably bound to TAR RNA in vitro and in vivo. Overexpression of wild type RHA strongly enhanced viral mRNA synthesis and virion production as well as HIV-1 long terminal repeat-directed reporter (luciferase) gene expression. Substitution of lysine for glutamate at residue 236 in dsRBD2 (RHA(K236E)) reduced its affinity for TAR RNA and impaired HIV-1 transcriptional activity. These results indicate that TAR RNA is a preferred target of RHA dsRBDs and that RHA enhances HIV-1 transcription in vivo in part through the TAR-binding of RHA.

The β-chemokine receptor CCR5 is considered to be an attractive target for inhibition of macrophage-tropic (CCR5-using or R5) HIV-1 replication because individuals having a nonfunctional receptor (a homozygous 32-bp deletion in the CCR5 coding region) are apparently normal but resistant to infection with R5 HIV-1. In this study, we found that TAK-779, a nonpeptide compound with a small molecular weight (Mr531.13), antagonized the binding of RANTES (regulated on activation, normal T cell expressed and secreted) to CCR5-expressing Chinese hamster ovary cells and blocked CCR5-mediated Ca2+signaling at nanomolar concentrations. The inhibition of β-chemokine receptors by TAK-779 appeared to be specific to CCR5 because the compound antagonized CCR2b to a lesser extent but did not affect CCR1, CCR3, or CCR4. Consequently, TAK-779 displayed highly potent and selective inhibition of R5 HIV-1 replication without showing any cytotoxicity to the host cells. The compound inhibited the replication of R5 HIV-1 clinical isolates as well as a laboratory strain at a concentration of 1.6–3.7 nM in peripheral blood mononuclear cells, though it was totally inactive against T-cell line-tropic (CXCR4-using or X4) HIV-1.

8 - Difluoromethoxy - 1 - ethyl - 6 - fluoro - 1,4 - dihydro - 7 - [4 - (2 - methoxyphenyl) - 1 - piperazinyl] - 4 - oxoquinoline - 3 - carboxylic acid (K-12) has recently been identified as a potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) transcription. In this study, we examined several combinations of K-12 and other antiretroviral agents for their inhibitory effects on HIV-1 replication in acutely and chronically infected cell cultures. Combinations of K-12 and a reverse transcriptase (RT) inhibitor, either zidovudine, lamivudine, or nevirapine, synergistically inhibited HIV-1 replication in acutely infected MT-4 cells. The combination of K-12 and the protease inhibitor nelfinavir (NFV) also synergistically inhibited HIV-1, whereas the synergism of this combination was weaker than that of the combinations with the RT inhibitors. K-12 did not enhance the cytotoxicities of RT and protease inhibitors. Synergism of the combinations was also observed in acutely infected peripheral blood mononuclear cells. The combination of K-12 and cepharanthine, a nuclear factor κB inhibitor, synergistically inhibited HIV-1 production in tumor necrosis factor alpha-stimulated U1 cells, a promonocytic cell line chronically infected with the virus. In contrast, additive inhibition was observed for the combination of K-12 and NFV. These results indicate that the combinations of K-12 and clinically available antiretroviral agents may have potential as chemotherapeutic modalities for the treatment of HIV-1 infection.

We have found novel piperazinyloxoquinoline derivatives to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in both acutely and chronically infected cells. 8-Difluoromethoxy-1-ethyl-6-fluoro-1,4-didehydro-7-[4-(2-met hoxyphenyl)-1-piperazinyl]-4-oxoquinoline-3-carboxylic acid (K-12), the most potent congener of the series, completely inhibited HIV-1 replication in acutely infected MOLT-4 cells at a concentration of 0.16 to 0.8 microM without showing any cytotoxicity. The compound completely suppressed tumor necrosis factor alpha (TNF-alpha)-induced HIV-1 expression in latently infected cells (OM-10.1) and constitutive viral production in chronically infected cells (MOLT-4/III(B)) at a concentration of 0.8 microM. K-12 could also inhibit HIV-1 antigen expression in OM-10.1 and MOLT-4/III(B) cells at this concentration. Northern blot analysis revealed that K-12 selectively prevented the accumulation of HIV-1 mRNA in MOLT-4/III(B) and TNF-alpha-treated OM-10.1 cells in a dose-dependent fashion. It was not inhibitory to HIV-1 Tat or the cellular transcription factors NF-kappaB and Sp1, suggesting that the piperazinyloxoquinoline derivatives are a group of HIV-1 transcription inhibitors with a unique mechanism of action.

Background We have previously found that the novel tetramethylnaphthalene derivative TMNAA selectively inhibits the proliferation of HTLV-1-infected T-cell lines but not HTLV-1-uninfected T-cell lines. Although its target molecule is still unknown, TMNAA did not affect NFB activity. Therefore, we further examined the antiproliferative activity of TMNAA against various T-cell lines in combination with cepharanthine (CEP), which is known to inhibit NFB.