Maria Seifer

Synthesis and biological evaluation of aryl-phospho-indole as novel HIV-1 non-nucleoside reverse transcriptase inhibitors.

A novel series of 3-aryl-phospho-indole (API) non-nucleoside reverse transcriptase inhibitors of HIV-1 was developed. Chemical variation in the phosphorus linker led to the discovery of 3-phenyl-methyl-phosphinate-2-carboxamide 14, which possessed excellent potency against wild-type HIV-1 as well as viruses bearing K103N and Y181C single mutants in the reverse transcriptase gene. Chiral separation of the enantiomers showed that only R enantiomer retained the activity. The pharmacokinetic, solubility, and metabolic properties of 14 were assessed.

In vitro tumorigenicity of hepatitis B virus DNA and HBx protein

Persistent infection by hepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma. This linkage may be caused by oncogenic HBV gene products. Our initial in vitro studies have revealed that a non-tumorigenic, fetal mouse hepatocyte line (FMH202-1), harboring simian virus 40 large tumor antigen (SV40TAg) as transgene, can be converted into a full-malignant phenotype by transfection with dimeric HBV-DNA Höhne, M., Schaefer, S., Seifer, M., Feitelson, M.A., Paul, D. and Gerlich, W.H. (1990) EMBO J 9, 1137-1145. The oncogenic effect was neither dependent on simultaneous expression of SV40TAg nor on the cell type, since HBV-transfected permanent mouse fibroblasts (LTK-) also displayed enhanced colony formation in soft agar. Transfection of FMH202-1 with the X region of HBV generated clones that also formed colonies in soft agar and were tumorigenic in nude mice. Growth in soft agar and induction of nude mice tumors both depended on high expression of HBx protein. Although HBx expression was stronger in X-transfected than HBV-transfected clones, the former did not grow well in soft agar, and the X-derived tumors developed more slowly. In the tumors, expression of HBx was almost shut off, but again high in the tumor-derived cell lines. Constitutive expression of c-fos was strongly enhanced in the X-transfected cell lines and tumors. Transfection of FMH202-1 with an isogenic HBx-deficient mutant fragment generated several clones, which expressed normal levels of HBx transcripts, but did not grow in soft agar.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenicity of the Gene S and Pre-S Domains in Hepatitis B Virions and HBsAg Filaments

The three morphological forms (20-nm particles, filaments, virions) of hepatitis B surface antigen (HBsAg) were isolated from serum of chronic virus carriers or from transfected cell lines. BALB/c mice and guinea pigs were immunized with the antigens and the antibody responses against the three antigenic domains of the viral envelope were assayed. The proportion of pre-S1, pre-S2 and gene S antibodies was similar to the molar proportion of the domains in the immunogens. The major gene S and pre-S1 epitopes were conformational, and the major epitopes of the pre-S2 domain were sequential. The immunogenicity of natural and recombinant antigens was identical. The proportion of subtype-specific antibodies was high. The results suggest that recombinant HBsAg filaments containing both subtypes ad and ay may be optimal hepatitis B vaccines.

Telbivudine, a nucleoside analog inhibitor of HBV polymerase, has a different in vitro cross-resistance profile than the nucleotide analog inhibitors adefovir and tenofovir

Telbivudine, a nucleoside analog inhibitor of the viral polymerase of hepatitis B virus (HBV), has been approved for the treatment of chronic HBV infection, along with the nucleoside inhibitors lamivudine and entecavir, and the nucleotide inhibitors adefovir and tenofovir. The resistance profiles of these agents were investigated via drug treatment of HepG2 cells stably transfected with wild-type or mutant HBV genomes bearing known resistance mutations. Telbivudine was not active against HBV strains bearing lamivudine mutations L180M/M204V/I but remained active against the M204V single mutant in vitro, potentially explaining the difference in resistance profiles between telbivudine and lamivudine. Against HBV genomes with known telbivudine-resistance mutations, M204I and L80I/M204I, telbivudine, lamivudine and entecavir lost 353- to >1000-fold activity whereas adefovir and tenofovir exhibited no more than 3-5-fold change. Conversely, against HBV cell lines expressing adefovir resistance mutations N236T and A181V, or the A194T mutant associated with resistance to tenofovir, telbivudine remained active as shown by respective fold-changes of 0.5 (N236T) and 1.0 (A181V and A194T). These in vitro results indicate that nucleoside and nucleotide drugs have different cross-resistance profiles. The addition of telbivudine to ongoing adefovir therapy could provide effective antiviral therapy to patients who develop adefovir resistance.

Assembly and antigenicity of hepatitis B virus core particles.

Recent studies in Xenopus oocytes and other systems have led to an understanding of the HBV capsid, or core particle, assembly process. Nascent HBV core polypeptides rapidly dimerize. Accumulation of free dimers to a signature concentration (approximately 0.8 microM) then triggers a highly cooperative capsid assembly reaction. This dimer-to-capsid transition is accompanied by a switch from HBe to HBc antigenicity and appears to be nucleated by interaction between core protein and RNA: deletion of a protamine-like RNA binding domain at the C-terminus of the core protein markedly increases the concentration of dimers needed to drive capsid assembly. The simple assembly pathway seen for HBV capsids mirrors that of R17 bacteriophage.

2'-C-Methyl branched pyrimidine ribonucleoside analogues : potent inhibitors of RNA virus replication

RNA viruses are the agents of numerous widespread and often severe diseases. Their unique RNA-dependent RNA polymerase (RDRP) is essential for replication and, thus, constitutes a valid target for the development of selective chemotherapeutic agents. In this regard, we have investigated sugar-modified ribonucleoside analogues as potential inhibitors of the RDRP. Title compounds retain ‘natural’ pyrimidine bases, but possess a β-methyl substituent at the 2′-position of the D- or L-ribose moiety. Evaluation against a broad range of RNA viruses, either single-stranded positive (ssRNA), single-stranded negative (ssRNA−) or double-stranded (dsRNA), revealed potent activities for D-2′-C-methyl-cytidine and -uridine against ssRNA+, and dsRNA viruses. None of the L-enantiomers were active. Moreover, the 5′-triphosphates of the active D-enantiomers were found to inhibit the bovine virus diarrhoea virus polymerase. Thus, the 2′-methyl branching of natural pyrimidine ribonucleosides transforms physiological molecules into potent, broad-spectrum antiviral agents that merit further development.

Replication of hepatitis B virus in transfected nonhepatic cells

Permanent murine fibroblasts (LTK-) were transfected with a dimer of hepatitis B virus (HBV) DNA and a neomycin resistance gene which were both linked to the simian virus 40 (SV40) early promoter/enhancer. One of the stably transfected clones, LTK4/36, which secreted HBsAg, HBeAg, and HBV DNA was further analyzed. It contained eight to nine copies of integrated HBV DNA per haploid genome and low amounts of episomal HBV DNA. The secreted viral DNA was covalently linked to protein and was associated with particles which had the characteristic density of natural virions from serum of human viremic carriers. The particles contained an endogenous DNA polymerase, small and middle surface proteins, but in contrast to natural virions very little core protein and large surface protein. Instead of core protein, they contained incompletely processed HBe protein which is colinear to core protein. The fibroblast-derived virions were less stable than virions from human carriers or from transfected hepatoma cells. After several days of storage, their DNA was only partially protected against DNase. Obviously, nonhepatic cells can express HBV-like particles, even if liver-dependent gene products like large surface protein and core protein are missing.

In vitro Activity and Resistance Profile of Samatasvir, a Novel NS5A Replication Inhibitor of Hepatitis C Virus

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is a clinically validated target for drugs designed to treat chronic HCV infection. This study evaluated the in vitro activity, selectivity, and resistance profile of a novel anti-HCV compound, samatasvir (IDX719), alone and in combination with other antiviral agents. Samatasvir was effective and selective against infectious HCV and replicons, with 50% effective concentrations (EC50s) falling within a tight range of 2 to 24 pM in genotype 1 through 5 replicons and with a 10-fold EC50 shift in the presence of 40% human serum in the genotype 1b replicon. The EC90/EC50 ratio was low (2.6). A 50% cytotoxic concentration (CC50) of >100 μM provided a selectivity index of >5 × 107. Resistance selection experiments (with genotype 1a replicons) and testing against replicons bearing site-directed mutations (with genotype 1a and 1b replicons) identified NS5A amino acids 28, 30, 31, 32, and 93 as potential resistance loci, suggesting that samatasvir affects NS5A function. Samatasvir demonstrated an overall additive effect when combined with interferon alfa (IFN-α), ribavirin, representative HCV protease, and nonnucleoside polymerase inhibitors or the nucleotide prodrug IDX184. Samatasvir retained full activity in the presence of HIV and hepatitis B virus (HBV) antivirals and was not cross-resistant with HCV protease, nucleotide, and nonnucleoside polymerase inhibitor classes. Thus, samatasvir is a selective low-picomolar inhibitor of HCV replication in vitro and is a promising candidate for future combination therapies with other direct-acting antiviral drugs in HCV-infected patients.

Cell type specific expression of pre S 1 antigen and secretion of hepatitis B virus surface antigen

SummaryProduction of the three hepatitis B surface (HBs) proteins was studied in a hepatoma cell line (PLC/PRF/5) and two HBs antigen secreting cell lines (HeLa and mouse L-cells), which had been transfected by a viral genome isolated by molecular cloning from PLC/PRF/5 chromosomal DNA. The DNA used for transfection contains the HBs-specific promoters and the enhancer which regulate the expression of HBs genes in the transfected cell lines. All three cell lines expressed well the small and middle HBs protein, but the larger pre S 1 containing protein was barely detectable in the L-cell. In vivo growth of the transfected HeLa cell as nude mouse tumour increased pre S 1 expression and suppressed secretion of HBsAg.

91 ANTIVIRAL ACTIVITY OF THE LIVER-TARGETED NUCLEOTIDE HCV POLYMERASE INHIBITOR IDX184 CORRELATES WITH TROUGH SERUM LEVELS OF THE NUCLEOSIDE METABOLITE IN HCV-INFECTED CHIMPANZEES

Background: ANA598 is a novel HCV non-nucleoside polymerase inhibitor currently in clinical development for the treatment of hepatitis C. Due to the high potential for developing resistance to any single direct antiviral used as monotherapy in hepatitis C, future therapies for HCV are expected to involve combinations of direct antivirals to increase antiviral potency and suppress viral resistance. The results from in vitro combination studies provide support for future clinical exploration of combination regimens that include ANA598. Methods: Combination studies were conducted using wt Huh7-Luc or M414T-containing 1b dicistronic replicons. Cells were cultured in the presence of compounds for 72 hours using both fixed ratios and checkerboard concentration matrices of test agents. Cultured media from PBMCs treated with the active metabolite of ANA773 were used to evaluate the combination effects of this agent with ANA598. The observed inhibitory activity of two agents in combination was compared to the combined action predicted from the dose responses for individual agents assuming Loewe additivity or Bliss independence. Results using CalcuSyn and internally developed analytical procedures were compared. Results: ANA598 was combined pairwise in vitro with IFN-a, the HCV NS3/4 protease inhibitor Telaprevir, the NS5B nucleoside polymerase inhibitor PSI-6130, or the TLR7 agonist ANA773 (currently in clinical development by Anadys). We have previously demonstrated that there is no overlap in viral mutations conferring resistance to these agents in vitro. Combinations were evaluated in both wt and mutant replicons containing the M414T mutation that confers resistance to palm site non-nucleoside NS5B inhibitors. No cytotoxicity was detected for any of the combinations tested. For each combination evaluated, the antiviral interaction between the compounds was determined to be additive to synergistic. Conclusions: The in vitro combination studies demonstrate that additive to synergistic antiviral effects are observed when ANA598 is combined with other anti-HCV agents having distinct mechanisms of action and nonoverlapping resistance profiles. Such combinations may produce a greater viral load reduction and potentially delay the emergence of drug resistance in vivo. Consequently, appropriately constructed combination regimens may provide clinical benefit by improving response rates and allowing non-responders to current therapies to be treated more effectively.