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Filibuvir (PF-00868554) is an investigational nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural 5B (NS5B) RNA-dependent RNA polymerase currently in development for treating chronic HCV infection. The aim of this study was to characterize the selection of filibuvir-resistant variants in HCV-infected individuals receiving filibuvir as short (3- to 10-day) monotherapy. We identified amino acid M423 as the primary site of mutation arising upon filibuvir dosing. Through bulk cloning of clinical NS5B sequences into a transient-replicon system, and supported by site-directed mutagenesis of the Con1 replicon, we confirmed that mutations M423I/T/V mediate phenotypic resistance. Selection in patients of an NS5B mutation at M423 was associated with a reduced replicative capacity in vitro relative to the pretherapy sequence; consistent with this, reversion to wild-type M423 was observed in the majority of patients following therapy cessation. Mutations at NS5B residues R422 and M426 were detected in a small number of patients at baseline or the end of therapy and also mediate reductions in filibuvir susceptibility, suggesting these are rare but clinically relevant alternative resistance pathways. Amino acid variants at position M423 in HCV NS5B polymerase are the preferred pathway for selection of viral resistance to filibuvir in vivo .

More effective and better-tolerated therapies are needed for chronic hepatitis C virus (HCV) infection. Among the direct-acting anti-HCV agents in development is the nonstructural 5B protein (NS5B polymerase) non-nucleoside inhibitor filibuvir. We investigated the antiviral activity, pharmacokinetics, safety, and tolerability of multiple doses of filibuvir in treatment-naive and treatment-experienced patients who were chronically infected with HCV genotype 1 in two phase 1b clinical studies (study 1 was a randomized, placebo-controlled dose escalation study and study 2 was a nonrandomized, open-label study). The filibuvir doses evaluated ranged from 200-1400 mg daily, and the duration of dosing ranged from 3-10 days. Genotypic changes in the NS5B nucleotide sequence following short-term filibuvir therapy were also assessed. Filibuvir potently inhibited viral replication in a dose-dependent manner. Mean maximum HCV RNA change from baseline ranged from −0.97 log10 IU/mL with filibuvir given at 100 mg twice daily to −2.30 log10 IU/mL with filibuvir given at 700 mg twice daily in treatment-naive patients. In treatment-experienced patients, an HCV RNA reduction of 2.20 log10 IU/mL was achieved with filibuvir given at 450 mg twice daily. Filibuvir was well tolerated in both studies. Adverse events were mild or moderate in severity. No discontinuations, serious adverse events, or deaths were reported. NS5B sequencing identified residue 423 as the predominant site of mutation after filibuvir dosing. Conclusion: Filibuvir administration resulted in significant reductions in HCV RNA concentrations at doses that were well tolerated in patients infected with HCV genotype 1. Filibuvir is currently being evaluated in combination with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients. (Hepatology 2011;)

MOZ-TIF2 and MOZ-CBP are leukemogenic fusion proteins associated with therapy-induced acute myeloid leukemia. These proteins are thought to subvert normal gene expression in differentiating hematopoietic progenitor cells. We have previously shown that MOZ-TIF2 inhibits transcription by CREB-binding protein (CBP)/p300-dependent activators such as nuclear receptors and p53. Here we have shown that MOZ-TIF2 associates with the RARβ2 promoter in vivo, resulting in altered recruitment of CBP/p300, aberrant histone modification, and down-regulation of the RARβ2 gene. In contrast, MOZ-TIF2 up-regulated transcription mediated by the MOZ/MYST3-dependent activator AML1/RUNX1. Both wild type MOZ and MOZ-TIF2 were found to colocalize with AML1, and MOZ-TIF2 was recruited to an AML1 target promoter. A MOZ-CBP fusion protein showed similar functions to MOZ-TIF2 in that it inhibited retinoic acid receptor-mediated transcription but enhanced AML1 reporter activation. Although it contains almost the entire CBP sequence, MOZ-CBP does not appear to associate with PML bodies. In summary, our results indicate that leukemogenic MOZ fusion proteins have differential effects on the activities of CBP-dependent and MOZ-dependent activators because of their ability to alter cofactor recruitment and chromatin modification at target promoters.

MOZ (monocytic leukaemia zinc finger protein; also known as ZNF220 or MYST3) is a member of the MYST family of protein acetyltransferases. Chromosomal translocations involving the MOZ gene are associated with AML (acute myeloid leukaemia), suggesting that it has a role in haematopoiesis. Recurrent reciprocal translocations fuse the MOZ gene [or the gene encoding MORF (MOZ-related factor); also known as MYST4] to genes encoding the nuclear receptor co-activators CBP [CREB (cAMP response element-binding protein)-binding protein], p300 or the p160 protein TIF2 (transcription intermediary factor 2). The resulting fusion proteins can transform haematopoietic progenitors in vitro, and induce myeloproliferative disease in mice. Recent insights into the molecular mechanisms underlying these effects indicate that MOZ fusion proteins interfere with the activities of transcription factors such as nuclear receptors, p53 and Runx proteins. Our studies suggest that subverting the function of cellular CBP and p300 proteins may play a key role in this process. Here we review the recent progress in understanding the role of MOZ fusion proteins in the aetiology of AML.

Chromosomal rearrangements associated with acute myeloid leukemia (AML) include fusions of the genes encoding the acetyltransferase MOZ or MORF with genes encoding the nuclear receptor coactivator TIF2, p300, or CBP. Here we show that MOZ-TIF2 acts as a dominant inhibitor of the transcriptional activities of CBP-dependent activators such as nuclear receptors and p53. The dominant negative property of MOZ-TIF2 requires the CBP-binding domain (activation domain 1 [AD1]), and coimmunoprecipitation and fluorescent resonance energy transfer experiments show that MOZ-TIF2 interacts with CBP directly in vivo. The CBP-binding domain is also required for the ability of MOZ-TIF2 to extend the proliferative potential of murine bone marrow lineage-negative cells in vitro. We show that MOZ-TIF2 displays an aberrant nuclear distribution and that cells expressing this protein have reduced levels of cellular CBP, leading to depletion of CBP from PML bodies. In summary, our results indicate that disruption of the normal function of CBP and CBP-dependent activators is an important feature of MOZ-TIF2 action in AML.

CREB-binding protein (CBP) and p300 contain modular domains that mediate protein-protein interactions with a wide variety of nuclear factors. A C-terminal domain of CBP (referred to as the SID) is responsible for interaction with the alpha-helical AD1 domain of p160 coactivators such as the steroid receptor coactivator (SRC1), and also other transcriptional regulators such as E1A, Ets-2, IRF3, and p53. Here we show that the pointed (PNT) domain of Ets-2 mediates its interaction with the CBP SID, and describe the effects of mutations in the SID on binding of Ets-2, E1A, and SRC1. In vitro binding studies indicate that SRC1, Ets-2 and E1A display mutually exclusive binding to the CBP SID. Consistent with this, we observed negative cross-talk between ERalpha/SRC1, Ets-2, and E1A proteins in reporter assays in transiently transfected cells. Transcriptional inhibition of Ets-2 or GAL4-AD1 activity by E1A was rescued by co-transfection with a CBP expression plasmid, consistent with the hypothesis that the observed inhibition was due to competition for CBP in vivo. Sequence comparisons revealed that SID-binding proteins contain a leucine-rich motif similar to the alpha-helix Aalpha1 of the SRC1 AD1 domain. Deletion mutants of E1A and Ets-2 lacking the conserved motif were unable to bind the CBP SID. Moreover, a peptide corresponding to this sequence competed the binding of full-length SRC1, Ets-2, and E1A proteins to the CBP SID. Thus, a leucine-rich amphipathic alpha-helix mediates mutually exclusive interactions of functionally diverse nuclear proteins with CBP.

We report a case of infection with Candida glabrata in which the organism became resistant to fluconazole and in which pre- and posttreatment isolates were available for comparison. The organism was cross-resistant to ketoconazole and itraconazole, in common with other azole-resistant yeasts. Fluconazole was a potent inhibitor of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) in lysates of cells from both susceptible and resistant cultures (50% inhibitory concentration, 0.2 microM), indicating that resistance was unrelated to changes in P-450DM. Instead, it appeared to arise from a permeability barrier to fluconazole, since resistant cells were unable to take up radiolabelled drug.