The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTPbinding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase.Hepatitis C virus (HCV) is the causative agent of the majority of chronic liver disease throughout the world. More than 170 million individuals are estimated to be infected with this virus (27). The size of the HCV epidemic and the limited efficacy of current therapy (based on the use of alpha interferon) have stimulated intense research efforts towards the development of antiviral drugs that are both better tolerated and more effective. The most widely established strategy for developing novel anti-HCV therapeutics aims at the identification of low-molecular-weight inhibitors of essential HCV enzymes.RNA-dependent RNA polymerase (RdRP) activity, carried out by the NS5B protein, is essential for virus replication (13) and has no functional equivalent in uninfected mammalian cells. It is thus likely that specific inhibitors of this enzyme can be found that block HCV replication with negligible associated toxicity. The NS5B RdRP has been expressed in a variety of recombinant forms (2, 4). The production of highly soluble forms of the enzyme (12, 24), devoid of the C-terminal membrane anchoring domain (23), has allowed considerable progress toward the determination of the enzyme's three-dimensional structure and mechanism of action. The crystal structure of NS5B revealed a classical "right hand" shape, showing the characteristic fingers, palm, and thumb subdomains (1,7,14). More recently, the three-dimensional structure of the HCV polymerase was solved in complex with RNA (20) as well as in a complex with nucleoside triphosphates (6). Three distinct nucleotide-binding sites were observed in the catalytic center of HCV RdRP whose geometry was remarkably similar to that observed in the initiation complex of the RNA phage ⌽6 RdRP (8), strengthening the proposal that the two enzymes initiate replication de novo by similar ...

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.Hepatitis C virus (HCV) chronically infects about 3% of the human population, causing a slowly evolving liver disease that leads to cirrhosis, liver failure, and occasionally hepatocellular carcinoma (39). Given the size of the HCV epidemic and the limited efficacy of the present therapy based on alpha interferon (16), the development of new, safer, and more effective drugs is of paramount importance and is presently an area of intensive research. The strategy most widely applied for developing novel anti-HCV therapeutics aims at identifying small molecule inhibitors of viral enzymes. The nonstructural protein 5B RNA-dependent RNA polymerase (NS5B RdRp) is an important target of drug discovery activities largely because it is essential for viral replication and also due to the clinical successes of inhibitors of other viral polymerases. In addition, the extensive structural and biochemical characterization of this enzyme provides the basis for drug design efforts as well as for elucidating the mechanism of action of inhibitors and for rapidly optimizing their potency.The NS5B protein was initially identified as an RdRp based on the presence of the signature GDD (Gly-Asp-Asp) motif characteristic for this class of enzymes (11). Its function was confirmed when an active form of the full-length protein was purified from baculovirus-infected insect cells (5). Subsequently, attempts to improve solubility, stability, and activity lead to the expression of C-terminal-truncated forms lacking the hydrophobic membrane anchor contained within the last 21 amino acids (1,17,25,33,35). In vitro, the enzyme shows little, if any, specificity for the HCV genome and can catalyze the synthesis of RNA by using a variety of homo-or heteropolymeric RNA templates both with and without a primer. In the absence of primer, NS5B can initiate RNA synthesis either by using the 3Ј-termi...

Huntington’s disease (HD) is a monogenetic neurodegenerative disorder that is caused by the expansion of a polyglutamine region within the huntingtin (HTT) protein, but there is still an incomplete understanding of the molecular mechanisms that drive pathology. Expression of the mutant form of HTT is a key aspect of diseased tissues, and the most promising therapeutic approaches aim to lower expanded HTT levels. Consequently, the investigation of HTT expression in time and in multiple tissues, with assays that accurately quantify expanded and non-expanded HTT, are required to delineate HTT homeostasis and to best design and interpret pharmacodynamic readouts for HTT lowering therapeutics. Here we evaluate mutant polyglutamine-expanded (mHTT) and polyglutamine-independent HTT specific immunoassays for validation in human HD and control fibroblasts and use to elucidate the CSF/brain and peripheral tissue expression of HTT in preclinical HD models.

Pathological conditions caused by reduced dosage of a gene, such as gene haploinsufficiency, can potentially be reverted by enhancing the expression of the functional allele. In practice, low specificity of therapeutic agents, or their toxicity reduces their clinical applicability. Here, we have used a high throughput screening (HTS) approach to identify molecules capable of increasing the expression of the gene Tbx1, which is involved in one of the most common gene haploinsufficiency syndromes, the 22q11.2 deletion syndrome. Surprisingly, we found that one of the two compounds identified by the HTS is the vitamin B12. Validation in a mouse model demonstrated that vitamin B12 treatment enhances Tbx1 gene expression and partially rescues the haploinsufficiency phenotype. These results lay the basis for preclinical and clinical studies to establish the effectiveness of this drug in the human syndrome.

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There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of β-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and β-globin genes by FACS with efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the β-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from β-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of βthalassemia and sickle cell anemia.

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Blocking Plasmodium falciparum human-to-mosquito transmission is essential for malaria elimination, nonetheless drugs killing the pathogenic asexual stages are generally inactive on the parasite transmissible stages, the gametocytes. Due to technical and biological limitations in high throughput screening of non-proliferative stages, the search for gametocyte-killing molecules so far tested one tenth the number of compounds screened on asexual stages. Here we overcome these limitations and rapidly screened around 120,000 compounds, using not purified, bioluminescent mature gametocytes. Orthogonal gametocyte assays, selectivity assays on human cells and asexual parasites, followed by compound clustering, brought to the identification of 84 hits, half of which are gametocyte selective and half with comparable activity against sexual and asexual parasites. We validated seven chemotypes, three of which are, to the best of our knowledge, novel. These molecules are able to inhibit male gametocyte exflagellation and block parasite transmission through the Anopheles mosquito vector in a standard membrane feeding assay. This work shows that interrogating a wide and diverse chemical space, with a streamlined gametocyte HTS and hit validation funnel, holds promise for the identification of dual stage and gametocyte-selective compounds to be developed into new generation of transmission blocking drugs for malaria elimination.