Eric Ferrari

Bio: Eric Ferrari is an academic researcher from Merck & Co.. The author has contributed to research in topics: NS5B & RNA polymerase. The author has an hindex of 17, co-authored 25 publications receiving 2269 citations. Previous affiliations of Eric Ferrari include Valeant Pharmaceuticals & Schering-Plough.

Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site

Abstract: Various classes of nucleotidyl polymerases with different transcriptional roles contain a conserved core structure. Less is known, however, about the distinguishing features of these enzymes, particularly those of the RNA-dependent RNA polymerase class. The 1. 9 A resolution crystal structure of hepatitis C virus (HCV) nonstructural protein 5B (NS5B) presented here provides the first complete and detailed view of an RNA-dependent RNA polymerase. While canonical polymerase features exist in the structure, NS5B adopts a unique shape due to extensive interactions between the fingers and thumb polymerase subdomains that serve to encircle the enzyme active site. Several insertions in the fingers subdomain account for intersubdomain linkages that include two extended loops and a pair of antiparallel alpha-helices. The HCV NS5B apoenzyme structure reported here can accommodate a template:primer duplex without global conformational changes, supporting the hypothesis that this structure is essentially preserved during the reaction pathway. This NS5B template:primer model also allows identification of a new structural motif involved in stabilizing the nascent base pair.

Antitumor Activity of SCH 66336, an Orally Bioavailable Tricyclic Inhibitor of Farnesyl Protein Transferase, in Human Tumor Xenograft Models and Wap-ras Transgenic Mice

Abstract: We have been developing a series of nonpeptidic, small molecule farnesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl protein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey systems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vincristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a therapeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials.

Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

Abstract: Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

De Novo Initiation of RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase

Abstract: RNA-dependent RNA polymerase (RdRp) encoded by positive-strand RNA viruses is critical to the replication of viral RNA genome. Like other positive-strand RNA viruses, replication of hepatitis C virus (HCV) RNA is mediated through a negative-strand intermediate, which is generated through copying the positive-strand genomic RNA. Although it has been demonstrated that HCV NS5B alone can direct RNA replication through a copy-back primer at the 3' end, de novo initiation of RNA synthesis is likely to be the mode of RNA replication in infected cells. In this study, we demonstrate that a recombinant HCV NS5B protein has the ability to initiate de novo RNA synthesis in vitro. The NS5B used HCV 3' X-tail RNA (98 nucleotides) as the template to synthesize an RNA product of monomer size, which can be labeled by ?gamma-(32)Pnucleoside triphosphate. The de novo initiation activity was further confirmed by using small synthetic RNAs ending with dideoxynucleotides at the 3' termini. In addition, HCV NS5B preferred GTP as the initiation nucleotide. The optimal conditions for the de novo initiation activity have been determined. Identification and characterization of the de novo priming or initiation activity by HCV NS5B provides an opportunity to screen for inhibitors that specifically target the initiation step.

Template/primer requirements and single nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase

Abstract: Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3′ terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3′ terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3′ terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique β-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.

Applications of Fluorine in Medicinal Chemistry

Abstract: The role of fluorine in drug design and development is expanding rapidly as we learn more about the unique properties associated with this unusual element and how to deploy it with greater sophistication. The judicious introduction of fluorine into a molecule can productively influence conformation, pKa, intrinsic potency, membrane permeability, metabolic pathways, and pharmacokinetic properties. In addition, 18F has been established as a useful positron emitting isotope for use with in vivo imaging technology that potentially has extensive application in drug discovery and development, often limited only by convenient synthetic accessibility to labeled compounds. The wide ranging applications of fluorine in drug design are providing a strong stimulus for the development of new synthetic methodologies that allow more facile access to a wide range of fluorinated compounds. In this review, we provide an update on the effects of the strategic incorporation of fluorine in drug molecules and applications in po...

Flaviviridae :T he Viruses and Their Replication

Abstract: FLAVIVIRUSES 1103 Background and Classification 1103 Structure and Physical Properties of the Virion 1104 Binding and Entry 1105 Genome Structure 1106 Translation and Proteolytic Processing 1107 Features of the Structural Proteins 1108 Features of the Nonstructural Proteins 1109 RNA Replication 1112 Membrane Reorganization and the Compartmentalization of Flavivirus Replication 1112 Assembly and Release of Particles from Flavivirus-infected Cells 1112 Host Resistance to Flavivirus Infection 1113

Antiherpesvirus drugs: a promising spectrum of new drugs and drug targets

Abstract: In the absence of effective vaccines to control herpesvirus infections, nucleosidic antiviral drugs have been the mainstay of clinical treatment since their development in the late 1970s. However, given the drawbacks of these drugs, including the increasing emergence of drug-resistant clinical isolates, new strategies for treating herpesvirus infections are warranted. A range of promising new drugs with novel molecular targets has been developed, but will they cure latent infections?

Replication of hepatitis C virus

Abstract: Hepatitis C virus (HCV) afflicts more than 170 million people worldwide causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The recent development of complete cell-culture systems for HCV has accelerated the pace of hepatitis research. Specifically, these techniques have provided new insights into the virus lifecycle that are reviewed here. This should pave the way for developing bespoke and effective antiviral therapies and vaccines. Exciting progress has recently been made in understanding the replication of hepatitis C virus, a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. The development of complete cell-culture systems should now enable the systematic dissection of the entire viral lifecycle, providing insights into the hitherto difficult-to-study early and late steps. These efforts have already translated into the identification of novel antiviral targets and the development of new therapeutic strategies, some of which are currently undergoing clinical evaluation.

Fluorine and Fluorinated Motifs in the Design and Application of Bioisosteres for Drug Design.

Abstract: The electronic properties and relatively small size of fluorine endow it with considerable versatility as a bioisostere and it has found application as a substitute for lone pairs of electrons, the hydrogen atom, and the methyl group while also acting as a functional mimetic of the carbonyl, carbinol, and nitrile moieties. In this context, fluorine substitution can influence the potency, conformation, metabolism, membrane permeability, and P-gp recognition of a molecule and temper inhibition of the hERG channel by basic amines. However, as a consequence of the unique properties of fluorine, it features prominently in the design of higher order structural metaphors that are more esoteric in their conception and which reflect a more sophisticated molecular construction that broadens biological mimesis. In this Perspective, applications of fluorine in the construction of bioisosteric elements designed to enhance the in vitro and in vivo properties of a molecule are summarized.