Inhibitors of HCV 5 Untranslated Region and Core Gene Ribozymes and Antisense Oligonucleotides. The 5' UTR, which encodes the HCV internal ribosome entry site (IRES), and the core gene encoding the nucleocapsid protein of HCV are highly conserved among HCV isolates, making them attractive targets for ribozyme- and antisense oligonucleotide-based antiviral strategies (301).

Ribozyme Pharmaceuticals (RPI) reported their design and synthesis of hammerhead ribozymes targeting various conserved sites in the 5' untranslated region (UTR). These ribozymes significantly reduced HCV 5' UTR-mediated expression in a 5' UTR-luceferase reporter system, as well as inhibited replication of an HCV-poliovirus chimera (302). Moreover, a nuclease resistant ribozyme, tar-

getting site 195, was selected for pharmacokinetics and tissue distribution studies after intravenous and subcutaneous administration in mice. The results showed that the ribozyme can be taken up and retained in the liver cells (303). RPI has recently completed their clinical trials of a 28-day safety and pharmacokinetic study of Heptazyme (LY 466700) following daily subcutaneous injections. Phase II clinical trials to study the drug's dose-ranging and efficacy makers in chronic HCV patients have been planned (RPI Press Release, September 28,2000).

Wu et al. reported that two hammerhead ribozymes, which were designed to target just upstream of the start codon of the viral transcript and the core, respectively, were capable of suppressing HCV-luciferase reporter gene expression in a cell-free system and in trans-fected Huh7 cells (304). The same two regions were also targeted by DNA analogs of ribozymes (305). The third approach used by the group was to apply antisense oligonucleo-tides directed against a sequence in the 5'UTR IRES region and a region of the UTR overlapping the core protein translational start site of HCV. They reported that the antisense oligo-nucleotides, in the form of asialoglycoprotein-polylysine complexes, could be delivered by receptor-mediated endocytosis and caused specific inhibition of HCV-directed protein synthesis, as monitored by the expression of luciferase activity, in cells (306).

Patients infected with HCV genotype lb have shown the poorest rate in response to interferon therapy. Kay et al. incubated total RNA from HCV l b positive human livers, containing plus and minus strands, with a library of hammerhead ribozymes, and thereby isolated several effective ribozymes directed against a conserved region of the plus and minus strand of the HCV genome (307). The ribozymes were found to reduce or eliminate the respective plus or minus strand HCV RNAs in cultured cells and from primary human hepa-tocytes obtained from infected patients (307). Other studies by Hayashi et al. were targeting the core region of HCV lb for the design of hammerhead ribozymes. They found that the ribozyme, whose cleavage site is located nearest to the initiation codon of the HCV ORF, showed the most efficient cleavage of the tar get RNA. On the other hand, the ribozyme with the cleavage site located farthest from the initiation codon blocked viral translation in a rabbit reticulocyte lysate most efficiently (308).

Hairpin ribozymes targeting HCV 5' UTR and capsid gene regions were reported by Barber et al. (309, 310). Because the 5' UTR contains considerable secondary structures, which could interfere with the cleavage activity of a ribozyme, the authors also prepared facilitator RNAs, trying to help to relax the secondary structure, and therefore, enhance the binding and activity of the ribozyme.

Different domains within the 5' UTR and core region have also been exploited as potential targets for inhibition of HCV translation by antisense oligonucleotides and oligode-oxynucleotides (ODNs).Isis Pharmaceuticals reported two phosphorothioate ODNs, ISIS 6095, which targeted a stem-loop structure within the 5' UTR known to be important for 1RES function (nt 260-2791, and ISIS 6547, which targeted sequences spanning the AUG used for initiation of HCV polyprotein translation (nt 330-3491, effectively inhibited HCV gene expression as monitored in transformed hepatocytes (311). Reduction of RNA levels and the subsequent protein levels by these phosphorothioate ODNs was associated with RNase H cleavage of the RNA strand of the oligonucleotide-RNA duplex. On the other hand, 2'-modified (e.g., 2'-0-methoxyethyl) phosphodiesters oligonucleotides inhibited HCV core protein synthesis with comparable potency to phosphorothioate ODNs by an RNase H-independent mechanism (311,312). In mice infected with an HCV-vaccinia virus recombinant, subcutaneous administration of ISIS 6547 and ISIS 14803 showed specific and dose-dependent inhibition of an HCV-lucif-erase reporter gene expression in the livers (313). (The two 20-base oligomers have the same sequence, but ISIS 14803 has 5-methyl-cytidine residues at all respective cytidine positions in ISIS 6547.) In March 2000, the company initiated clinical trials with ISIS 14803 in patients who failed interferon or interferon-plus-ribavirin therapy (314). In addition, investigators of Isis Pharmaceuticals reported a new probing strategy by using hybridization affinity screening and RNase H cleavage anal-

lysis on a fully randomized sequence DNA oligonucleotides (10-mer) library to identify energetically preferred hybridization sites on folded target RNA (315). The hypothesis is that binding-optimized shorter oligomers (10-15-ers) may have equivalent or greater affinity and specificity for hybridization site than longer ones (e.g., 20-mers).

Caselmann et al. reported that a phospho-rolhioale ODNs complementary to nucleotides 326-348 spanning the 3' end of the UTR and the start codon of the polyprotein precursor was very efficient in inhibiting HCV gene expression (316, 317). Moreover, strong inhibition of HCV gene expression was still remained with modified oligomers having ethylphosphonate or benzylphosphonate modifications located at the termini (316, 318). Inhibition correlated with induction of H activity. Furthermore, these oligomers could be coupled with cholesterol or bile acid to enhance their lipophilicity for improved liver specific delivery (319).

An independent investigation conducted by Vidalin et al. identified a domain within 5' UTR, which contains the conserved pyrimi-dine-rich tract (nt 103-138), as a new region susceptible to ODN inhibition (320). They also evaluated a-anomer phosphodiesters ODNs. It was found that a-ODNs inhibited HCV translation as efficiently as their /3-ODNs counterparts. Wands et al. demonstrated that translation of HCV RNAs was efficiently inhibited by antisense RNA when the HCV core-luciferase cDNA was co-transfected with antisense RNA-producing contracts in Huh7 cells (321).

2,4,1.2 inhibitors of HCV internal Ribo-some Entry Site. As a crucial RNA genomic structure required by HCV for initiation of translation, HCV internal ribosome entry site (IRES) has become an attractive target for therapeutic intervention. A small yeast RNA (a 60-nt-long RNA called inhibitor RNA or IRNA), which has previously shown to selectively block internal initiation of translation programmed by poliovirus RNA, has also shown to block HCV IRES-mediated translation in transient transfection of hepatoma cells (Huh-7) and a hepatoma cell line consti-tutively expressing IRNA. In these cells, it was further shown that replication of chimeric po liovirus containing the HCV IRES element was blocked (322). Site-directed mutagenesis studies suggested that the secondary structure of IRNA might be important in its competing with viral IRES structural elements for the binding of cellular proteins required for IRES-mediated translation (323).

Using an in uitro assay in which IRES-dependent translation of luciferase function could be selectively suppressed by the presence of inhibitor, BioChem Pharma and OSI Pharmaceuticals reported the findings of HCV IRES inhibition by phenazine and phenazine-like molecules by screening a compound library and fungal extracts (324). The hit compound (136) is also known as neutral red,

which is a dye used commonly in antiviral assays. The central ring seemed crucial for activity because the open ring analogs exhibited much lower or no activity. Polar substituents at positions 2 and 8 were also important for the inhibitory activity (324). Finally, Eisai Co. has patented several low molecular weight inhibitors, such as compounds (137) and (138) (see references in Refs. 324-326).

2.4.2 HCV NS3 Protein. NS3 is a multifunctional protein in which the N-terminal (ca. 180 amino acids) encodes a serine protease responsible for a distinct temporal hierarchy event of cleavage of NS3/4A, NS4A/4B, NS4B/ 5A, and NS5A/5B junctions, generating four

mature viral non-structural proteins, including NS4A, NS4B, NS5A, and NS5B. The remaining C-terminal two-thirds (ca. 450 amino acids) of the NS3 protein encode a nucleic acid-stimulated nucleoside triphosphatase (NTPase) and a helicase activities (327).

The interaction between the NS3 and its cofactor NS4A is required for the proteolytic activity of NS3. NS4A is a relatively small protein (54 amino acids). Binding of NS4A brings about several conformational changes, resulting in the stabilization of the conformation of the N-terminal domain of the protease and optimization of the alignment of the catalytic triad of His-57, Asp-81, and Ser-139 (327, 328). The structure of the isolated NS3 protease domain either in the presence or in the absence of the cofactor peptide was resolved by X-ray crystallography and by NMR spectroscopy.

A zinc ion is coordinated tetrahedrally by Cys-97, Cys-99, Cys-145, and His-149 at a site located remote from the active site. Interestingly, these residues are conserved in all known HCV genotypes. The zinc ion is believed to be required for structural integrity and activity of the enzyme (329,330).

The NS3 serine protease has been regarded as one of the preferred targets for the development of anti-HCV agents because it presents three potential targets for antiviral design: (1) the enzyme active site, (2)the structural zinc-binding site, and (3) the NS4A binding site (327). However, the interaction between NS3

and NS4A is considered an unlikely target for the development of inhibitors because this region involves a very large surface area and the two components are tightly intercalated (328, 331).

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