Short Capped Oligonucleotides To

initiate influenza viral mRNA synthesis, the viral associated RNA-dependent RNA polymerase binds to the cap structure at the 5' ends of host cell RNA polymerase II transcripts and then a virally encoded endonucle-ase cleaves the capped 5'-termini to provide caps for the 5'-termini of the viral mRNAs and to serve as primers for transcription by the viral RNA-dependent RNA polymerase (RNA transcriptase). The PB2 polymerase protein mediates both the binding and the endonu-cleolytic cleavage of capped mRNAs, Conceptually, the 5'-capped short RNA fragments by design are potential decoys of cap-dependent transcription. Takaku et al. reported the synthesis of short RNA molecules (8-13 ntds long) with a 5'-capped structure (m7GpppGm) using T7 RNA polymerase (141a). These short RNAs were tested, with or without liposomal encapsulation, for their inhibitory effect by a CAT-ELISA assay using the clone 76 cells, showing that the 9-ntd-long RNA molecule (m7GpppGmAAUACUCA) had the highest inhibitory activity. Furthermore, these RNA molecules exhibited higher inhibitory activity than that of the antisense phosphorothioate oligonucleotide complementary to the AUG initiation codon of PB2 mRNA.

Because the influenza virus employs the cap embezzled from the host cell, the virus is not sensitive to the inhibitory effect of S-ad-enosylhomocysteine (SAH) hydrolase inhibitors, which interfere S-adenosylmethionine (SAM)-dependent methylation reactions, leading to inhibition of a broad range of DNA and RNA viruses (142) (see Section 2.9.1).

2.1.3.4 2,4-Dioxobutanoic Acid Derivatives. As discussed above, an intrinsic property of influenza virus RNA-dependent RNA poly-merase (RNA transcriptase) is its cap-dependent endonucleolytic cleavage activity. Through a random screening, Tomassini and a group of Merck investigators have identified L-735882 (43) a 4-substituted 2,4-dioxobutanoic acid, as a specific inhibitor of cap-dependent endonu-

activity of the transcriptase and with antiviral activity against both influenza A and B viruses in cell culture (143). Compound (44), a synthetic analog of (43) with sub-micromo-lar antiviral activity, was found to be the most soluble in water and readily adsorbed into the

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nasopharyngeal track in a mouse challenge model. When instilled intranasally into infected mice, (44), at its highest water-soluble dose, caused a 3.9-log reduction of the virus titers in nasal washes (143).

2.1.3.5 2,6-Diketopiperazine Derivatives. Tomassini et al. also reported the finding of flutimide (45), a natural product isolated from

a fungus, which inhibited the cleavage cf capped RNA by influenza virus endonuclease, with an IC„ of 6.8 pM (144) .The SARanalysis with several synthetic analogs indicated that both the N-hydroxy and olefin groups were re-

quired for activity (145). The most potent analogs were compounds (46) and (47), both having IC50 of —0.9 yM. Flutimide inhibited influenza virus infection of MDCK cells with an EC50 of 5.9 yM without any toxicity at 100 fiM concentrations. Compounds (46) and (47) seemed to be more potent in the antiviral assay; however, they showed cytotoxicity to the cells at >10 yM concentrations (145).

2.1.3.6 BMY-26270 and Analogs. Krystal et al. of Bristol-Myers Squibb identified BMY-26270 (48) through a high-throughput in vitro transcription assay from the company's chemical collection (146). This compound selectively inhibited influenza transcriptases of both A and B viruses with an IC50 ~ 40 julM. When compared with related compounds selected from the chemical collection, it was suggested that the hydroxamic acid and phenol moieties, as well as their topological relationship, are essential for the activity. Two related N-hydroxy-imides, BMY-183355 (49) and BMY-183021 (50) where the relatively acidic amine NH presumably functions as an iso-of the phenolic hydrogen, also demonstrated an IC50 - 50 Notably, these

compounds inhibited endonuclease activity preferentially over capped RNA binding activity. However, they showed significant cytotoxicity in cell culture.

2.1.3.7 MI Zinc Finger Peptides. The matrix protein (Ml) is a major structural component of the influenza virion. Ml can bind to RNA directly and inhibit its own polymerase; it was proposed that this activity might be because of the presence of a zinc finger motif. Judd et al. reported that peptide 6, a synthetic based on the zinc finger region of the Ml protein sequence of influenza A/PR/8/34 (H1N1) centered around residues 148-166, was 103-fold more effective in polymerase inhibition than was Ml, and greater than 103fold more effective, on a molar basis, than ribavirin and amantadine again virus A/PR/ 8/34 by measuring the inhibition of viral cyto-pathic effect in MDCK cells (147). Little or no in vitro antiviral activity could be seen if the peptide was added later than 1 h after virus challenge, suggesting that the peptide inhibits virus at an early stage in viral replication, presumably through inhibition of the polymerase. Pre-treatment with the peptide also significantly protected the cells from viral challenge. Because the Ml sequence representing peptide 6 is highly conserved among type A influenza viruses, peptide 6 exhibited in vitro antiviral activity against a wide range of type A influenza viruses representing

H1N1, H2N2, and H3N2 subtypes. Interestingly, it was also active against two type B influenza viruses (B/Lee/40 and B/Shanghai/4/ 94). Because peptide 6 shows therapeutic effect, it is possible that, with coordination of zinc, the peptide can assume a compact size and readily enters the cells. Derivatives with alternations in the finger loop, tail length, or residues involved in coordination of zinc showed reduced or abolished antiviral activity. When tested in a mouse model of influenza infections, peptide 6, administered intrana-sally beginning 4 h pre- or 8 h post-virus exposure to an H1N1 virus (A/PR/8/34) or an H3N2 virus (A/Victoria/3/75), was effective in preventing death, reducing the arterial oxygen decline, inhibiting lung consolidation, and reducing virus titers (titer reduction not seen with A/Victoria/3/75) in the lungs of infected animals (148).

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