Phosphorothioate analogs of (2'-5')(A)4: agonist and antagonist activities in intact cells

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Abstract
Metabolically stable phosphorothioate tetramer analogues of (2''-5'')(A)n with Rp and/or Sp chirality in the 2''-5''-phosphodiester linkages constitute a new class of antiviral agents since they mimic the effects of interferons. Three of the diastereomeric 5''-monophosphates (i.e., pRpRpRp, pSpRpRp, and pRpSpSp) bind to and activate RNase L from extracts of HeLa cells. However, the pSpSpSp (2''-5'')-(A)4-phosphorothioate is unique in that it binds to, but cannot activate, RNase L to cleave rRNA. When microinjected into the cytoplasm of HeLa cells followed by virus infection, the pRpRpRp, pSpRpRp, and pRpSpSp (2''-5'')(A)4-phosphorothioates demonstrate antiviral activity, as does (2''-5'')(A)4ox-red, an active (2''-5'')(A)n analogue. When microinjected simultaneously with (2''-5'')(A)nox-red, the pSpSpSp (2''-5'')(A)4-phoshorothioate inhibits activation of RNase L in HeLa cells, thereby blocking direct protection of vesicular stomatitis virus. The agonist and antagonist properties of pRpRpRp and pSpSpSp, respectively, are transient probably as a consequence of the hydrolysis of the 5''-monophosphate and formation of the less active (2''-5'')(A)4-phoshorothioate cores. The possible use of these (2''-5'')(A)4-phosphorothioates as tools for dissecting the biological significance of the (2''-5'')(A)n system or in antiviral chemotherapy is discussed.