ABSTRACT
2',5'-Oligoadenylate (2-5A) derivatives have been designed to act distal to the human immunodeficiency virus-1 (HIV-1)-induced blockade in the 2-5A synthetase/RNase L antiviral pathway. Stereochemical modification of individual internucleotide linkages of the 2-5A molecule was accomplished by phosphoramidite and phosphotriester chemical syntheses. Phosphorothioate/phosphodiester trimer and tetramer 2-5A derivatives revealed differences in the stereodynamics of activation of RNase L and inhibition of HIV-1 replication. The first and second internucleotide linkages are critical for activation of recombinant, human RNase L; A(Rp)ApA, A(Sp)ApA and ApA(Rp)A are agonists (IC50 = 2 x 10(-7), 2 x 10(-6) and 8 x 10(-6) M); ApA(Sp)A is an antagonist. The second and third internucleotide linkages are crucial for activation of murine RNase L; ApA(Rp)A, ApA(Rp)ApA, and ApApA(Rp)A are agonists (IC50 = 5 x 10(-7) M); ApA(Sp)A, ApA(Sp)ApA, and ApApA(Sp)A are antagonists. Inhibition of HIV-1-induced syncytia formation by the phosphorothioate/phosphodiester derivatives is specific for derivatives with substitution at the 2',3'-terminus. ApA(Rp)A, ApA(Sp)A, ApApA(Rp)A, and ApApA(Sp)A are potent inhibitors of HIV-1-induced syncytia formation (80-, 10-, 40-, and 15-fold more inhibitory, respectively, than solvent control). HIV-1 infection results in enhanced uptake and accumulation of ApA(Rp)A and ApA(Sp)A (7- and 10-fold, respectively). These stereochemically modified 2-5A derivatives are taken up preferentially by HIV-1-infected cells and show promise in anti-HIV-1 chemotherapy.
Subject(s)
Adenine Nucleotides/chemical synthesis , Adenine Nucleotides/pharmacology , Antiviral Agents/pharmacology , Endoribonucleases/metabolism , HIV-1/physiology , Virus Replication/drug effects , Adenine Nucleotides/metabolism , Animals , Antiviral Agents/chemical synthesis , Base Sequence , Cell Division/drug effects , Cell Line , Cell Survival/drug effects , DNA Primers , Endoribonucleases/biosynthesis , Enzyme Activation , Escherichia coli , Giant Cells/drug effects , HIV-1/drug effects , Humans , Indicators and Reagents , Kinetics , L Cells , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Restriction Mapping , Structure-Activity RelationshipABSTRACT
The combination of 2'-OH protection in ribonucleosides by the p-nitrophenylethylsulfonyl (NPES) group with the 3'-(beta-cyanoethyl) (N,N-diisopropyl)-phosphoramidite function reveals a new approach to oligoribonucleotide synthesis. The corresponding adenosine and guanosine derivatives have been applied to automated solid phase synthesis with good success.
