Inhibition of HIV-1 Replication and Activation of RNase L by Phosphorothioate/Phosphodiester 2′,5′-Oligoadenylate Derivatives

Robert W Sobol,Earl E Henderson,Ning Kon,Jie Shao,Patricia Hitzges,Eli Mordechai,Nancy L Reichenbach,Ramamurthy Charubala,Helga Schirmeister,Wolfgang Pfleiderer,Robert J Suhadolnik

doi:10.1074/jbc.270.11.5963

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.

Highlights

2',5'-0ligoadenylate (2-5A) derivatives have been designed to act distal to the human immunodeficiency virus-1 (HIV-1)-induced blockade in the 2-5A synthetase/
It has been established that the growth cycle of HIV-1 and 2-5A metabolism, i.e. high levels of 2-5A and activated RNase L, correlate with failure of infected cells to release HIV-1 (Schroder et aZ., 1989, 1994)
Our approach to circumvent the HIV-1 Tatinduced blockade of the 2-5A synthetaselRNase L antiviral pathway has been to augment the depleted intracellular 2-5A pool with 2-5A derivatives that are nuclease-resistant, are non-toxic, activate RNase L, and inhibit HIV-1 replication (Montefiori et aZ., 1989; Muller et aZ., 1991; Sobol et al, 1993; Schroder et al, 1994)

Summary

Introduction

2',5'-0ligoadenylate (2-5A) derivatives have been designed to act distal to the human immunodeficiency virus-1 (HIV-1)-induced blockade in the 2-5A synthetase/. 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. Inhibition of HIV-1-induced syncytia formation by the phosphorothioate/phosphodiester derivatives is specific for derivatives with substitution at the. 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 ofApA(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

Methods

Results

Conclusion