Inhibition of human immunodeficiency virus activity by phosphorodithioate oligodeoxycytidine.

Abstract

Phosphorothioate oligodeoxynucleotides exert a sequence-independent cytoprotective effect against human immunodeficiency virus type 1 (HIV-1). We now report that phosphorodithioate-containing oligodeoxycytidines are very potent inhibitors of HIV-1 reverse transcriptase in vitro, as they exhibit an increasing inhibitory effect with length and number of phosphorodithioate internucleotide linkages. This inhibitory effect can be at least 30-fold greater with phosphorodithioate oligodeoxycytidine than for the corresponding phosphorothioate analog of similar length. In cell culture, phosphorodithioate oligodeoxycytidines are active inhibitors of syncytia formation and effectively inhibit de novo infection of target cells by HIV-1. Moreover, comparative experiments show that a deoxycytidine phosphorodithioate 14-mer is as effective an inhibitor of de novo infection as a phosphorothioate-containing 28-mer. Such potent inhibition by oligomers of relatively short length makes dithioate analogs an additional class of potential therapeutic agents against acquired immunodeficiency syndrome.Over the past several years, oligodeoxynucleotides and their analogs have been reported to inhibit viral replication and the expression of oncogenes in cell culture (1, 2). Although the pioneering work of Zamecnik and Stephenson (3, 4) used normal oligodeoxynucleotides, recent research has focused on derivatives ( Fig. 1) that have certain desirable features, including nuclease resistance and rapid transport into cells (5-8). Generally these oligomers appear to function by inhibiting translation and splicing, but other inactivation mechanisms have also been found (9).During studies with oligodeoxynucleotides as inhibitors of human immunodeficiency virus type 1 (HIV-1), Matsukura et al. discovered that phosphorothioate oligodeoxycytidines exhibited potent antiviral activity (10); in these oligomers, one of the nonbridging oxygens at each linkage in oligodeoxycytidine, (dC)", has been replaced with sulfur, and individual oligomers are designated S(dC),. Inhibition of HIV-1 reverse transcriptase (RT) (11) or other steps in its life cycle such as binding, fusion, and entry (5, 10) were suggested as possible sites of action. Subsequent studies corroborated these observations and demonstrated that phosphorothioates are more effective than phosphoramidates and methylphosphonates as sequence-nonspecific inhibitors of HIV-1 replication (12).These developments prompted us to examine oligodeoxycytidine analogs bearing one or more phosphorodithioate linkages (Fig. 1) MATERIALS AND METHODSOligodeoxycytidines. , phosphorodithioate-containing (16, 17, 23), and unmodified oligodeoxycytidines (24) were synthesized and characterized by conventional procedures.Inhibition of Purified EHV-1 RT. Twenty-microliter assay mixtures contained 50 mM Tris (pH 8.3); 10 mM MgCl2; 50 mM KCI; 5 mM dithiothreitol; 1.0 ,uM preannealed primed template, 5'-32P-labeled d(GpApTpTpCpApGpCpTpApGpTpCpCpA) primer and d(CpApApApCpTpGpTpGpApTpApCpGpApTpGpGpApCpTpApGpCpTpGpAp...

Phosphorothioate oligonucleotides, suramin and heparin inhibit DNA-dependent protein kinase activity

“…It has been reported that S-oligos with a chain length longer than 15 could bind and inhibit the proteins, and that the inhibitory effect reached a plateau level with a chain length of 28 (Gao et al, 1992;Marshall et al, 1992). In the present study, we reported that the inhibitory effect of S-oligos on DNA-PK activity is sequence-independent and is dependent on the chain length.…”

“…The inhibitory effect reached a plateau level with a chain length of 36, which is almost the same length as previously reported. Marshall et al (1992) reported that the inhibitory effect of oligonucleotides on HIV-1 reverse transcriptase could be at least 30-fold greater with phosphorodithioate oligonucleotides, which have two sulphur at each site of internucleotide linkages, than with phosphorothioate oligonucleotides. Benimetskaya et al (1995) reported that binding of phosphorothioate oligonucleotides to proteins is independent on P-chirality at the internucleotide linkage sites.…”

“…Such proteins include heparinbinding proteins such as basic fibroblast growth factor (bFGF), acid fibroblast growth factor (aFGF), Kaposi's growth factor (FGF-4) (Guvakova et al, 1995), and DNA polymerases (Gao et al, 1992). S-oligo also inhibits the functions of other proteins such as CD4 , gp120 (Stein et al, 1991), Mac-1 (Benimetskaya et al, 1997), RNase H (Gao et al, 1992), human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (Marshall et al, 1992), herpes simplex virus (HSV) type 2-induced DNA polymerase (Gao et al, 1989), and HIV-1 integrase (Tramontano et al, 1998).…”

Azasugar-Containing Phosphorothioate Oligonucleotide (AZPSON) DBM-2198 Inhibits Human Immunodeficiency Virus Type 1 (HIV-1) Replication by Blocking HIV-1 gp120 without Affecting the V3 Region

“…These suggested mechanisms include blocking of viral adsorption (Luganini et al, 2008;Wyatt et al, 1994) and/or inhibition of HIV-1-specific enzymes, such as reverse transcriptase (Marshall and Caruthers, 1993;Marshall et al, 1992) or integrase (Jing and Hogan, 1998;Jing et al, 2000). P = S oligodeoxycytidine [poly (SdC) 28 ] interacts specifically with the positively-charged V3 loop of HIV-1 gp120 (Vaillant et al, 2006), possibly resulting in the inhibition of HIV-1 replication.…”

Newly Designed Six-Membered Azasugar Nucleotide-Containing Phosphorothioate Oligonucleotides as Potent Human Immunodeficiency Virus Type 1 Inhibitors

“…The mechanisms suggested have included adsorption blocking (9,16,47,50,51,53) and inhibition of HIV-1-specific enzymes, such as reverse transcriptase (29)(30)(31) or integrase (21-23, 36, 42). However, most studies of the antiviral mechanisms of PAS ONs, as well as recent studies with small interfering RNA (siRNA) against HIV-1 (3,5,7,35,38,39,49), have been conducted by transfection or viral vector-mediated delivery (4,17,27,33,34), rather than simple treatment of the infected culture.…”