Inhibition of acquired immunodeficiency syndrome virus by oligodeoxynucleoside methylphosphonates.

Abstract

Antisense oligodeoxynucleotides containing internucleoside methylphosphonate linkages were examined for their ability to inhibit human immunodeficiency virus (HIV)-induced syncytium formation and virus expression. HIV inhibitory activity was found to be dependent on both chain length and the number of phosphonate residues. Introduction of 18 phosphonate groups in an oligomer of chain length 20 significantly increased HIIV inhibitory activity relative to the parent oligonucleotide, whereas 5 such groups showed little or no increase in the HIV inhibition capacity. Methylphosphonate-linked oligomers are more stable to nuclease degradation and hence could be potentially useful in the treatment of acquired immunodeficiency syndrome.With the identification of human immunodeficiency virus (HIV) type 1 as the etiological agent of acquired immunodeficiency syndrome (AIDS), a number of approaches are being explored to develop therapeutic agents for the treatment of AIDS and AIDS-related complex (1). A molecular approach to inhibit HIV-1 replication involves the use of complementary antisense oligonucleotides that can selectively bind to defined sequences of viral RNA. Antiviral activity of specific antisense oligonucleotides was first observed in studies with Rous sarcoma virus (2, 3). The antisense oligomer approach has since been applied to inhibit a number of RNA and DNA viruses including HIV (4-6), vesicular stomatitis virus (7-9), herpes simplex virus type 1 (7, 10), and influenza virus (11). Attempts have been made to improve the activity ofantisense oligonucleotides by increasing their stability to nucleases, their cellular uptake, and their hybridization with complementary RNA. These studies have involved replacing the internucleoside phosphates by methylphosphonates (7,10), phosphorothioates (5), or phosphoramidates (22) or by modifying the ends of the molecule (2, 4, 6, 9, 11). In this paper, we show that methylphosphonate analogues are potent inhibitors of HIV-1 replication in cell culture and, with their added resistance to nucleases, could be potentially useful in the treatment of AIDS. MATERIALS AND METHODSChemical Synthesis. Oligonucleotides were synthesized by using an automated DNA synthesizer (Biosearch model 8600, San Rafael, CA). Phosphodiester bonds were constructed by using the standard phosphoramidite protocol of the manufacturer except that the oxidation step was performed before the capping reaction. Methylphosphonate linkages were assembled from nucleoside methylphosphonamidites by using the method previously reported (12). Oligonucleotides were cleaved from the support in concentrated ammonium hydroxide at room temperature for 2 hr. The supernatant was taken to dryness, and the acyl protecting groups were removed by treatment with ethylenediamine/ethanol, 1:1 (vol/vol) at room temperature for 4 hr. Solvent was evaporated under reduced pressure followed by coevaporation of two additional aliquots of ethanol.Oligonucleotides with mostly phosphodiester linkages were purified in the same way...

Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues.

“…The cells were incubated in culture medium in a humidified atmosphere containing 5% CO2 at 37°C. After 4 days the cells and supernatant were examined for the level of HIV-1 expression by measuring syncytia (MOLT-3 cells), the expression of viral antigens p24 and p17, reverse transcriptase activity, as well as cell viability, as reported earlier (10,11,18,19). Twenty-four hours after viral infection, the cells were washed, then the oligomer was added, and the experiment was carried on for 4 days thereafter before termination.…”

“…Inhibition of HIV-1 expression in H9 or MOLT-3 cells in the presence of oligonucleotides was carried out as reported earlier (10,11,18,19). Antisense oligomers were added at different concentrations only once, either at 0 hr (within a few seconds of virus addition) or 24 or 48 hr after addition of virus to the cells.…”

“…However, relatively short half-lives of normal oligonucleotides (9) in serum and in cells due to the presence of nucleases, and possibly the low permeability of these charged molecules into normal cells, limit their potential usefulness in vivo. To overcome this limitation, we and others (10)(11)(12)(13)(14)(15) have studied internucleotide phosphate backbone-modified oligonucleotides, such as methylphosphonates (3,10) as well as phosphorothioates and various phosphoramidates (11,12), for their antiviral activities against HIV.…”

Nuclease resistant methylphosphonate-DNA/LNA chimeric oligonucleotides

“…[32][33][34] In a first attempt of combining the desirable properties of methylphosphate linkages and LNA monomers, we reported synthesis, satisfactory RNA binding properties and increased 3 0 -exonucleolytic stability of ONs containing a single methylphosphonate-LNA (MP-LNA, Fig. 1) monomer.…”

“…[29][30][31] MP-DNA displays stabilization towards nucleolytic degradation but unfortunately also significantly reduced binding affinity towards complementary RNA. [32][33][34] In a first attempt of combining the desirable properties of methylphosphate linkages and LNA monomers, we reported synthesis, satisfactory RNA binding properties and increased 3 0 -exonucleolytic stability of ONs containing a single methylphosphonate-LNA (MP-LNA, Fig. 1) monomer.…”