T30177, an oligonucleotide stabilized by an intramolecular guanosine octet, is a potent inhibitor of laboratory strains and clinical isolates of human immunodeficiency virus type 1

Abstract

T30177, an oligonucleotide composed of only deoxyguanosine and thymidine, is 17 nucleotides in length and contains single phosphorothioate internucleoside linkages at its 5 and 3 ends for stability. This oligonucleotide does not share significant primary sequence homology with or possess any complementary (antisense) sequence motifs to the human immunodeficiency virus type 1 (HIV-1) genome. T30177 inhibited replication of multiple laboratory strains of HIV-1 in human T-cell lines, peripheral blood lymphocytes, and macrophages. T30177 was also found to be capable of inhibiting multiple clinical isolates of HIV-1 and preventing the cytopathic effect of HIV-1 in primary CD4 ؉ T lymphocytes. In assays with human peripheral blood lymphocytes there was no observable toxicity associated with T30177 at the highest concentration tested (100 M), while the median inhibitory concentration was determined to be in the range of 0.1 to 1.0 M for the clinical isolates tested, resulting in a high therapeutic index for this drug. In temporal studies, the kinetics of addition of T30177 to infected cell cultures indicated that, like the known viral adsorption blocking agents dextran sulfate and Chicago sky blue, T30177 needed to be added to cells during or very soon after viral infection. However, analysis of nucleic acids extracted at 12 h postinfection from cells treated with T30177 at the time of virus infection established the presence of unintegrated viral cDNA, including circular proviral DNA, in the treated cells. In vitro analysis of viral enzymes revealed that T30177 was a potent inhibitor of HIV-1 integrase, reducing enzymatic activity by 50% at concentrations in the range of 0.050 to 0.09 M. T30177 was also able to inhibit viral reverse transcriptase activity; however, the 50% inhibitory value obtained was in the range of 1 to 10 M, depending on the template used in the enzymatic assay. No observable inhibition of viral protease was detected at the highest concentration of T30177 used (10 M). In experiments in which T30177 was removed from infected cell cultures at 4 days post-HIV-1 infection, total suppression of virus production was observed for more than 27 days. PCR analysis of DNA extracted from cells treated in this fashion was unable to detect the presence of viral DNA 11 days after removal of the drug from the infected cell cultures. The ability of T30177 to inhibit both laboratory and clinical isolates of HIV-1 and the experimental data which suggest that T30177 represents a novel class of integrase inhibitors indicate that this compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.One relatively new approach used in the development of antiviral therapeutics for human immunodeficiency virus type 1 (HIV-1) is the use of oligonucleotides designed as antisense agents (24,26,31). While much effort is being spent on rationally designed oligonucleotides such as antisense agents, there have also been recent findings of multiple alternative mechanisms by which oligonucleotides can i...

Antiviral and Biological Properties of Dimethoxytrityl-Linked Guanine-Rich Oligodeoxynucleotides that Inhibit Replication by Primary Clinical Human Immunodeficiency virus Type 1 Isolates

“…This effectiveness was contradictory to the previous determination that well-known adsorption and fusion inhibitors such as dextran sulphate and soluble CD4 are more effective against laboratory strains than primary clinical isolates (Daar et al, 1990;Meylan et al, '1994), but seemed to agree with the results published by Ojwang et al (1995). Wyatt et al (1994) reported that the hyperstructure of a short G-rich ODN with a phophorothioate backbone was essential for preventing cell-to-cell infection and acute infection.…”

“…For example, ISIS5320, consisting of an 8-mer G-rich sequence (5' TTGGGGTT 3') with a phosphorothioate backbone, which is folded into an intramolecular G-tetrad, exerts anti-HIV activity by preventing the binding of the V3 loop of HIV to CD4 (Wyatt et al, 1994). Furthermore, T30177, an ODN composed entirely of deoxyguanosine and thymidine modified with a single phosphorothioate internucleoside linkage at its 5' and 3' ends, inhibits replication of both laboratory strains and clinical isolates by inhibiting HIV-1 integrase activity and/or by blocking the interaction between gp120 and CD4 (Ojwang et al, 1994(Ojwang et al, , 1995.…”

Is antisense an appropriate nomenclature or design for oligodeoxynucleotides aimed at the inhibition of HIV-1 replication?

“…T30177 is a 17-mer ODN that comprises only deoxyguanosine and thymidine residues stabilized by an intramolecular guanosine octet. 20 T30177 contains single phosphorothioate internucleoside linkages at its 5' and 3' ends, and has shown strong anti-HIV activity in infected cells on the basis of its 3-dimensional structure.…”

Development of Resistance against Diketo Derivatives of Human Immunodeficiency Virus Type 1 by Progressive Accumulation of Integrase Mutations

“…For most of the IN inhibitors that did show antiviral activity in cell culture, it was not unambiguously demonstrated that the integration step was targeted. Moreover, for two classes of IN inhibitors with antiviral activity, the G quartets (24) and L-chicoric acid derivatives (32), it has been demonstrated by means of selecting and sequencing drug-resistant HIV-1 strains that viral entry and not integration is the main antiviral target in cell culture (13,27). In contrast, Ojwang et al (24) reported that G quartets generated an increase in two-LTR circles and a decrease in integrated DNA at concentrations that inhibit viral replication.…”