Joshua O. Ojwang

Intramolecular G-quartet Motifs Confer Nuclease Resistance to a Potent Anti-HIV Oligonucleotide

We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5′-g*tggtgggtgggtggg*t-3′, where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 μM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with tof 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (tfrom 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.

Inhibition of herpes simplex virus in culture by oligonucleotides composed entirely of deoxyguanosine and thymidine

Oligodeoxynucleotides (ODNs) composed entirely of deoxyguanosine and thymidine, but not specifically designed to act as antisense agents, were able to significantly inhibit herpes simplex virus growth in acute infection assay systems. The guanosine/thymidine (GT) ODNs which demonstrated this antiviral activity contained either natural phosphodiester (PO) or phosphorothioate (PS) modified internucleoside linkages. In all experiments, the antiviral activity of the ODNs was enhanced when the backbone was modified to contain the PS linkages. When present during the time of virus addition, the ODNs were able to block the adsorption of virus to Vero cells. In this assay the PS-containing ODNs had ID50 values of approximately 0.020 microM for HSV-2 and of 0.3 microM for HSV-1. When these same PS-containing ODNs were used against HSV-2 in single-cycle viral yield assays, designed to minimize the effects due to external blockage of virus, the ID50 values rose to 0.2 microM. Analysis of viral DNA obtained 14 h post-HSV-2 infections in the single-cycle assay, revealed a decrease in replicated viral DNA in cells treated with PS-ODNs. Analysis of viral mRNA obtained 4 h post-HSV-2 infection revealed, in cells treated with the PS-ODNs, a decrease in measurable HSV-2 alpha- and beta-mRNAs. Although the mechanism of action of the antiviral activity (beyond adsorption blocking) is not fully understood, the toxicity of these compounds was low, giving high therapeutic indices for the GT-rich PS-ODNs. The good therapeutic index of GT-ODNs make this a class of compounds which warrant investigation as therapeutic agents to be used against herpes viruses.

Inhibition of episomal hepatitis B virus DNA in vitro by 2,4-diamino-7- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-pyrrolo[2,3-d]pyrimidine.

The nucleoside analog 2,4-diamino-7-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (T70080) and several related compounds were evaluated for anti-hepatitis B virus (HBV) activity by using cultured 2.2.15 cells. T70080 reduced episomal viral replication in these cells by 50% at a concentration of 0.7 microgram/ml. At the same time, T70080 reduced cellular proliferation by 50% at a concentration in excess of 100 micrograms/ml, yielding a therapeutic index of > 143. In cells cultured for 12 days in the presence of 10 or 50 micrograms of T70080 per ml and then with drug-free medium, for an additional 12 days, viral DNA replication was completely inhibited initially but resumed between 6 and 12 days post-drug removal. In view of the potent anti-HBV activity shown, T70080 is a good candidate for further evaluation as a treatment of human HBV infection.

Inhibition of Friend Murine Leukemia virus activity by guanosine/thymidine oligonucleotides

Oligonucleotides consisting of only deoxyguanosine and deoxythymidine were stable in culture and were able to significantly inhibit Friend Murine Leukemia Virus (FMLV) production in acute cell culture assay systems. The oligonucleotides did not share homology with, or possess any complementary (antisense) sequence motifs to the FMLV genome. The guanosine/thymidine-containing oligonucleotides (GTOs) which demonstrated anti-FMLV activity in acute infection assays were synthesized with natural phosphodiester (PD) linkages (backbones). The observed antiviral activities of these oligonucleotides increased significantly when the PD backbone was replaced with a phosphorothioate (PT) backbone. Experiments designed to investigate a potential antiviral mechanism of action demonstrated that oligonucleotides tested were capable of blocking virus adsorption. In addition, GTOs with PD backbones were competitive inhibitors of FMLV reverse transcriptase (RT). When the same experiments were performed using oligonucleotides with PT backbones, all compounds tested demonstrated significant competitive inhibition of FMLV RT. The measured inhibitory activity of all compounds tested in culture assays was enhanced by at least a factor of 10 when the PD linkages were replaced with PT. The enhanced antiviral activity exhibited by the sulfur group on the oligonucleotide backbone, and the lack of any designed, sequence-specific interactions, suggest that a large percentage of the reported antiviral activity of oligonucleotides containing a phosphorothioate backbone is due to factors other than rationally designed, sequence-specific interactions. The ability of GTOs to inhibit FMLV in culture, potentially via a number of different mechanisms, makes this a class of compounds which warrants investigation as therapeutic agents to be used against retroviral infections.

Thiazolo[4,5-d]pyrimidines. Part II. Synthesis and anti-human cytomegalovirus activity in vitro of certain acyclonucleosides and acyclonucleotides derived from the guanine analogue 5-aminothiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione

The synthesis and in vitro antiviral activity of certain hydroxyalkoxymethyl, hydroxyalkyl, hydroxyalkenyl and phosphonoalkenyl derivatives of the guanine congener 5-aminothiazolo-[4,5-d]pyrimidine-2,7(3H,6H)-dione are reported. The compounds of this study were selected for their structural similarity to acyclonucleosides with known anti-herpesvirus activity. 5-Amino-3-[(Z)-4-hydroxy-2-buten-1-yl]thiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione was the only member of the series to display significant in vitro activity against human cytomegalovirus (HCMV); however, this compound did not inhibit other herpesviruses, human immunodeficiency virustype 1 or murine cytomegalovirus. It was found to have a cytotoxicity profile similar to that of ganciclovir (DHPG). The antiviral effect was found to be sensitive to the initial viral input and the time of addition during the virus replication cycle. Significantly, the compound was found to have equal anti-HCMV activity, against standard virus strains, to DHPG, but also showed potent activity against DHPG-resistant virus strains, except for a strain mutated in the UL97 (phosphotransferase) gene.

Inhibition of Tumor Necrosis Factor Alpha (TNFα) Expression and Function In Vitro by Modified Antisense Oligonucleotides

Abstract Antisense oligonucleotides containing C-5 hexynyl/propynyl modified pyrimidines were synthesized using solid phase phosphoramidite chemistry. These modified oligonucleotides were found to have significant inhibitory activity against TNFα production in vitro.