In vitro antiretroviral activity and in vitro toxicity profile of SPD754, a new deoxycytidine nucleoside reverse transcriptase inhibitor for treatment of human immunodeficiency virus infection.

SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates, its activity in combination with other antiretrovirals, and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L, T215Y/F, plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V, Y115F, and M184V plus one other NAM) or stavudine (V75T/M, M41L, T215F/Y, and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold, respectively (these changes gave values comparable to or less than the corresponding values for zidovudine, lamivudine, abacavir, and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades, including A/G, B, C, D, A(E), D/F, F, and H. SPD754 showed additive effects in combination with other NRTIs, tenofovir, nevirapine, or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 microM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro, SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity.

Parallel synthesis of isatin-based serine protease inhibitors.

The synthesis of N-functionalised isatins using parallel, solution synthesis is described. Functionalised polymers were employed as stoichiometric and catalytic reagents as well as purification media in the exercise, and the derivatives were screened against a panel of serine proteases; high percentage inhibition was observed in several cases.

Drug resistance and drug combination features of the human immunodeficiency virus inhibitor, BCH-10652 [(+/-)-2'-deoxy-3'-oxa-4'-thiocytidine, dOTC].

The heterosubstituted nucleoside analogue dOTC [( )-2'-deoxy-3'-oxa-4'-thiocytidine, BCH-10652] is a racemic compound structurally related to 3TC (lamivudine), but has the oxygen and sulphur in the furanosyl ring transposed. Both the enantiomers (-)dOTC (BCH-10618) and (+)dOTC (BCH-10619) had equivalent activity against wild-type strains of HIV-1 in C8166 T-cells (EC50 1.0-10.0 microM) and in PBMCs (EC50 0.1-3.0 microM). Investigation of the activity of dOTC and its enantiomers against laboratory strains of HIV-1 with defined resistance to 3TC, AZT (zidovudine), ddl (didanosine), PMEA (adefovir), nevirapine and saquinavir indicated that sensitivity was maintained (<3-fold change in EC50) in all cases, with the exception of HIV-1RF 3TC-resistant viruses. The degree of resistance recorded for dOTC (four- to sevenfold), (-)dOTC (five- to eightfold) and (+)dOTC (five- to >18-fold) against these M1841 or M184V mutants, was significantly less than that recorded for 3TC (>100-fold). In addition, the inhibitory effect of the compounds against clinical isolates of HIV-1 recovered from patients with suspected resistance to 3TC and AZT was investigated. Clinical isolates were genotyped using the Murex Line Probe Assay (LiPA) and subgrouped into wild-type, 3TC-resistant and dual 3TC/AZT-resistant, as well as undefined or mixed genotype populations. Compared with the mean EC50 values obtained with genotypically and phenotypically wild-type clinical isolates, the mean EC50 values calculated for isolates phenotypically resistant to 3TC or 3TC and AZT were only 2.6-, 1.6- and 8.2-fold higher for dOTC, (-)dOTC and (+)dOTC, respectively. When the rate of emergence of virus resistant to dOTC and its enantiomers in vitro was investigated, virus resistant to (+)dOTC was readily selected for (<10 passages), and a methionine (ATG) to isoleucine (ATA) amino acid change at codon 184 was identified. In contrast, virus resistant to dOTC and (-)dOTC took longer to appear (15-20 passages), with a methionine (ATG) to valine (GTG) amino acid change at position 184 identified in both cases. In addition, virus passaged 20 times in the presence of dOTC also had a partial lysine (AAA) to arginine (AGA) exchange at position 65. These viruses showed only low-level resistance to dOTC and its enantiomers, but were highly resistant to 3TC. The antiviral effects of dOTC in combination with the nucleoside RT inhibitors AZT, 3TC, d4T (stavudine) and ddl, the non-nucleoside RT inhibitor nevirapine and the protease inhibitors saquinavir, ritonavir and indinavir was investigated. Two-way drug combination assays were carried out in peripheral blood mononuclear cell (PBMC) cultures by measuring the reduction in p24 viral antigen levels, and data was analysed using the MacSynergy II program. dOTC in combination with 3TC or d4T showed a moderate synergistic effect while all other combinations had an additive interaction.

A novel high throughput screening assay for HCV NS3 helicase activity.

A novel assay for measurement of Hepatitis C virus (HCV) NS3 helicase activity was developed using Flashplate technology. This assay involves the use of a DNA duplex substrate and recombinant HCV NS3 produced in Escherichia coli. The DNA duplex consisted of a pair of oligonucleotides, one biotinylated, the other radiolabeled at their respective 5' termini. This DNA duplex was immobilized, via the biotin molecule, on the surface of a neutravidin-coated SMP103 Flashplate (NEN Life Science Products). Helicase activity results in the release of the radiolabeled oligonucleotide, which translates in signal reduction with respect to control wells. Biochemical characterization of the HCV NS3 helicase activity was performed using this assay. We demonstrated that the NS3-mediated unwinding is proportional to both the amount of DNA substrate in the well, and to the NS3 concentration in the reaction. Most of the NS3-mediated unwinding was achieved in the initial 60 min of incubation. As expected the reactions were ATP-dependent and found to be affected by the concentration of MgCl(2), MnCl(2), KCl, EDTA, and by pH. We found this assay to be highly reproducible since only slight variation was observed when a total of 68 helicase reactions were performed on one plate. Therefore, this Flashplate helicase assay is fast, convenient and reproducible. These criteria make it suitable for high throughput screening of potential NS3 helicase inhibitors.

Antiviral properties of a series of 1,6-naphthyridine and 7, 8-dihydroisoquinoline derivatives exhibiting potent activity against human cytomegalovirus.

A series of 1,6-naphthyridine (L. Chan, H. Jin, T. Stefanac, J. F. Lavallee, G. Falardeau, W. Wang, J. Bedard, S. May, and L. Yuen, J. Med. Chem. 42:3023-3025, 1999) and isoquinoline (L. Chan, H. Jin, T. Stefanac, W. Wang, J. F. Lavallee, J. Bedard, and S. May, Bioorg. Med. Chem. Lett. 9:2583-2586, 1999) analogues exhibiting a high level of anti-human cytomegalovirus (HCMV) activity were investigated in a series of studies aimed at better understanding the mechanism of action of some representatives of this class of compounds. In vitro antiviral profiling revealed that these compounds were active against a narrow spectrum of viruses, essentially the human herpesviruses and type 2 rhinovirus. In HCMV assays, a 39- to 223-fold lower 50% inhibitory concentration was obtained for compound A1 than for ganciclovir against strains AD 169 and Towne. In addition, ganciclovir, foscarnet, cidofovir, and BDCRB (2-bromo-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole)-resistant HCMV strains remained susceptible to 1,6-naphthyridines and 7, 8-dihydroisoquinolines tested in this study, supporting the view that a novel mechanism of action could be involved. Drug combination studies showed a small but significant synergistic antiviral effect between compound B2 and ganciclovir. Cytotoxicity profiling of representative compounds under various cell growth conditions indicated a generally similar cytotoxic effect, relative to ganciclovir, in log-phase growing cells. However, in stationary cells, a relatively higher level of toxicity was observed than that for control compound. Effect of time of drug addition showed that the anti-HCMV activity of compound A1, ganciclovir, and cidofovir was lost at approximately the same time (72 h postinfection), indicating that the compound was affecting events at the early and late stage of virus replication. This interpretation is also supported by reduction of de novo synthesis of pp65 tegument protein and lack of any effect of the compound on viral adsorption. A reduction of the HCMV enhancer-promoter-directed luciferase expression was also observed in a stably transfected cell line when compound A1 was present at relatively high concentrations.

Antiviral activity of 2'-deoxy-3'-oxa-4'-thiocytidine (BCH-10652) against lamivudine-resistant human immunodeficiency virus type 1 in SCID-hu Thy/Liv mice.

Oral administration of 2'-deoxy-3'-oxa-4'-thiocytidine (BCH-10652), a nucleoside analog structurally similar to lamivudine (3TC), caused dose-dependent inhibition of viral replication in SCID-hu Thy/Liv mice infected with human immunodeficiency virus type 1 NL4-3 and with an NL4-3 clone containing the M184V mutation in reverse transcriptase that confers resistance to 3TC. These experiments demonstrate the utility of this mouse model for evaluating drug resistance and for performing direct comparisons between antiviral compounds in vivo.

Stability-activity relationships of a family of G-tetrad forming oligonucleotides as potent HIV inhibitors. A basis for anti-HIV drug design.

Recently, we have demonstrated that T30695, a G-tetrad-forming oligonucleotide, is a potent inhibitor of human immunodeficiency virus, type I (HIV-1) integrase and the K(+)-induced loop folding of T30695 plays a key role in the inhibition of HIV-1 integrase (Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). Here we have modified T30695 by introducing a hydrophobic bulky group, propynyl dU, or a positively charged group, 5-amino dU, into the bases of T residues of the loops, and by substitution of the T-G loops by T-T loops. Physical measurements have demonstrated that the substitution of propynyl dU or 5-amino dU for T in the T residues of the loops did not alter the structure of T30695, and these derivatives also formed an intramolecular G-quartet structure, which is an essential requirement for anti-HIV activity. Measured IC(50) and EC(50) values show that these substitutions did not induce an apparent decrease in the ability to inhibit HIV-1 integrase activity and in the inhibition of HIV-1 replication in cell culture. However, the substitution of T-T loops for T-G loops induced a substantial decrease in both thermal stability and anti-HIV activity. The data analysis of T30695 and the 21 derivatives shows a significant, functional correlation between thermal stability of the G-tetrad structure and the capacity to inhibit HIV-1 integrase activity and between thermal stability of the G-tetrad structure and the capacity to inhibit HIV-1 replication, as assessed with the virus strains HIV-1 RF, IIIB, and MN in cell culture. This relationship between thermostability and activity provides a basis for improving the efficacy of these compounds to inhibit HIV-1 integrase activity and HIV-1 replication in cell culture.

The hepatitis C virus NS5B RNA-dependent RNA polymerase activity and susceptibility to inhibitors is modulated by metal cations.

OBJECTIVES: The aim of this study was to understand the effect of metal cations on the hepatitis C virus (HCV) NS5B in vitro RNA-dependent RNA polymerase (RdRp) activity and its susceptibility to various inhibitors. METHODS: A recombinant full-length HCV NS5B protein was expressed in insect cells and purified to homogeneity. RdRp activity was assessed using standard filtration or polyacrylamide gel-based assays. RESULTS: Efficient inhibition of the HCV NS5B RdRp activity by gliotoxin, as well as by various substrate analogs, occurs in the presence of Mn2+, but not of Mg2+. Assays performed in the presence of both cofactors suggest that, in vitro, the enzyme's affinity for Mn2+ is higher than that for Mg2+. In addition, the RdRp activity, displayed in the presence of heteropolymeric templates, is significantly increased when the metal cofactor consists of Mn2+. Finally, steady state kinetics showed that the velocity of the reaction, as well as the affinity of the enzyme for its substrate, could both be affected by the nature of the divalent metal cation used.

Mechanism of action and in vitro activity of 1',3'-dioxolanylpurine nucleoside analogues against sensitive and drug-resistant human immunodeficiency virus type 1 variants.

(-)-Beta-D-1',3'-Dioxolane guanosine (DXG) and 2,6-diaminopurine (DAPD) dioxolanyl nucleoside analogues have been reported to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1). We have recently conducted experiments to more fully characterize their in vitro anti-HIV-1 profiles. Antiviral assays performed in cell culture systems determined that DXG had 50% effective concentrations of 0.046 and 0.085 microM when evaluated against HIV-1(IIIB) in cord blood mononuclear cells and MT-2 cells, respectively. These values indicate that DXG is approximately equipotent to 2', 3'-dideoxy-3'-thiacytidine (3TC) but 5- to 10-fold less potent than 3'-azido-2',3'-dideoxythymidine (AZT) in the two cell systems tested. At the same time, DAPD was approximately 5- to 20-fold less active than DXG in the anti-HIV-1 assays. When recombinant or clinical variants of HIV-1 were used to assess the efficacy of the purine nucleoside analogues against drug-resistant HIV-1, it was observed that AZT-resistant virus remained sensitive to DXG and DAPD. Virus harboring a mutation(s) which conferred decreased sensitivity to 3TC, 2',3'-dideoxyinosine, and 2',3'-dideoxycytidine, such as a 65R, 74V, or 184V mutation in the viral reverse transcriptase (RT), exhibited a two- to fivefold-decreased susceptibility to DXG or DAPD. When nonnucleoside RT inhibitor-resistant and protease inhibitor-resistant viruses were tested, no change in virus sensitivity to DXG or DAPD was observed. In vitro drug combination assays indicated that DXG had synergistic antiviral effects when used in combination with AZT, 3TC, or nevirapine. In cellular toxicity analyses, DXG and DAPD had 50% cytotoxic concentrations of greater than 500 microM when tested in peripheral blood mononuclear cells and a variety of human tumor and normal cell lines. The triphosphate form of DXG competed with the natural nucleotide substrates and acted as a chain terminator of the nascent DNA. These data suggest that DXG triphosphate may be the active intracellular metabolite, consistent with the mechanism by which other nucleoside analogues inhibit HIV-1 replication. Our results suggest that the use of DXG and DAPD as therapeutic agents for HIV-1 infection should be explored.

Achieving antisense inhibition by oligodeoxynucleotides containing N(7)-modified 2'-deoxyguanosine using tumor necrosis factor receptor type 1.

Antisense oligodeoxynucleotides (ODNs) are being explored as therapeutic agents for the treatment of many disorders including viral infections, cancers, and inflammatory disorders. In addition, antisense technology can be of great benefit to those attempting to assign function to the multitude of new genes being uncovered in the genomics initiative. However, the demonstration that the gene-regulating effects produced by antisense-designed ODNs are attributable to an antisense mechanism of action requires carefully designed experimentation. Critical to the assignment of an antisense mechanism of action is the availability of nuclease-stable ODNs, inside cells, that have a high binding affinity with the target mRNA and modulate gene functions in a sequence-dependent manner. To help us achieve a goal of sequence-specific antisense activity we designed antisense ODNs containing C(5)-propyne-modified 2'-deoxyuracil and N(7)-propyne-modified 7-deaza-2'-deoxyguanosine bases and partially modified (phosphorothioate) internucleoside linkages. These modified ODNs were found to have enhanced binding affinity to their target mRNA sequences as well as reduced sequence-independent side effects. We used these ODNs to specifically inhibit p55 tumor necrosis factor receptor type 1 expression and tumor necrosis factor alpha-mediated functions in culture assays.

Anti-human immunodeficiency virus type 1 activity, intracellular metabolism, and pharmacokinetic evaluation of 2'-deoxy-3'-oxa-4'-thiocytidine.

The racemic nucleoside analogue 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC) is in clinical development for the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection. dOTC is structurally related to lamivudine (3TC), but the oxygen and sulfur in the furanosyl ring are transposed. Intracellular metabolism studies showed that dOTC is phosphorylated within cells via the deoxycytidine kinase pathway and that approximately 2 to 5% of dOTC is converted into the racemic triphosphate derivatives, which had measurable half-lives (2 to 3 hours) within cells. Both 5'-triphosphate (TP) derivatives of dOTC were more potent than 3TC-TP at inhibiting HIV-1 reverse transcriptase (RT) in vitro. The K(i) values for dOTC-TP obtained against human DNA polymerases alpha, beta, and gamma were 5,000-, 78-, and 571-fold greater, respectively, than those for HIV RT (28 nM), indicating a good selectivity for the viral enzyme. In culture experiments, dOTC is a potent inhibitor of primary isolates of HIV-1, which were obtained from antiretroviral drug-naive patients as well as from nucleoside therapy-experienced (3TC- and/or zidovudine [AZT]-treated) patients. The mean 50% inhibitory concentration of dOTC for drug-naive isolates was 1.76 microM, rising to only 2.53 and 2.5 microM for viruses resistant to 3TC and viruses resistant to 3TC and AZT, respectively. This minimal change in activity is in contrast to the more dramatic changes observed when 3TC or AZT was evaluated against these same viral isolates. In tissue culture studies, the 50% toxicity levels for dOTC, which were determined by using [(3)H]thymidine uptake as a measure of logarithmic-phase cell proliferation, was greater than 100 microM for all cell lines tested. In addition, after 14 days of continuous culture, at concentrations up to 10 microM, no measurable toxic effect on HepG2 cells or mitochondrial DNA replication within these cells was observed. When administered orally to rats, dOTC was well absorbed, with a bioavailability of approximately 77%, with a high proportion (approximately 16.5% of the levels in serum) found in the cerebrospinal fluid.

Anti-HIV-1 activities of 1,3-dioxolane guanine and 2,6-diaminopurine dioxolane.

DXG and its prodrug DAPD have been demonstrated to be effective inhibitors of HIV-1 in various cells. The EC50s for DXG were 0.032 microM in CBMCs and 0.05 microM in MT-4 cells, which were generally equipotent as 3TC. 3TC-resistant, but not AZT-resistant, HIV-1 had minimum diminished sensitivity to the compounds. Both DXG and DAPD were non-toxic to cells up to 500 microM.

A high throughput colorimetric cell proliferation assay for the identification of human cytomegalovirus inhibitors.

A colorimetric assay based on the cleavage of the tetrazolium salt WST-1 has been developed for human cytomegalovirus (HCMV) antiviral susceptibility testing and adapted to a microtiter plate format. Optimal conditions were determined and the standard routine assay was calibrated with a viral input of 0.05-0.10 plaque forming unit (p.f.u.)/cell with a density of 2000 cells/well in a 96-well microtiter plate for an incubation period of 7 days. Ganciclovir (9-(2-hydroxy-1(hydroxymethyl) ethyoxymethyl) guanine; DHPG), and cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine; HPMPC) were used as positive control test compounds to validate the assay. The effective EC50 concentration values obtained with the two antiviral compounds in the present assay were in good agreement with plaque reduction assay results performed in parallel experiments. This method presents the advantage of being easy and rapid to perform, reliable, reproducible, and convenient for use in a high throughput screening capacity.

Pyrido [1,2a] indole derivatives identified as novel non-nucleoside reverse transcriptase inhibitors of human immunodeficiency virus type 1.

Pyrido [1,2a] indole derivatives were identified as potent inhibitors of human immunodeficiency virus type 1 (HIV-1) replication during a random screening programme. The compounds showed no antiviral activity against HIV-2 or in cells chronically infected with HIV-1, but had good inhibitory effect against purified HIV-1 reverse transcriptase (RT) in an in vitro assay. They were therefore classified as non-nucleoside RT inhibitors (NNRTI). The synthesis of additional compounds of the same class revealed a structure-activity relationship. The most potent compound of the series, BCH-1, had similar antiviral activity to the licensed NNRTI nevirapine against laboratory strains of HIV-1 cultured in cell lines and primary clinical isolates of HIV-1 cultured in peripheral blood mononuclear cells. However, BCH-1 showed greater cytotoxicity, providing a narrow selectivity index in the order of 35. BCH-1 had equivalent antiviral activity against viruses resistant to the nucleoside RT inhibitors zidovudine, didanosine and lamivudine and maintained better activity (less than threefold change in IC50) than nevirapine against viruses resistant to a range of NNRTIs with the single amino acid changes L100I, K103N, E138K or Y181C in the RT. Viruses with single V106A or Y188C amino acid changes showed five- and 10-fold resistance to BCH-1, respectively, in contrast to nevirapine, which had a > 100-fold change in IC50. However, virus with both V106A and Y188C amino acid changes showed higher level resistance (> 15-fold) to BCH-1. Virus with > 10-fold resistance to BCH-1 was rapidly selected for after growth in increasing concentrations of compound and was shown to be cross-resistant to nevirapine. Sequencing of this virus revealed two amino acid changes at positions 179 (V to D) and 181 (Y to C) in the RT. BCH-1 represents a new class of NNRTI, which may act as a lead to identify more selective compounds.

Convenient approaches to the synthesis of oligonucleotide macrocycles containing non-nucleotide linkers.

Two convenient, practical routes to the synthesis of non-nucleotide bridged cyclic oligonucleotides have been developed. The first procedure included circularization of oligonucleotides by template-directed ligation on solid phase, while the second procedure involved preparation of a circular oligomer by non-template chemical ligation of a linear precursor in solution. Using these approaches, a series of single- and double-stranded cyclic oligonucleotides with non-nucleotide bridges has been synthesized.

Human immunodeficiency virus glycoprotein gp120 as the primary target for the antiviral action of AR177 (Zintevir).

The human immunodeficiency virus (HIV) inhibitor AR177 (T30177, Zintevir) has been identified as a potent inhibitor of HIV integrase in vitro. The compound is currently the subject of clinical phase I/II trials. However, the primary target for the mechanism of action in vivo has not been identified unequivocally. We have found that AR177 inhibits syncytium formation between MOLT-4 cells and HUT-78 cells persistently infected with the HIV-1IIIB or NL4-3 strain, at a 50% effective concentration of 3 microg/ml, roughly 3-fold higher than the concentration required to inhibit HIV replication. Furthermore, flow cytometric analysis has shown that AR177 at 25 microg/ml interferes with the binding of the monoclonal antibody 9284 (directed to the V3 loop of gp120) on HIVIIIB-infected HUT-78 cells, pointing to inhibition of virus binding or virus fusion as the mechanism of action of AR177. To precisely characterize the site/target of intervention by AR177, we have selected HIV-1 (NL4-3) strains resistant to AR177. The binding of the AR177-resistant strain, unlike the parental HIV-1 NL4-3 strain, could not be inhibited by AR177. The resistant phenotype was associated with the emergence of mutations in the gp120 molecule. DNA sequence analysis revealed the presence of the K148E, Q278H, K290Q, and F391I mutations and a deletion of 5 amino acids (FNSTW) at positions 364-368 in the V4 region of the resistant strain but not of the wild-type HIV strain. Selection of resistant strains, although it takes a relatively long time to develop, may also select for strains with lower replicative capacity. No mutations were found in the integrase enzyme gene. Our data argue against HIV integrase being the primary target for the mechanism of anti-HIV action of AR177.

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 virus type 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.

Ion selective folding of loop domains in a potent anti-HIV oligonucleotide.

Previously, we have described inhibition of HIV-1 infection by T30177, 5'-(GTGGTGGGTGGGTGGGT)-3', an oligonucleotide that is a potent inhibitor of HIV-1 integrase in vitro (Mazumder et al. (1996) Biochemistry 35, 13762). Here a family of oligonucleotides, analogs of T30177, has been studied. On the basis of thermal denaturation, we show that a folded structure of T30177 is much more stable than that of the thrombin binding aptamer, which only differs with T30177 in the loop sequence. Sequence changes reveal that loop interactions are solely responsible for this observed stability difference. In the presence of K+ ion, the fold of T30695, a designed 16mer derivative, is indeed more stable than T30177. Loop folding within T30695 is very ion selective. Quantitative analysis of thermal denaturation suggests that the loops of T30695, 5'-(GGGTGGGTGGGTGGGT)-3', and T30177 confer the ability to coordinate three equivalents of K+ ion (one bound to the core octet and two bound to the loops); however, the thrombin binding aptamer is shown to bind only one K+ equivalent. Folding kinetics and CD titration demonstrate that K+-induced folding of T30695 and T30177 is a two-step process, consistent with a sequential model in which a first equivalent of K+ binds to the octet core, followed by slow K+-induced rearrangement of the loop domains. Comparing structural stability with the capacity of the folded oligomers to inhibit the HIV-1 integrase enzyme in vitro or HIV-1 infection in cell culture, we have found that the folding and activity data are highly correlated, suggesting that formation of an orderly, ion-coordinated loop structure similar to that in T30177 or T30695 may be a prerequisite for both integrase inhibition and anti-HIV-1 activity.

Mode of interaction of G-quartets with the integrase of human immunodeficiency virus type 1.

Oligonucleotides that can form a highly stable intramolecular four-stranded DNA structure containing two stacked guanosine-quartets (G-quartets) have been reported to inhibit the replication of the human immunodeficiency virus type 1 (HIV-1) in cell culture. Two possible mechanisms for the observed antiviral activity have been proposed: interference with virus adsorption to the cell and/or inhibition of HIV-1 integrase. We investigated the molecular interaction of G-quartet-containing oligonucleotides with HIV-1 integrase in comparison with random oligonucleotides and dextran sulfate. The prototypical G-quartet-containing oligonucleotide, T30177 (Zintevir), inhibited the overall integration reaction with an IC50 value of 80 nM. A random oligonucleotide was 10-fold less potent, but dextran sulfate was more potent, with an IC50 value of 7 nM. We developed novel kinetic assays to dissect the overall integration reaction in three steps: the formation of the initial stable complex (ISC), the 3'-processing reaction, and the DNA strand-transfer step. We then analyzed the kinetics of the ISC formation and 3'-processing. The rate constant determined for the conversion of ISC into the cleaved product was 0.08 +/- 0.01 min-1. T30177 did not inhibit 3'-processing or DNA strand transfer, whereas dextran sulfate inhibited DNA strand transfer to some extent. Binding studies using surface plasmon resonance technology revealed that both T30177 and dextran sulfate were capable of preventing the binding of integrase to specific DNA. We propose a model in which the interaction of HIV-1 integrase with G-quartets results in the inhibition of the formation of the ISC between integrase and substrate DNA. Finally, we selected for an HIV-1 strain that was resistant to T30177 in cell culture. DNA sequence analysis revealed mutations in the envelope glycoprotein gp120 but not in the integrase gene. Although gp120 seems to be the main target for the antiviral activity in cell culture of G-quartets, the study of their specific inhibition of HIV-1 integrase may lead to the development of effective integrase inhibitors.

Sequence-specific inhibition of the tumor necrosis factor-alpha receptor I gene by oligodeoxynucleotides containing N7 modified 2'-deoxyguanosine.

Tumor necrosis factor-alpha (TNF-alpha) is a highly pleiotropic cytokine produced mainly by activated macrophages. This cytokine has been found to mediate the growth of certain tumors, the replication of HIV-1, septic shock, cachexia, graft-versus-host disease, and autoimmune diseases. The binding of TNF-alpha to the p55 tumor necrosis factor receptor type I (TNFRI) is considered one of the initial steps responsible for the multiple physiologic effects mediated by TNF-alpha. The role of TNF-alpha as an inflammatory mediator through TNFRI makes both of these genes attractive targets for intervention in both acute and chronic inflammatory diseases. We have designed antisense oligodeoxynucleotides (ODNs) containing chemically modified purine and pyrimidine bases that specifically inhibit TNFRI expression and functions. These ODNs were designed to hybridize to the 3'-polyadenylation signal region of the TNFRI gene. In cell-based assays, gene-specific antisense inhibition occurred in a dose-dependent fashion at submicromolar concentrations in the presence of cellular uptake enhancing agents. Within ODN sets with a common pattern of stabilizing backbone substitution, the inhibition of the gene expression is found to be correlated with the affinity of the ODNs for their cognate mRNA target sites, providing direct evidence for an antisense mechanism of action. In addition, events triggered by the binding of TNF-alpha to TNFRI, such as the production of IL-6 and IL-8, were significantly reduced by treatment of cells with the anti-TNFRI ODN. Therefore, antisense ODNs can be used to control biologic processes mediated by TNF-alpha and may be useful as therapeutic agents to treat conditions resulting from overproduction of TNF-alpha.