Triple helix-forming oligodeoxyribonucleotides targeted to the human tumor necrosis factor (TNF) gene inhibit TNF production and block the TNF-dependent growth of human glioblastoma tumor cells.

Synthetic oligodeoxyribonucleotides (ODNs) designed to selectively inhibit the transcription or translation of specific genes are being used to modulate the activity of the targeted gene. Because multiple copies of mRNA can be transcribed from one actively expressed gene, ODNs that target double-stranded DNA and form triple helices upon binding with the gene itself have an advantage over ODNs that target the gene product (mRNA) in an antisense fashion. For the present studies, we designed four different triple helix-forming phosphodiester ODNs (TFOs) targeted to the tumor necrosis factor (TNF) gene and examined their effect on production of TNF and on cellular growth of tumors in which TNF acts as an autocrine growth factor. The ODNs J-109-50 and J-108-57 were designed to interact with polypurine oligonucleotides corresponding to the binding sites for nuclear factors kB (-237 to -208) and Sp1 (-58 to -33), respectively; J111-51 was designed to interact with a polypurine oligonucleotide in the third intron (+1429 to +1456) of the TNF gene. To enhance the cellular penetration and prevent degradation by cellular nucleases, the TFOs were modified at their 3' ends by either a cholesterol side chain or a propanolamine blocking group. Treatment of the human promonocytic cell line THP-1 with TNF-TFOs at a nontoxic concentration (2 microM) reduced the production of TNF. All of the TNF-TFOs tested were effective, and control-irrelevant TFOs were ineffective in inhibiting TNF production. The activity of the most efficacious TNF-TFOs also correlated with a decrease in TNF mRNA as observed by using reverse transcriptase PCR assays. In several tumors in which TNF acts as an autocrine growth factor, we examined the antiproliferative activity of J111-51. We found that in the human glioblastoma tumor cell line U-251, TNF-induced growth was blocked by J111-51 in a dose-dependent manner. Thus, overall results demonstrate that oligonucleotides directed to the specific regions of TNF can be designed, which may have a potential in cancer therapy.

Transactivation of human immunodeficiency virus by herpesviruses.

We examined the interaction of human immunodeficiency virus (HIV) and herpes group viruses. For this purpose, a chimeric plasmid (pLTR-CAT) was constructed in which the long terminal repeat (LTR) sequences derived from a molecular clone of HIV were fused to a bacterial chloramphenicol acetyltransferase gene (CAT). Transient expression assays in transfected tissue culture cells were used to monitor the activity of the LTR. Basal levels of CAT activity were measured in HeLa and human lung fibroblast (HLF) cells transfected with pLTR-CAT. When HeLa or HLF cells transfected with pLTR-CAT were infected with herpesviruses, HIV LTR-directed expression of the CAT gene was detected. An enhancement of the HIV LTR-directed expression of CAT was observed for herpes simplex virus (HSV)-1 and HSV-2, cytomegalovirus and varicella zoster virus. Enhanced CAT expression directed by the LTR was also shown by cotransfection of recombinant plasmids containing two non-overlapping regions of HSV-1, a fragment from HSV-2 which is non-colinear with the regions used from HSV-1, the immediate early gene of pseudorabies virus and the adenovirus early gene EIA. HIV LTR-directed expression may be a useful model for studying the effects on HIV of various infectious agents known to be present in individuals with AIDS or HIV infection.

Inhibition of human immunodeficiency virus type 1 activity in vitro by oligonucleotides composed entirely of guanosine and thymidine.

Oligonucleotide compounds composed of only deoxyguanosine and deoxythymidine were able to significantly inhibit human immunodeficiency virus type -1 (HIV-1)-induced syncytium formation and virus production (as measured by p24 core antigen expression) in an acute infection assay system. The oligonucleotides did not share any homology with or possess any complementary (antisense) sequence motifs to the HIV-1 genome. The guanosine/thymidine-containing oligonucleotides (GTOs) that showed this anti-HIV activity contained natural phosphodiester (PD) linkages (backbones) between the nucleosides. One of the PD oligonucleotide sequence motifs tested was capable of inhibiting HIV-1-induced syncytium formation and p24 production with a median effective dose in culture (ED50) in the submicromolar range. In addition, oligonucleotides tested were able to significantly suppress HIV-1 p24 levels > or = 7 days after removal of the drug from the infected cell culture medium. The growth inhibition properties (toxicity) of this genre of oligonucleotides was determined to be well above the ED50 values yielding high selective indexes. In vitro results showed that GTOs with PD backbones were potent competitive inhibitors of HIV-1 reverse transcriptase. These same molecules were capable of blocking the interaction between gp120 and CD4. All measured activities of these molecules were increased by factors of 10-500 when the PD backbone was replaced with a PT backbone in a sequence-dependent manner. The enhanced antiviral activity displayed by the sulfur group on the oligonucleotide backbone and the lack of any sequence-specific interactions suggest that a percentage of antiviral activity of oligonucleotide-based therapeutics is due to mechanisms other than those originally postulated for oligonucleotides. The good selective index of GTOs coupled with the prolonged suppression of HIV-1 in culture after removal of oligonucleotides from the infected cell culture make this a class of compounds that warrant investigation as therapeutic agents to be used against HIV-1.

Synthesis and anti-DNA viral activities in vitro of certain 2,4-disubstituted-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d d pyrimidine nucleosides.

Several novel 2,4-disubstituted-7-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)pyrrolo[2,3-d]pyrimidines have been synthesized and evaluated for their anti-human cytomegalovirus (HCMV), anti-hepatitis B virus (HBV), and anti-herpes simplex virus (HSV) activities in vitro. These nucleosides were prepared starting from 2-amino-4-chloro-7-(2-deoxy-2-fluoro- 3,5-di-O-benzoyl-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (3), which in turn was synthesized by direct glycosylation of the sodium salt of 2-amino-4-chloropyrrolo[2,3-d]pyrimidine (1) with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha-D-arabinofuranosyl bromide (2). Displacement of the 4-chloro group of 3 with OH, NH2, NHOH, SH, and SeH nucleophiles furnished the corresponding nucleosides 6-8, 12, and 14, respectively. The 3'-deoxygenation of 2-amino-4-chloro-7- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (4) and subsequent amination gave 2,4-diamino-2',3'-dideoxy derivative 19. Catalytic hydrogenation of 3 followed by debenzoylation afforded 2-aminopyrrolo[2,3-d]pyrimidine nucleoside 23. Among the compounds evaluated for their ability to inhibit the growth of HCMV (strain AD169) in MRC-5 cells using a plaque reduction assay, only 7 was significantly active in vitro with a 50% inhibitory concentration (IC50) of 3.7 micrograms/mL (TI > 125), whereas the IC50 value of ganciclovir (DHPG) was 3.2 micrograms/mL. Strain D16 of HCMV was more resistant to 7 (IC50 11 micrograms/mL) than the AD169 strain. When 7 was tested in combination with DHPG, the resultant anti-HCMV activity was found to be moderately synergistic with no evidence of antagonism. Nucleoside 7 also reduced episomal HBV replication in human hepatoblastoma 2.2.15 cells with an IC50 of 0.7 micrograms/mL (TI > 143). Development of cells harboring HBV which had become resistant to the drug was not observed with 7. Compound 7 also exhibited significant activity against herpes simplex virus types 1 and 2 (IC50 of 4.1 and 6.3 micrograms/mL, respectively) in Vero cells.

Effect of beta-enantiomeric and racemic nucleoside analogues on mitochondrial functions in HepG2 cells. Implications for predicting drug hepatotoxicity.

A group of enantiomeric nucleoside analogues with beta-D or beta-L configuration, which represent potential candidates for the treatment of hepatitis B virus (HBV) infection, were incubated in human hepatoblastoma HepG2 cells at concentrations between 0.1 and 10 microM for 4-14 days. Then the effect on mitochondrial DNA (mtDNA) content, lactic acid production, lipid droplet formation, and mitochondrial morphology were evaluated. No effect on lactic acid production was detected in cells treated with beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine (beta-L-FTC), beta-D-2',3'-dideoxy-5-fluoro-3'-thiacytidine (beta-D-FTC), racemic cis 2',3'-dideoxy-5-fluoro-3'thiacytidine [(+/-)-FTC], and 2,4-diamino-7-(2,3-dideoxy-2-fluoro-beta-D-arabinofuranosyl) pyrrolo[2',3'-d]pyrimidine (T70178), whereas a slight increase was associated with beta-D-2-hydroxymethyl-5-(2,6-diaminopurin-9-yl)-1,3-dixolane++ + (beta-D-DAPD) and 4-amino-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimi dine -5-thiocarboxamide (T70182) at 10 microM. A concentration-dependent increase in lactic acid production was observed in cells exposed to beta-D-2',3'-dideoxy-3'-thiacytidine [(+)-BCH-189], racemic cis 2',3'-dideoxy-3'-thiacytidine [(+/-)-BCH-189], beta-D-2',3'-dideoxy-5-fluorocytidine (beta-D-FddC), beta-L-2',3'-dideoxy-5-fluorocytidine (beta-L-FddC), beta-D-2-hydroxymethyl-5-(5-fluorocytosin-I-yl)-1,3,-dioxolane (beta-D-FDOC), 2,4-diamino-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) pyrrolo[2,3-d]pyrimidine (T70080), and 4-amino-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo [2,3-d]pyrimidine (T70179). Inhibition on mtDNA content was demonstrated to be concentration-dependent with (+)-BCH-189, beta-D-FddC, and T70080, whereas 3TC, (+/-)-BCH-189, beta-L-FTC, beta-D-FTC, (+/-)-FTC, beta-L-FddC, beta-D-DAPD, T70178, T70179, and T70182 had no effect. beta-D-FDOC resulted in a marked inhibition of mtDNA synthesis at 10 microM but not at lower concentrations. Cells treated with 3TC, (+/-)-BCH-189, beta-L-FTC, beta-D-FTC, (+/-)-FTC, beta-L-FddC, beta-D-DAPD, T70178, T70179, and T70182 did not show morphological changes compared with the control. In contrast, increased cytoplasmic lipid droplets associated with a loss of cristae in mitochondria were detected in cells treated with either beta-D-FDOC, beta-D-FddC, or T70080, (+)-BCH-189 treatment resulted in loss of cristae in mitochondria. In summary, 3TC, beta-L-FTC, beta-D-FTC, (+/-)-FTC, beta-D-DAPD, T70178, and T70182 exhibited a relatively safe profile, supporting their further development.

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.

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.

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.

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.

Verrucous carcinoma of the vulva associated with an unusual type 6 human papillomavirus.

An unusually rapid growing vulvar verrucous carcinoma found associated with human papillomavirus type 6 is described. Both human papillomavirus structural antigens and deoxyribonucleic acid (DNA) sequences were detected in the tumor biopsy. The human papillomavirus genome was isolated and compared with the genome of human papillomavirus type 6b, which was cloned from an anogenital wart. The two genomes were extremely homologous in genomic organization and DNA sequence, except for a region on the viral genome containing the virus control elements. A discussion of this observation and how it could relate to the importance of viral subtypes follows.

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.

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.

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.

Potassium-induced loop conformational transition of a potent anti-HIV oligonucleotide.

Spectroscopic, thermal denaturation and kinetic studies have revealed that DNA oligonucleotides 5'-d(GGGTGGGTGGGTGGGT) (T30695) and 5'-d(GTGGTGGGTGGGTGGGT) (T30177) from extremely stable intramolecular G-tetrads via a two-step process that involves the binding of one K+ ion to a central pair of G-quartets and two additional K+ ions, presumably, to the loops (Jing et al., (1997) Biochemistry in press). In that these oligonucleotides are potent HIV-1 inhibitors and among the most active HIV-1 integrase inhibitors yet identified, we have sought to further characterize the K(+)-induced folding process for the purpose of rational chemical modification of these anti-HIV agents. Our NMR investigation demonstrates that in the presence of Li+ ions, T30695 forms an unimolecular tetrad fold, stabilized by a pair of syn-anti-syn-anti G-quartets comprising a central core. The NMR spectrum of T30695 as a function of K+ titration reveals a well-defined transition that saturates upon addition of three K+ ions per oligomer. During this process, the initial Li(+)-dependent G-quartet structure converts into a highly symmetrical, stable form (the NMR detected melting transition temperature is increased by approximately 20 degrees C). The conformation of the G-quartet core remains unchanged, while the loosely structured loop residues become organized in a fashion which is stabilized by K+ ion binding and by interactions with the core. To explain these data, we propose a model wherein K+ binding to the loops induces structural rearrangement, to yield a planar array of loop bases in proximity to the underlying G-quartets. By reference to closely related homologues, which lack activity as an HIV-1 or integrase inhibitor, the possibility is discussed that this ion-coordinated loop structure is crucial to the biological activity of T30695.

Nucleic acid hybridization as a diagnostic tool for the detection of human papillomaviruses.

At this time nucleic acid hybridization tests are the most sensitive and reproduceable methods for the detection and differentiation of HPV types in clinical samples. The hybridization method of choice depends on the information desired and the availability of the proper diagnostic nucleic acid probes. Assuming most of the HPV nucleic acid probes become readily available in the near future, then the most sensitive test for screening clinical specimens--albeit the most laborious--will be the Southern blot procedure. As more information covering the involvement of HPV infections with the progression of lesions from benign to malignant is compiled, the need to know the particular subtype or status of HPV integration may become more or less important in the screening of clinical samples. If this information becomes less important, or unnecessary for a simple screening procedure, then dot blot hybridization may prove to be a much easier method for obtaining the information desired. If the sensitivity of in-situ hybridization using non-radioactive probes increases, then this method would become the fastest, easiest, and cleanest technique for the screening of a large number of clinical samples where limited information is desired. The ideal test for the future would be automated. In order to automate a test for HPV infections, the test design must become much simpler, and in order to design a simpler test, more information will be needed concerning the biology of HPV infections, how they cause benign or malignant cellular proliferation, and how the host immune system responds to HPV infections.

Studies on the transmission of viral disease via the CO2 laser plume and ejecta.

While recent reports have noted the presence of viral DNA sequences in the laser plume, no significant effort has been made to study transmission of the virus in vivo via airborne laser debris. Studies were undertaken to identify potential hazards to operating room occupants in gynecologic laser surgery. ACO2 laser in the continuous wave mode using a power density of 666 W/cm2 was fired through a 5-cm metal cylinder at virus-infected tissues. Airborne particulate debris, 100-200 microns, was removed from the cylinder's inner surfaces. In one instance, deposition of the debris was found on the surgeon's eyeglasses 1 m from the site of impact despite the use of a smoke evacuator. The first set of studies involved confirmed human papillomavirus (HPV) lesions of the human female lower genital tract. Specimens were collected for electron microscopy and Southern Blot viral hybridization. Additional cervical electron microscopy specimens were recovered from the speculum during pulsed CO2 laser treatment at 13 W average power during conization. Electron microscopy of the vulvar debris revealed only anucleate keratinized squamous epithelial cells. Cervical specimens demonstrated similar cells with nearly instantaneous vaporization of intracellular water and apparent condensation of cellular carbon. HPV Southern Blot testing revealed insufficient quantities of DNA for that technique. The second set of studies involved bovine papillomavirus lesions from dairy cattle. The debris was transmitted to susceptible animals. The bovine studies failed to demonstrate the transmission of disease in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-phase approach for the expression of high-affinity human anti-human immunodeficiency virus immunoglobulin Fab domains in Escherichia coli.

We describe here a two-phase approach for the development of high-affinity human anti-HIV immunoglobulin Fab domains in a bacterial expression system. The first phase of this technique involves the generation of human hybridoma cell lines producing high-affinity antibodies (MAbs). Anti-HIV-1 human MAbs from peripheral blood lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and from an HIV-1-seronegative volunteer immunized with HIV-1 rgp160. One MAb (T15G1), derived from the blood of the seropositive donor, was specific for HIV-1 gp41, recognized gp41 on the surface of HIV-1-infected cells and bound this antigen with an apparent dissociation constant of 4 x 10(-10) M. A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gp120 with a dissociation constant on the order of 8 x 10(-10) M, but was unable to recognize cell surface antigen. In the second phase of this technique the Fab domains of these two MAbs were molecularly cloned into a bacterial expression vector. mRNA was isolated from the M7B5 and T15G1 hybridoma cell lines and used as a template for the production of cDNA. The cDNA was amplified using the polymerase chain reaction (PCR) technique, and then fused, in frame, into a bacterial expression vector. The recombinant Fabs (rFabM7B5 and rFabT15G1) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ). DNA sequencing was used to define the gamma chain isotypes and the VH and VL chain gene usage. The binding specificities of rFabM7B5 and rFabT15G1 were indistinguishable from their respective intact MAbs.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Inhibition of high affinity basic fibroblast growth factor binding by oligonucleotides.

Oligonucleotides can be used to inhibit the binding of basic fibroblast growth factor to cells. Though standard phosphodiester oligonucleotides show a slight inhibition of binding, the oligonucleotides with phosphorothioate internucleoside linkages have inhibition levels equivalent to that of the polyanion heparin. Variations in sequence of the oligonucleotides does lead to differences in the inhibitory action of the oligonucleotides. This inhibition of basic fibroblast growth factor by phosphorothioate oligonucleotides may account for much of the published data on inhibition of various genes by proposed antisense oligonucleotides and needs to be taken into account when considering the mechanism of action of oligonucleotides in biological systems.