Inhibition of respiratory syncytial virus replication by antisense oligodeoxyribonucleotides.

Oligodeoxyribonucleotides targeted against respiratory syncytial virus (RSV) genomic RNA inhibited RSV replication in cell culture by an apparent antisense mechanism. HEp-2 cells were infected with RSV strain A2 and incubated in the presence of oligonucleotides. Virus replication was measured by enzyme-linked immunosorbent assay (ELISA), virus yield assay, or production of specific RSV mRNAs. Using ELISA, 50% effective concentration (EC50) values were about 0.5-1 microM for an antisense oligonucleotide targeted to the start of the NS2 gene. All oligonucleotides inhibited virus antigen production as measured by ELISA. In all assays, this antisense oligonucleotide was more potent than: (1) a control oligonucleotide containing the reverse sequence; (2) oligonucleotides targeted at RSV mRNA; (3) a random sequence oligonucleotide; and (4) ribavirin. Reverse transcriptase polymerase chain reaction (PT-PCR) showed sequence specific depletion of the genomic RNA target following treatment of cells with the antisense oligonucleotide. Specific cleavage of the genomic target RNA has been detected at the antisense oligonucleotide binding site, suggesting that cellular Rnase H participates in the reaction. These results indicate that antisense oligonucleotides targeted against RSV genomic RNA can effectively inhibit RSV replication and may have therapeutic value.

Antisense oligodeoxynucleotides against the BZLF1 transcript inhibit induction of productive Epstein-Barr virus replication.

Expression of the Epstein-Barr virus (EBV) BZLF1 gene product, ZEBRA, in latently infected cells is sufficient to induce the viral lytic cycle. The use of oligodeoxynucleotides complementary to the BZLF1 transcript was studied to inhibit this induction of productive viral replication. For this purpose, we employed oligodeoxynucleotides complementary to the translation initiation codons and their flanking sequences. Incubation of Akata cells with the 25-mer phosphodiester (PO)- or phosphorothioate (PS)-antisense oligodeoxynucleotides for 3 h before stimulation with anti-immunoglobulin G antibodies (anti-IgG) partially inhibited the anti-IgG-mediated induction of ZEBRA synthesis. Both the PO- and PS-antisense oligodeoxynucleotide treatments also suppressed the productive EBV replication (as measured by linear DNA production) in a dose-dependent manner, with much greater efficiency than did PO and PS-oligodeoxynucleotides with sense, reverse or random sequences of the same length. Another 20-mer antisense oligodeoxynucleotide complementary to sequences downstream of the translation initiation codons showed a similar inhibitory effect on EBV replication. However, the inhibition was considerably lower when the cells were treated with oligodeoxynucleotides complementary to sequences upstream of the start codons. These results indicate that BZLF1 antisense oligodeoxynucleotides inhibit the viral activation in a sequence-specific fashion. In the virus-producer cell line P3HR-1, the same PS-antisense oligodeoxynucleotides also partially suppressed the spontaneous viral replication after 6-10 days, substantially more than the PS-random oligodeoxynucleotides. Inhibition of BZLF1 appears to be sufficient to suppress the induction of EBV replication.

Anti-AIDS agents--XIX. Neotripterifordin, a novel anti-HIV principle from Tripterygium wilfordii: isolation and structural elucidation.

A new kaurane type diterpene lactone, neotripterifordin (1), has been isolated from the roots of Tripterygium wilfordii. The structure of 1 was elucidated by spectroscopic methods, which included the concerted application of a number of 2-D NMR techniques including 1H-1H COSY, phase-sensitive NOESY, HETCOR, and long-range HETCOR. Compound 1 showed potent anti-HIV replication activity in H9 lymphocyte cells with an EC50 of 25 nM and TI of 125.

Anti-AIDS agents, 11. Betulinic acid and platanic acid as anti-HIV principles from Syzigium claviflorum, and the anti-HIV activity of structurally related triterpenoids.

Betulinic acid [1] and platanic acid [2], isolated from the leaves of Syzigium claviforum, were found to be inhibitors of HIV replication in H9 lymphocyte cells. Evaluation of anti-HIV activity with eight derivatives of 1 revealed that dihydrobetulinic acid [3] was also a potent inhibitor of HIV replication. The C-3 hydroxy group and C-17 carboxylic acid group, as well as the C-19 substituents, contribute to enhanced anti-HIV activity. The inhibitory activity of these compounds against protein kinase C (PKC) was also examined, since a correlation between anti-HIV and anti-PKC activities has been suggested. However, there was no apparent correlation between anti-HIV activity and the inhibition of PKC among these compounds.

Anti-AIDS agents, 10. Acacetin-7-O-beta-D-galactopyranoside, an anti-HIV principle from Chrysanthemum morifolium and a structure-activity correlation with some related flavonoids.

An active anti-HIV principle, acacetin-7-O-beta-D-galactopyranoside, has been isolated from Chrysanthemum morifolium. Seven additional flavonoids isolated from this plant, 13 known related flavonoids, and 14 synthetic flavonoids were also evaluated as inhibitors of HIV replication in H9 cells. A known flavone, chrysin, was found to be the most promising compound in this series. Flavonoids with hydroxy groups at C-5 and C-7 and with a C-2-C-3 double bond were more potent inhibitors of HIV growth. In general, the presence of substituents (hydroxyl and halogen) in the B-ring increased toxicity and/or decreased activity.

Anti-aids agents, 6. Salaspermic acid, an anti-HIV principle from Tripterygium wilfordii, and the structure-activity correlation with its related compounds.

Salaspermic acid [1], an inhibitor of HIV reverse transcriptase and HIV replication in H9 lymphocyte cells, was isolated from the roots of Tripterygium wilfordii for the first time. The structure of 1 derived from spectral data was established unequivocally by an X-ray analysis of crystals of the monohydrate. A structure-activity correlation of 1 with ten related compounds indicated that the acetal linkage in ring A and the carboxyl group in ring E of 1 may be required for the anti-HIV activity.

Anti-AIDS agents, 4. Tripterifordin, a novel anti-HIV principle from Tripterygium wilfordii: isolation and structural elucidation.

A new kaurane-type diterpene lactone, tripterifordin [1], has been isolated from the roots of Tripterygium wilfordii. The structure of 1 was elucidated by spectroscopic methods, including the concerted application of a number of 2D nmr techniques that involved the 1H-1H COSY, heteronucleus-detected variants of the heteronuclear chemical shift correlation (HETCOR), phase-sensitive NOESY, and long-range HETCOR. Compound 1 shows anti-HIV replication activity in H9 lymphocyte cells with an EC50 of 1 microgram/ml.

Anti-AIDS agents, 3. Inhibitory effects of colchicine derivatives on HIV replication in H9 lymphocyte cells.

A series of colchicine and isocolchicine derivatives were evaluated as inhibitors of HIV replication in H9 lymphocytes. Colchicine showed only very slight inhibition in the absence of toxicity, as measured by the therapeutic index (IC50/EC50). None of the derivatives inhibited HIV replication in the absence of toxicity.

Anti-AIDS agents, 2: Inhibitory effects of tannins on HIV reverse transcriptase and HIV replication in H9 lymphocyte cells.

Nine tannins, including gallo- and ellagitannins, were evaluated as potential inhibitors of HIV replication. 1,3,4-Tri-O-galloylquinic acid [1], 3,5-di-O-galloyl-shikimic acid [2], 3,4,5-tri-O-galloylshikimic acid [3], punicalin [6], and punicalagin [7] inhibited HIV replication in infected H9 lymphocytes with little cytotoxicity. Two compounds, punicalin and punicacortein C [8], inhibited purified HIV reverse transcriptase with ID50 of 8 and 5 microM, respectively. Further studies with H9 lymphocytes indicated that chebulagic acid [5] and punicalin did not inactivate virus directly. However, 1,3,4-tri-O-galloylquinic acid and 3,5-di-O-galloylshikimic acid were more effective inhibitors under those conditions. All tannins appear to inhibit virus-cell interactions. Thus, inspite of their anti-RT activity, the mechanism by which tannins inhibit HIV may not be associated with this enzyme.

Anti-AIDS agents, 1. Isolation and characterization of four new tetragalloylquinic acids as a new class of HIV reverse transcriptase inhibitors from tannic acid.

Four new tetragalloylquinic acids, 3,5-di-O-galloyl-4-O-digalloylquinic acid, 3,4-di-O-galloyl-5-O-digalloylquinic acid, 3-O-digalloyl-4,5-di-O-galloylquinic acid, and 1,3,4,5-tetra-O-galloylquinic acid, were isolated and characterized from a commercial tannic acid as a new class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitor. Compounds 2, 3, and 4 inhibit HIV RT activity 90, 89, and 84% at 100 microM and 73, 70, and 63% at 30 microM, respectively. Compounds 2-5 also inhibit the HIV growth in cells in the range of 61-70% with low cytotoxicity at 25 microM. The HIV cell growth inhibitory effects of these compounds at 25 microM and 6.25 microM (44-57%) are comparable to their effects against the HIV RT at 30 microM and 10 microM, respectively. The inhibitory effect of 3 against DNA polymerases indicates that the selective antiviral action of 3 is determined by more than its action with HIV RT.

DNA strand scission by bleomycin group antibiotics.

Certain properties of the bleomycin analogs deglycobleomycin A2 and decarbamoylbleomycin A2 have been characterized. In common with bleomycin A2, both deglycobleomycin A2 and decarbamoylbleomycin A2 were found to mediate DNA degradation in the presence of Fe(II) + O2. Both analogs were found to have essentially the same sequence selectivity for DNA strand scission as bleomycin A2 when a 5'-[23P]-end-labeled linear duplex DNA derived from SV40 DNA was employed as a substrate. Product analysis for the three analogs was carried out by assay for malondialdehyde (precursors) after digestion of calf thymus DNA, and also by hplc analysis of the digestion products formed from the dodecanucleotide d(CGCTTTAAAGCG). All three Fe(II) X bleomycin A2 analogs produced the same products, albeit not in the same relative amounts.

Analysis of products formed during bleomycin-mediated DNA degradation.

By the use of DNA, copolymers of defined nucleotide composition, and a synthetic dodecanucleotide having putative bleomycin cleavage sites in proximity to the 5'- and 3'-termini, the products formed concomitant with DNA strand scission have been isolated and subjected to structural identification and quantitation via direct comparison with authentic synthetic samples. The products of DNA strand scission by Fe(II)-bleomycin include oligonucleotides having each of the four possible nucleoside 3'-(phosphoro-2''-O-glycolates) at their 3'-termini, as well as the four possible base propenals. At least for 3-(adenin-9'-yl)propenal and 3-(thymin-1'-yl)propenal, the products formed were exclusively of the trans configuration.

DNA damage induced by bleomycin in the presence of dibucaine is not predictive of cell growth inhibition.

Growth inhibition and cell killing by bleomycin are believed to be related to the ability of this antibiotic to cleave chromosomal DNA. Because bleomycin has an intracellular site of action, its ability to cross biological membranes must be critical to its overall effectiveness as an antitumor agent. The local anesthetic dibucaine acts to enhance membrane fluidity; therefore, the reported ability of this local anesthetic to modulate bleomycin effects on KB cells was investigated. Cells were treated with various bleomycin congeners in the presence or absence of dibucaine for 24 h. Dibucaine enhanced the inhibition of cell growth mediated by bleomycin A2, demethylbleomycin A2, bleomycin B2, and isobleomycin A2. N-Acetylbleomycin A2 did not inhibit cell growth in the absence of dibucaine, but it was inhibitory in the presence of dibucaine. Cells treated simultaneously for analysis of DNA breakage on alkaline sucrose gradients revealed that breakage was also enhanced in the presence of dibucaine. The degree of enhancement varied with dose and bleomycin congener. N-Acetylbleomycin A2 did not induce DNA breakage in either the absence or the presence of dibucaine. While growth inhibition and net DNA breakage correlated reasonably well in the absence of dibucaine for each bleomycin analogue tested, proportionality was lost in the presence of dibucaine, and very little DNA breakage was present when growth inhibition was complete. These observations imply that, at least in the presence of dibucaine, bleomycin may mediate growth inhibition at some locus in addition to chromosomal DNA and, also, that a given net amount of bleomycin analogue induced DNA damage per se does not produce a specific degree of growth inhibition.

Bleomycin may be activated for DNA cleavage by NADPH-cytochrome P-450 reductase.

In the presence of NADPH and O2, NADPH-cytochrome P-450 reductase was found to activate Fe(III)-bleomycin A2 for DNA strand scission. Consistent with observations made previously when cccDNA was incubated in the presence of bleomycin and Fe(II) + O2 or Fe(III) + C6H5IO, degradation of DNA by NADPH-cytochrome P-450 reductase activated Fe(III)-bleomycin A2 produced both single- and double-strand nicks with concomitant formation of malondialdehyde (precursors). Cu(II)-bleomycin A2 also produced nicks in SV40 DNA following activation with NADPH-cytochrome P-450 reductase, but these were not accompanied by the formation of malondialdehyde (precursors). These findings confirm the activity of copper bleomycin in DNA strand scission and indicate that it degrades DNA in a fashion that differs mechanistically from that of iron bleomycin. The present findings also-establish the most facile pathways for enzymatic activation of Fe(III)-bleomycin and Cu(II)-bleomycin, provide data concerning the nature of the activated metallobleomycins, and extend the analogy between the chemistry of cytochrome P-450 and bleomycin.