[Gene therapy for AIDS].
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
M Matsukura
Publications and source records attributed to M Matsukura.
[Regulation of gene expression by antisense molecule].
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Animals↗
Central alpha-adrenoceptor subtypes involved in the emetic pathway in cats.
The intracerebroventricular (i.c.v.) injection of clonidine, xylazine, adrenaline and methoxamine elicited dose-dependent vomiting in cats in that order of potency. The vomiting induced by clonidine, xylazine and adrenaline was antagonized by i.c.v. yohimbine and phentolamine possessing alpha 2-adrenoceptor-blocking activity, but not by prazosin showing alpha 1-adrenoceptor-blocking activity. In contrast, methoxamine-induced vomiting was antagonized by prazosin, but not by yohimbine. The vomiting induced by xylazine and adrenaline was not prevented by i.c.v. 6-hydroxydopamine treatment, but was prevented by i.c.v. reserpine treatment. Ablation of the area postrema with some damage to extremely adjacent areas abolished the vomiting induced by each alpha-adrenoceptor agonist. These results indicate that both central alpha 1- and alpha 2-adrenoceptors are involved in the emetic pathway in cats, although alpha 2-adrenoceptors seem to have the main role. It is also suggested that monoamines, and in particular 5-hydroxytryptamine in the brain, are involved in the regulation of alpha-adrenoceptor-mediated vomiting.
Adrenergic alpha-Agonists↗
Inhibition of human immunodeficiency virus activity by phosphorodithioate oligodeoxycytidine.
Phosphorothioate oligodeoxynucleotides exert a sequence-independent cytoprotective effect against human immunodeficiency virus type 1 (HIV-1). We now report that phosphorodithioate-containing oligodeoxycytidines are very potent inhibitors of HIV-1 reverse transcriptase in vitro, as they exhibit an increasing inhibitory effect with length and number of phosphorodithioate internucleotide linkages. This inhibitory effect can be at least 30-fold greater with phosphorodithioate oligodeoxycytidine than for the corresponding phosphorothioate analog of similar length. In cell culture, phosphorodithioate oligodeoxycytidines are active inhibitors of syncytia formation and effectively inhibit de novo infection of target cells by HIV-1. Moreover, comparative experiments show that a deoxycytidine phosphorodithioate 14-mer is as effective an inhibitor of de novo infection as a phosphorothioate-containing 28-mer. Such potent inhibition by oligomers of relatively short length makes dithioate analogs an additional class of potential therapeutic agents against acquired immunodeficiency syndrome.
Antiviral Agents↗
First conference on antisense DNA and RNA in Japan.
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DNA, Antisense↗
Oligodeoxyribonucleotide phosphorothioate fluxes and localization in hematopoietic cells.
An antisense oligonucleotide phosphorothioate, previously shown to inhibit HIV-1 viral expression in chronically infected H9 cells, was fluorescently labeled to study oligonucleotide fluxes and localization within living cells. Observations based on flow cytometry and fluorescence microscopy show the following: within around 0.5-2 h, an apparent steady-state distribution of the oligonucleotide is achieved in which the intracellular oligonucleotide concentration is less than that present in the external medium; following oligonucleotide uptake and resuspension of the cells in oligonucleotide-free medium, an oligonucleotide efflux, with a time constant similar to that for uptake, is observed (although a significant fraction of the phosphorothioate remains within the cell); cellular uptake as a function of the external oligonucleotide concentration is nonlinear, being more efficient at lower concentrations (less than 2 microM); and a predominant oligonucleotide localization within the cell nucleus and perinuclear organelles is observed.
Base Sequence↗
Synthesis and antiarrhythmic activity of 2,2-dialkyl-1'-(N-substituted aminoalkyl)-spiro-(chroman-4,4'-imidazolidine)-2',5'-diones.
A novel series of 2,2-dialkyl-1'-(N-substituted aminoalkyl)-spiro-[chroman-4,4' -imidazolidine]-2',5'-diones was synthesized and evaluated for antiarrhythmic activity in chloroform- or/and aconitine-induced ventricular arrhythmia in mice. Among these compounds, (-)-6-chloro-2,2-dimethyl-1'-[3-(4-hydroxypiperidino)propyl] -spiro-[chroman-4,4'-imidazolidine]-2',5' -dione was found to be more effective than reference agents and was selected for further development.
Animals↗
Synthesis and cardiovascular activity of phenylalkylamine derivatives. I. Potential specific bradycardic agents.
A series of acyclic amide derivatives of N-(omega-aminoalkyl)-N-methylhomoveratrylamine was synthesized and evaluated for their bradycardic activity in isolated guinea pig right atria. Among these compounds, (E)-N-[3-[N'-[2-(3,4-dimethoxyphenyl)ethyl]-N'-methylamino]propyl]- 4-[3,4-(methylenedioxy)phenyl]-3-butenamide (35) was the most potent in vitro and was also found to show dose-dependent bradycardia without remarkable reduction of left ventricular dp/dtmax or mean aortic pressure in anesthetized dogs.
Animals↗
Abasic oligodeoxyribonucleoside phosphorothioates: synthesis and evaluation as anti-HIV-1 agents.
The syntheses and anti-HIV-1 evaluations of two, abasic oligodeoxyribonucleotide phosphorothioate analogs, d[Cps(Eps)26C] and d[Cps(Vps)26C] (where E and V derive from 1,2-dideoxy-D-ribofuranose and (+/-)-butane 1, 3-diol, respectively), are described.
Antiviral Agents↗
Cyclobut-A and cyclobut-G, carbocyclic oxetanocin analogs that inhibit the replication of human immunodeficiency virus in T cells and monocytes and macrophages in vitro.
Two newly synthesized carbocyclic oxetanocin analogs, (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]adenine (cyclobut-A) and (+/-)-9-[(1 beta,2 alpha,3 beta)-2,3-bis(hydroxymethyl)-1-cyclobutyl]guanine (cyclobut-G) were tested for activity against the infectivity of human immunodeficiency virus (HIV) in vitro. A number of other carbocyclic oxetanocin analogs failed to exert good antiretroviral effects. Both cyclobut-A and cyclobut-G protected CD4+ ATH8 cells against the infectivity and cytopathic effect of HIV type 1 (HIV-1) and suppressed proviral DNA synthesis in ATH8 cells exposed to HIV-1 in vitro at concentrations of 50 to 100 microM. These compounds also inhibited the in vitro infectivity of another human pathogenic retrovirus, HIV-2. Furthermore, both compounds completely suppressed the replication of a monocytotropic strain of HIV-1 in monocytes and macrophages at concentrations as low as 0.5 microM, as assessed by inhibition of HIV-1 p24 gag protein production. We also found that 2'-deoxyguanosine readily reversed the antiretroviral activity of cyclobut-G in our system, whereas the activity of cyclobut-A was hardly reversed by 2'-deoxyadenosine or 2'-deoxycytidine. We noted, however, that these compounds inhibited the proliferation of peripheral blood mononuclear cells at concentrations of greater than or equal to 100 microM in vitro. Although both cyclobut-A and cyclobut-G appear to have a certain level of in vitro toxicity, our observations may have theoretical and clinical implications in understanding the structure-activity relationships of antiretroviral agents active against HIV.
Adenine↗
Phosphoroselenoate oligodeoxynucleotides: synthesis, physico-chemical characterization, anti-sense inhibitory properties and anti-HIV activity.
Oligodeoxynucleotides with a phosphorus atom in which one of the non-bridging oxygen atoms is substituted by selenium were prepared and investigated with respect to their antisense properties. A general synthesis of phosphoroselenoate analogs of oligonucleotides is described using potassium selenocyanate as the selenium donor. The compounds, characterized by 31P NMR, were shown to decompose to phosphate with a half-life of ca. 30 days. Melting temperatures of duplexes between poly(rA) or poly(rI) with oligo(dT) and oligo(dC), respectively, indicate diminished hybridization capability of phosphoroselenoate oligomers relative to both the unmodified phosphodiester oligomers and the phosphorothioate congeners. A phosphoroselenoate 17-mer is a sequence specific inhibitor of rabbit beta-globin synthesis in wheat germ extract and in injected Xenopus oocytes. In contrast phosphoroselenoate analogs are potent non-sequence specific inhibitors in rabbit reticulocyte lysate. In vitro HIV assays were carried out on a phosphoroselenoate sequence and compared with a phosphorothioate analogue that has previously been shown to exhibit anti-HIV activity (Matsukura et al., Proc. Natl. Acad. Sci. (1987) 84, 7706-7710). The phosphoroselenoate was somewhat less active, and was much more toxic to the cells.
Animals↗
Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev (art/trs) in chronically infected cells.
In this report, we demonstrate the sequence-specific suppression of viral expression in T cells chronically infected with human immunodeficiency virus 1 (HIV-1), using antisense phosphorothioate oligodeoxynucleotides. As a target for antisense intervention, we used the HIV-1 gene rev, which is essential for viral replication and regulates the expression of virion proteins, in part, by affecting the splicing of the viral mRNA. A phosphorothioate oligomer complementary to the initiation sequence of HIV-1 rev had a significant and selective inhibitory effect on the production of several viral proteins in chronically HIV-1-infected T cells and drastically reduced the unspliced (genomic) viral mRNA transcripts, with relative sparing of smaller (spliced) transcripts. By contrast, the antisense sequence with unmodified normal phosphodiester linkages as well as phosphorothioate oligomers containing sense, random, homopolymeric sequences, or antisense sequence with N3-methylthymidine residues did not have an inhibitory effect on viral expression. Thus, sequence specificity and nuclease resistance were critical for the anti-viral-gene regulatory effect of the antisense molecules. The altered HIV-1 mRNA profile induced by the antisense phosphorothioate oligomer suggests that the mechanism for the inhibition of viral expression is due to an interference with the regulatory gene, rev, by translation arrest.
Base Sequence↗
Phosphorothioate oligodeoxynucleotides are potent sequence nonspecific inhibitors of de novo infection by HIV.
Phosphorothioate homo-oligodeoxynucleotides have recently been found to protect ATH-8 cells against the cytopathic effect of de novo infection by HIV. The effect is dose and chain-length dependent, with a maximum effect seen for 21-28-mers. We have now synthesized a series of phosphorothioate oligomers with mixed-based sequences and found that all of them have a dose-dependent cytoprotective effect that is maximal at an oligomer concentration of about 1-2 microM. The least effective sequences contain only A or T, and the most effective sequences have 40% GC content or greater. The results also confirm the length effect, namely that 21-mers are more cytoprotective than 14-mers.
Antiviral Agents↗
[In vitro anti-HIV activity of phosphorothioate alpha-anomeric oligodeoxynucleotides].
Oligonucleotide analogs consisting exclusively of alpha-anomeric deoxynucleoside units bridged with phosphorothioate linkages have been synthesized and tested in vitro as antiviral agents against human immunodeficiency virus (HIV) in human T cells. Two 28-mers, an homopolymer alpha-S-dC28 and an oligomer alpha-S-anti-rev complementary to the initiation site of the regulatory viral gene rev exhibited antiviral activities comparable to those reported for the corresponding beta-anomeric phosphorothioate analogs. In contrast, a nuclease-resistant homopolymer, alpha-dC28 was inactive. Their preliminary results would indicate that the origin of oligonucleotide phosphorothioate anti-HIV activity is not exclusively correlated with their higher nuclease resistance.
Base Sequence↗
Synthesis of phosphorothioate analogues of oligodeoxyribonucleotides and their antiviral activity against human immunodeficiency virus (HIV).
Nuclease-resistant phosphorothioate analogues of oligodeoxynucleotides (oligos) were synthesized by sulfurization of either internucleoside phosphite linkages, in a repetitive manner during chain extension, or internucleoside hydrogen phosphonate linkages, in a single step following chain assembly. These analogues were tested as antiviral agents against human immunodeficiency virus (HIV). In a cytopathic effect inhibition assay using HIV-uninfected susceptible T cells (tetanus toxoid-specific normal T cells) co-cultured with irradiated chronically HIV-infected cells, phosphorothioate oligomers inhibited the cytopathic effect and replication of several isolates of HIV-1 and HIV-2. Thus phosphorothioate analogues of oligos could inhibit cell-to-cell transmission of the virus as well as the infection by cell-free virus particles and also could inhibit a variety of isolates of human retroviruses.
Antiviral Agents↗
Adenallene and cytallene: acyclic-nucleoside analogues that inhibit replication and cytopathic effect of human immunodeficiency virus in vitro.
Although several antiretroviral compounds are already known, almost no acyclic nucleoside derivatives lacking an oxacyclopentane have been reported to exert significant inhibition against human immunodeficiency virus type 1 (HIV-1) in vitro. We found two unsaturated acyclic nucleoside derivatives, adenallene [9-(4'-hydroxy-1',2'-butadienyl)adenine] and cytallene [1-(4'-hydroxy-1',2'-butadienyl)cytosine], that protect various CD4+ T-cell lines from the infectivity and cytopathic effect of HIV-1. These compounds inhibit the expression of HIV-1 gag-encoded protein and suppress viral DNA synthesis at concentrations that do not affect functions of normal T cells in vitro. They also inhibit the in vitro infectivity of another human retrovirus, HIV-2. Further in vitro analyses of the anti-HIV-1 activity revealed that the presence of two cumulated double bonds between the 1' and 2' carbons and between the 2' and 3' carbons confers antiretroviral activity in certain pyrimidine or purine derivatives containing a four-carbon chain. We have also found that the 4'-hydroxyl group is critical for the in vitro anti-HIV activity of adenallene. Our observations may provide structure-activity relationships for acyclic nucleoside analogues and may be of value in developing a new class of experimental drugs for the therapy of HIV-related diseases.
Adenine↗
Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus.
Nuclease-resistant phosphorothioate analogs of certain oligodeoxynucleotides have been tested in vitro as antiviral agents against human immunodeficiency virus (HIV) in human T cells. Phosphorothioate analogs complementary to HIV sequences, as well as noncomplementary analogs including homooligomers, exhibited potent antiviral activity. The antiviral activity was related to the base composition of the analogs, and longer phosphorothioates were more effective than shorter ones. A 28-mer phosphorothioate oligodeoxycytidine (S-dC28) at a concentration of 1 microM exhibited potent antiviral activity and inhibited de novo viral DNA synthesis as shown by Southern blot analysis. However, S-dC28 failed to inhibit gag expression in chronically infected T cells assessed by immunofluorescent assay at concentrations up to 25 microM. An N3-methylthymidine-containing phosphorothioate analog, which does not hybridize efficiently in vitro to complementary normal DNA, showed no antiviral activity. A 14-mer phosphorothioate oligodeoxycytidine (S-dC14) synergistically enhanced the antiviral activity of 2',3'-dideoxyadenosine, an anti-HIV nucleoside. Therefore, phosphorothioate analogs of oligodeoxynucleotides could represent a unique class of experimental therapeutic agents against the acquired immunodeficiency syndrome and related diseases. However, their mechanism of action is likely to be complex.
Base Sequence↗
Long-term inhibition of human T-lymphotropic virus type III/lymphadenopathy-associated virus (human immunodeficiency virus) DNA synthesis and RNA expression in T cells protected by 2',3'-dideoxynucleosides in vitro.
We report that 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine inhibit retroviral DNA synthesis and mRNA expression in T cells exposed to the virus that causes acquired immunodeficiency syndrome, and afford such cells long-term protection in vitro under conditions of substantial viral excess. Both 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine appear to completely block reverse transcription from viral RNA to viral DNA. Viral mRNA expression is also not detected in cells protected by the drugs throughout 30 days of culture following exposure to the virus. Purine and pyrimidine analogues as 2',3'-dideoxynucleoside-5'-triphosphate serve as substrates for the human T-lymphotropic virus type III/lymphadenopathy-associated virus reverse transcriptase to elongate a DNA chain by one residue, after which the chain is terminated. Cloned normal helper/inducer T cells exposed to a cytopathic dose of the virus, but protected by the drugs, respond normally to specific antigen in vitro. These results suggest that the drugs could be promising agents for further studies in the experimental treatment of patients infected with retroviruses.
Antiviral Agents↗