Structural characterization of partially thiolated poly(cytidylic acid)

Ho, Yau-Kwan; Fiel, Robert J.; Aradi, J.; Bardos, Thomas J.

doi:10.1021/bi00592a017

Cited by 7 publications

(5 citation statements)

References 19 publications

(20 reference statements)

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“…The inhibitory potential was increased when the modified nucleotide content was higher. Due to the nature of the chemical modification the distribution of the thiolated bases within the oligonucleotide must be random as was found in the case of partially 5-thiolated poly(C) [7].…”

Section: S Tdkos J Aradi/febs Letters 396 (1996) 43~16mentioning

confidence: 99%

“…The most thoroughly studied inhibitory template analogs tre 5-mercaptopyrimidine-containing polynucleotides [7][8][9].…”

Section: • Introductionmentioning

confidence: 99%

“…Their acivities are not strictly dependent on specific nucleic acid seluences. In addition to the nucleoside and non-nucleoside 'ype inhibitors the modified oligonucleotides are another class ,~f RT inhibitors.The most thoroughly studied inhibitory template analogs tre 5-mercaptopyrimidine-containing polynucleotides [7][8][9].['hey are potent inhibitors of the replication of HIV in prinary human lymphocytes [10]. The poly(U60,hsSU40) proved o be a potent competitive inhibitor of RT [11].…”

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confidence: 99%

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(s⁴dU)₃₅: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

Tőkés

Aradi

1996

FEBS Letters

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.~ey wordr." Inhibition; Reverse transcriptase; Human ~mmunodeficiency virus; Oligo 4-thio-2'-deoxyuridylate: ~mtitemplate • IntroductionReverse transcriptase (EC 2.7.7.49) represents one of the main targets in the development of the chemotherapy against HIV, the etiological agent of the acquired immunodeficiency .yndrome [1]. Therefore, a large number of nucleoside and non-nucleoside RT inhibitors have been studied recently as i~otential agents against AIDS [2][3][4][5].Chemically modified oligo-and polynucleotides have also ~een studied and characterized as inhibitory template (or '~rimer) analogs for DNA polymerases including RT. They rove been termed antitemplates [6]. The antitemplates interact vith proteins (i.e. template dependent nucleic acid polynerases) rather than nucleic acids (antisense effect). Their acivities are not strictly dependent on specific nucleic acid seluences. In addition to the nucleoside and non-nucleoside 'ype inhibitors the modified oligonucleotides are another class ,~f RT inhibitors.The most thoroughly studied inhibitory template analogs tre 5-mercaptopyrimidine-containing polynucleotides [7][8][9].['hey are potent inhibitors of the replication of HIV in prinary human lymphocytes [10]. The poly(U60,hsSU40) proved o be a potent competitive inhibitor of RT [11]. The phos~horothioate-linked and phosphorodithioate-linked oligoleoxycytidylate analogs are also potent competitive inhibitors ff viral DNA polymerases [12][13][14][15]. A pentose modified oligomcleotide, 13-4'-thio-oligouridylate, has been shown recently o interact with HIV-1 RT [16]. It was shown earlier that poly(s4U), a base modified polyribonucleotide, inhibits the virion-associated transcriptase of influenza A [17].In this paper we report the HIV-1 RT inhibitory activity of oligonucleotides containing s4dUMP. Materials and methods MaterialsThe unlabeled deoxyribonucleotides dTTP, dGTP ('Ultrapure') as well as poly(A) and poly(C) were Pharmacia products. The labelled nucleotides [aH]dTTP and [3H]dGTP were from Amersham. All other reagents were of the highest quality commercially available.HIV-1 RT was purchased from Worthington (Freehold, NJ). Its concentration was 8 U/gl, Lot No. 63E792N. The alkaline phosphatase (grade I) and phosphodiesterase (snake venom nuclease from Crotalus durissus) were purchased from Boehringer Mannheim (Germany).The template-primers were poly(A)'(dT)a~i and poly(C)'(dGh6, prepared as described earlier [1 I].The oligodeoxycytidylates were synthesized on a Pharmacia Gene Assembler Plus DNA synthesizer in our laboratory and purified by two precipitations with n-butanol. Synthes& and character&ation of s4dU containing oligonucleotides,( dCx,s4 dUv ),~ The thiolated oligonucleotides were prepared by treating 1.5-3.5 mg of (dC)n (n = 10, 20, 25, 30, 35, 40), dissolved in aqueous pyridine, with liquid H2S [18,19] for 2-10 days at 35°C or 50°C, depending on the desired degree of thiolation. The modified oligonucleotides were isolated by ethanol precipitation as described for modified 5S rRNA [20], or ...

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Section: S Tdkos J Aradi/febs Letters 396 (1996) 43~16mentioning

confidence: 99%

“…The most thoroughly studied inhibitory template analogs tre 5-mercaptopyrimidine-containing polynucleotides [7][8][9].…”

Section: • Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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(s⁴dU)₃₅: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

Tőkés

Aradi

1996

FEBS Letters

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“…Previous reports from this laboratory described the preparation and properties of a new type of enzyme inhibitors, termed antitemplates (4), which were obtained by the introduction of a reactive 5-mercapto group into some of the cytosine and/or uracil bases of synthetic or natural RNA and DNA molecules (7,8,22,24,34). These partially thiolated polynucleotides were found to inhibit various RNA and DNA polymerases by effectively competing with the functional templates or primers for their binding sites on the enzymes and thus blocking their functions (6,29).…”

mentioning

confidence: 99%

“…It was then dissolved in 10 mM Tris-HCl buffer (pH 7.4) and purified by the appropriate size exclusion chromatography to give an approximately 60% yield-of the partially thiolated oligo-or polynucleotide, which appeared to be homogeneous (by polyacrylamide gel electrophoresis, as above). The percent thiolation (percentage of 5-mercaptopyrimidines per total bases) was determined by measuring the difference in the A334 in the presence and absence of dithiothreitol and using a semiempirical equation as described previously (22). The accuracy of this determination is ±5% of the mean value.…”

mentioning

confidence: 99%

Structure-activity relationships and mode of action of 5-mercapto-substituted oligo- and polynucleotides as antitemplates inhibiting replication of human immunodeficiency virus type 1

Bardos

Schinazi

Ling

et al. 1992

Antimicrob Agents Chemother

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Introduction of a reactive 5-mercapto group into some of the cytosine and/or uracil bases of various oligoand polynucleotides by partial thiolation resulted in several potent inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) in primary human lymphocytes. These compounds exhibited little if any toxicity against uninfected peripheral blood mononuclear cells and showed 15 to 75 times higher antitemplate activity against a p66/p5i HIV-1 recombinant reverse transcriptase (RT) than against the DNA polymerase a from human lymphocytes. In contrast, the unthiolated oligo-and polynucleotides are void of antitemplate activity, and their apparent inhibitory effect on HIV-1 closely paralleled their toxicity for the cells. Partially thiolated poly(dC) (MPdC) was the most potent of all the compounds tested against HIV-1 in peripheral blood mononuclear cells (50% effective concentration, 1.8 ,ug/ml or 0.019 ,uM), while showing low cytotoxicity (>100 ,ug/ml). The corresponding unmodified poly(dC) showed no anti-HIV-1 activity at 50 ,ug/ml but had pronounced cytotoxicity. MPdC was also a potent inhibitor of HIV-1 RT (50% inhibitory concentration, 0.30 ,ug/ml). The inhibitory activities of thiolated homooligo(dCs) against both HIV-1 replication and HIV-1 RT increased with increasing chain length. The heterooligonucleotides included in this study were designed as structural analogs of portions of the natural primer of HIV-1 RT, i.e., tRNArYs. An 18-mer analog of the 3' terminus, complementary (antisense) to the primer-binding site of the HIV-1 genome, was attached to an oligo(dC) tail and 5-thiolated; this increased its activity and decreased its toxicity. This compound will serve as a new lead in the development of more effective antitemplates against HIV-1.

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Selectivity of antitemplates as inhibitors of deoxyribonucleic acid polymerases

Cavanaugh

Hughes

et al. 1982

Biochemical Pharmacology

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Structural characterization of partially thiolated poly(cytidylic acid)

Cited by 7 publications

References 19 publications

(s⁴dU)₃₅: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

(s⁴dU)₃₅: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

Structure-activity relationships and mode of action of 5-mercapto-substituted oligo- and polynucleotides as antitemplates inhibiting replication of human immunodeficiency virus type 1

Selectivity of antitemplates as inhibitors of deoxyribonucleic acid polymerases

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Structural characterization of partially thiolated poly(cytidylic acid)

Cited by 7 publications

References 19 publications

(s4dU)35: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

(s4dU)35: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

Structure-activity relationships and mode of action of 5-mercapto-substituted oligo- and polynucleotides as antitemplates inhibiting replication of human immunodeficiency virus type 1

Selectivity of antitemplates as inhibitors of deoxyribonucleic acid polymerases

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(s⁴dU)₃₅: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase

(s⁴dU)₃₅: a novel, highly potent oligonucleotide inhibitor of the human immunodeficiency virus type 1 reverse transcriptase