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

Abstract

.~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 ...

Antiretroviral effect of 4-thio-uridylate against human immunodeficiency virus type 1

“…In addition to conformational changes in cell surface receptors, coreceptors of HIV and viral envelope proteins, redox changes in these proteins are also required for successful HIV-1 entry and infection. Several inhibitors of protein disulfide isomerase (PDI) as well as cell-surface-thiol interacting agents including thioredoxin, influence HIV infection [14,15] indicating that redox processes active in viral entry could be potential targets for treatment of HIV infection, as we and others reported earlier [14][15][16][17][18].…”

Inhibition of human telomerase by oligonucleotide chimeras, composed of an antisense moiety and a chemically modified homo‐oligonucleotide

“…The (s 4 dU) 35 (abbreviated as X 35 ; X represents a 4‐thio‐deoxyuridylate unit, structure shown in Fig. 1 ) was a competitive inhibitor of HIV‐RT and human telomerase with respect to the functioning RT template‐primer and telomerase substrate primer [13,15]. The inhibitory features of X 35 prompted us to develop chimera oligonucleotides, composed of a 13mer phosphodiesther antisense sequence (AS), designed to target hTR template region and a 3′ or 5′ X n ( n = 4, 8, 16 and 24) moiety, which is thought to interact with the protein subunit of human telomerase.…”

“…Chemically modified oligonucleotides used for inhibition studies, Cy5‐labeled molecules and primers used in TP‐TRAP assay were synthesized by standard phosphoramidite chemistry and purified using anion‐exchange chromatography as described previously [13,14]. (s 4 dU) n and the chimeras were prepared by H 2 S treatment (10 days at 55 °C) of the corresponding (dC) n or (dC) n ‐extended AS/SCR [15]. The starting oligonucleotide of 3′‐blocked (s 4 dU) 8 AS was synthesized using 3′‐Spacer C3 CPG (Glen Research).…”

“…Measurement of reverse transcriptase activity was performed as we described earlier [15]. Inhibitors were preincubated with 0.1 U HIV reverse transcriptase (Amersham) in a reaction mixture containing [Me‐ 3 H]dTTP.…”

Modified Telomeric Repeat Amplification Protocol: A Quantitative Radioactive Assay for Telomerase without Using Electrophoresis

“…Therefore an oligonucleotide inhibitor of HIV reverse transcriptase, developed in our laboratory (24), was chosen as a model inhibitor to show the usefulness of the assay for characterization of a telomerase inhibitor.…”