HIV-1 Integrase: High-Level Production and Screening Assay for the Endonucleolytic Activity

Billich, Andreas; Schauer, Michael; Frank, Stefan; Rosenwirth, Brigitte; Billich, S.

doi:10.1177/095632029200300206

Cited by 30 publications

(22 citation statements)

References 19 publications

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“…2 and Table 1, column A). These compounds include the topoisomerase inhibitors doxorubicin and actinomycin D (19,27), aurintricarboxylic acid (22), suramin (21), dihydroxynapthaquinone (27), three flavones (Fig. 4, compounds 6-8) (14), curcumin (15), and AZTMP (24).…”

Section: Methodsmentioning

confidence: 99%

Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules.

Farnet

Wang²,

Lipford³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

114

123

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To replicate, HIV-1 must integrate a cDNA copy of the viral RNA genome into a chromosome of the host. The integration system is a promising target for antiretroviral agents, but to date no clinically useful integration inhibitors have been identified. Previous screens for integrase inhibitors have assayed inhibition of reactions containing HIV-1 integrase purified from an Escherichia coli expression system. Here we compare action of inhibitors in vitro on purified integrase and on subviral preintegration complexes (PICs) isolated from lymphoid cells infected with HIV-1. We find that many inhibitors active against purified integrase are inactive against PICs. Using PIC assays as a primary screen, we have identified three new anthraquinone inhibitors active against PICs and also against purified integrase. We propose that PIC assays are the closest in vitro match to integration in vivo and, as such, are particularly appropriate for identifying promising integration inhibitors.Following binding of HIV-1 to a sensitive cell, the viral and cellular membranes fuse and the viral core particle is released into the cytoplasm. There the viral genomic RNA is reverse transcribed, yielding a double-stranded DNA copy of the viral RNA genome. Next, the complex of viral cDNA and proteins, the "preintegration complex" (PIC), migrates to the nucleus and covalently attaches the viral cDNA to a chromosome of the host. Integration completes the formation of a provirus, which contains all the information necessary to direct the synthesis of the viral RNAs and proteins required for the formation of new virions (for reviews, see refs. 1 and 2). Fig. 1A summarizes the DNA breaking and joining reactions involved in integration in vivo. The blunt ends of the linear product of reverse transcription are cleaved to remove two nucleotides from each 3' end. The recessed 3' ends are then joined to protruding 5' ends of breaks made in the target DNA to yield an integration intermediate (Fig. 1A, II). The points of joining of the two DNA strands are staggered by five base pairs. The DNA between the two points of joining then melts, yielding gaps at each junction between host and viral DNA.

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Section: Methodsmentioning

confidence: 99%

Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules.

Farnet

Wang²,

Lipford³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

114

123

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“…Among them, polyanionic sulfonate, suramin and dextran sulfate were the first active integrase inhibitors to be investigated in vitro [167,168]. Several sulfonamides (2-mercaptobenzenesulfonamides), aromatic disulfides and diarylsulfones have been demonstrated to inhibit integrase function at low micromolar range [143,169].…”

Section: Sulfated Compoundsmentioning

confidence: 99%

“…Several classes of DNA-binding compounds have been evaluated against integrase, including doxorubicin, mitoxantrone, phenanthroline-cuprous complex, neutropsin and bisdistamycin, based on data from wellknown DNA-binding proteins such as cellular topoisomerases [167,168,174,175]. Although they inhibit integrase activity at low concentrations, cytotoxicity and non-specificity are the main limitations in the use of these compounds [167,168,[174][175][176]. In order to develop selective HIV-1 integrase inhibitors, some groups have developed an elegant strategy using DNA-binding proteins combined with ODN that have the propensity to form specific DNA triple-helix.…”

Section: Dna Intercalatorsmentioning

confidence: 99%

Targeting HIV-1 integrase

Sayasith

Sauvé

Yelle³

2001

Expert Opinion on Therapeutic Targets

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Human immunodeficiency virus Type 1 (HIV-1) integrase is an essential enzyme for the obligatory integration of the viral DNA into the infected cell chromosome. As no cellular homologue of HIV integrase has been identified, this unique HIV-1 enzyme is an attractive target for the development of new therapeutics. Treatment of HIV-1 infection and AIDS currently consists of the use of combinations of HIV-1 inhibitors directed against reverse transcriptase (RT) and protease. However, their numerous side effects and the rapid emergence of drug-resistant variants limit greatly their use in many AIDS patients. In principle, inhibitors of the HIV-1 integrase should be relatively non-toxic and provide additional benefits for AIDS chemotherapy. There have been many major advances in our understanding of the molecular mechanism of the integration reaction, although some critical aspects remain obscure. Several classes of compounds have been screened and further scrutinised for their inhibitory properties against the HIV integrase; however, there are currently no useful inhibitors available clinically for the treatment of AIDS patients. This review describes the current knowledge of the biological functions of the HIV-1 integrase and reports the major classes of integrase inhibitors identified to date.

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“…47 Systematic screening of potential inhibitors has been undertaken using mostly purified IN-based assays. From such screens several IN inhibitor classes have now been identified, including [48][49][50][51][52][53][54] hydroxylated aromatic compounds such as aurintricarboxylic acids, 55 bis-catechols, 56 caffeic acid phenethyl ester (CAPE), 57 flavones and flavanoids, 58 curcumin, 59,60 tyrphostins, 61 lignanolides, 62 coumarin derivatives, 63 cosalanes, 64 hydrazide derivatives, 65 depside and depsidones, 66 strylquinoline derivatives, 67 and lamellarins. 68 Also some peptides have been described as IN inhibitors.…”

Section: Introductionmentioning

confidence: 99%

A New Class of HIV-1 Integrase Inhibitors: The 3,3,3‘,3‘-Tetramethyl-1,1‘-spirobi(indan)-5,5‘,6,6‘-tetrol Family

et al. 2000

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Integration is a required step in HIV replication, but as yet no inhibitors of the integration step have been developed for clinical use. Many inhibitors have been identified that are active against purified viral-encoded integrase protein; of these, many contain a catechol moiety. Though this substructure contributes potency in inhibitors, it is associated with toxicity and so the utility of catechol-containing inhibitors has been questioned. We have synthesized and tested a systematic series of derivatives of a catechol-containing inhibitor (1) with the goal of identifying catechol isosteres that support inhibition. We find that different patterns of substitution on the aromatic ring suffice for inhibition when Mn 2+ is used as a cofactor. Importantly, the efficiency is different when Mg 2+ , the more likely in vivo cofactor, is used. These data emphasize the importance of assays with Mg 2+ and offer new catechol isosteres for use in integrase inhibitors.

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HIV-1 Integrase: High-Level Production and Screening Assay for the Endonucleolytic Activity

Cited by 30 publications

References 19 publications

Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules.

Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules.

Targeting HIV-1 integrase

A New Class of HIV-1 Integrase Inhibitors: The 3,3,3‘,3‘-Tetramethyl-1,1‘-spirobi(indan)-5,5‘,6,6‘-tetrol Family

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