Inhibitors of human immunodeficiency virus integrase ( retrovirus / AIDS / topoisomerase / zinc ringer )

In an effort to further extend the number of targets for development of antiretroviral agents, we have used an in vitro integrase assay to investigate a variety of chemicals, including topoisomerase inhibitors, antimalarial agents, DNA binders, naphthoquinones, the flavone quercetin, and caffeic acid phenethyl ester as potential human immunodericiency virus type 1 integrase inhibitors. Our results show that although several topoisomerase inhibitors-including doxorubicin, mitoxantrone, ellipticines, and quercetin-are potent integrase inhibitors, other topoisomerase inhibitors-such as amsacrine, etoposide, teniposide, and camptothecin-are inactive. Other intercalators, such as chloroquine and the bifunctional intercalator ditercalinium, are also active. However, DNA binding does not correlate closely with integrase inhibition. The intercalator 9-aminoacridine and the polyamineDNA minor-groove binders spermine, spermidine, and distamycin have no effect, whereas the non-DNA binders primaquine, 5,8-dihydroxy-1,4-naphthoquinone, and caffeic acid phenethyl ester inhibit the integrase. Caffeic acid phenethyl ester was the only compound that inhibited the integration step to a substantially greater degree than the initial cleavage step of the enzyme. A model of 5,8-dihydroxy-1,4-naphthoquinone interaction with the zinc finger region of the retroviral integrase protein is proposed. Although human immunodeficiency virus (HIV) integrasemediated integration of HIV DNA into the host genome is essential to the virus life cycle, to date pharmacologic antiretroviral research has neglected this enzyme, focusing principally on agents that inhibit other virally encoded enzymes, such as HIV reverse transcriptase or HIV protease (1). In vitro systems using oligonucleotide substrates and purified integrase protein have been invaluable for investigating the DNA substrate requirements and chemical mechanisms of the reactions catalyzed by the HIV integrase (2-11). The development of an in vitro assay of integrase function now permits rapid testing of a large number of compounds as potential inhibitors of the HIV integrase. We report here an investigation of the effects of many such inhibitors on the cleavage and strand-transfer reactions; to our knowledge the only previous study is the demonstration (12) that aurintricarboxylic acid and its relatives inhibit integrase-promoted cleavage. Development of a clinically tolerable inhibitor of the HIV integrase could have profound implications for antiretroviral therapy, including potential synergy with currently available reverse transcriptase inhibitors, as well as prevention of a chronic carrier state. MATERIALS AND METHODS Materials. HIV-1 integrase protein (3.5 pmol per reaction), produced via an Escherichia coli expression vector as described (13), was obtained from R. Craigie (Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases) and stored at -70°C in 1 M NaCl/20mM Hepes, pH 7.6/1 mM EDTA/1 mM dithiothreitol/20% glycerol (wt/vol). Caffeic acid phenethyl ester (CAPE) was brought to our attention by Dezider Grunberger (Columbia University, New York), who supplied the compound. Doxorubicin, 5-iminodaunorubicin, mitoxantrone, ellipticine, ellipticinium, 9-aminoacridine, amsacrine, ditercalinium, ethidium, camptothecin, 9-aminocamptothecin, 10,11-methylenedioxycamptothecin, etoposide (VP-16), teniposide (VM-26), and quercetin were obtained through the Developmental Therapeutics Program, National Cancer Institute. Hydroxyrubicin and adriamycinone were obtained through Waldemar Priebe (M. D. Anderson Hospital, Houston). Naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 5-hydroxy-1,4-naphthoquinone, and dihydroxyanthraquinone were purchased from Aldrich. Chloroquine, primaquine, quinacrine, and amodiaquine were obtained through Sigma. Hydroxychloroquine was from SterlingWinthrop Research Institute. Mefloquine was from Hoffmann-La Roche. Oligonucleotide Substrate. Oligonucleotides were obtained from Midland Certified Reagent (Midland, TX), and were HPLC-purified before use. The following complementary oligonucleotides were used as substrates: AE118: 5'-GTGTGGAAAATCTCTAGCAGT-3' and AE117: 5'-ACTGCTAGAGATTTTCCACAC-3' (2). AE118 (1 .l of 0.1 mg/ml) was 5'-radiolabeled by treating it with [32P]ATP and polynucleotide kinase at 37°C for 45 min followed by 15 min at 85°C to inactivate the kinase. The oligonucleotide was subsequently slowly hybridized by mixing with 1 Al of AE117 at 0.4 mg/ml. Unincorporated nucleotides were separated from labeled oligonucleotide by passage through a G-25 quick spin column (Boehringer Mannheim). HIV Integrase Assay. The stock enzyme (0.44 mg/ml) was first diluted 1:3 in protein storage buffer 1 M NaCl/20 mM Hepes, pH 7.6/1 mM EDTA/1 mM dithiothreitol/20% (wt/ vol) glycerol. Subsequent enzyme dilution was at 1:20 in reaction buffer (25 mM Mops, pH 7.2/7.5 mM MnCl2/bovine serum albumin at 100 ,g/ml/10 mM 2-mercaptoethanol) to give 50 mM NaCl/1 mM Hepes/50 ,uM EDTA/50 uM dithiothreitol/10% (wt/vol) glycerol/7.5 mM MnCl2/bovine serum albumin at 0.1 mg/ml/10 mM 2-mercaptoethanol/25 mM Mops, pH 7.2. Reaction volume was 16 ,ul. All reactions were done in 10% dimethyl sulfoxide to enhance the reaction efficiency and as a universal drug solvent. Reactions were for 1 hr with 0.3 pmol of 32P-labeled double-stranded oligonucleotide. Reactions were stopped by adding 16 ,ul ofMaxamAbbreviations: DHNQ, dihydroxynaphthoquinone; CAPE, caffeic acid phenethyl ester; top2, topoisomerase II; HIV, human immunodeficiency virus. *To whom reprint requests should be addressed. 2399 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 90 (1993)