![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
-Hydroxytropolones
Laboratory of Molecular Pharmacology (E.A.S., A.A.J., C.M.) and HIV and AIDS Malignancy Branch (D.A.D., R.Y.), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Received October 25, 2005; accepted January 17, 2006
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
-hydroxytropolones) were found to inhibit HIV-1 integrase. A structure-activity relationship investigation with several tropolone derivatives from The National Cancer Institute compound repository demonstrated that the 7-hydroxy group is essential for integrase inhibition. -Hydroxytropolones preferentially inhibit strand transfer and are inhibitory both in the presence of magnesium or manganese. Lack of inhibition of disintegration in the presence of magnesium coupled with results from different cross-linking assays suggests -hydroxytropolones as interfacial inhibitors. We propose that -hydroxytropolones chelate the divalent metal (Mg2+ or Mn2+) in the enzyme active site. The most active compound against HIV-1 integrase in biochemical assays [2,4,6-cycloheptatrien-1-one, 2,7-dihydroxy-4-isopropyl (NSC 18806) IC50 = 4.8 ± 2.5 µM] exhibits weak cytoprotective activity against HIV-1IIIB in a cell-based assay. -Hydroxytropolones represent a new family of inhibitors for the development of novel drugs against HIV infection.
). The replication steps of HIV, a member of the retrovirus family, are well known and can therefore be targeted rationally (for general review, see De Clercq, 2005). After HIV binding to the host cell, viral single-stranded RNA genomes are released into the cell and serve as templates for the virus-encoded reverse transcriptase to synthesize double-stranded DNA copies bearing the long terminal repeats (LTRs) at both ends (Turner and Summers, 1999). The viral linear DNA is integrated into the host genome in a reaction catalyzed by the viral enzyme integrase (IN). Integration is essential for viral replication because integrated viral DNA (provirus) serves as a template for the synthesis of new viruses after processing by the host cell transcription-translation machines (Brown, 1990; Fesen et al., 1993; Asante-Appiah and Skalka, 1997; Van Maele and Debyser, 2005).
Antiviral therapy currently uses a combination of reverse transcriptase and HIV protease inhibitors. Inhibitors of virus fusion to the host cells have recently been developed (Barbaro et al., 2005; De Clercq, 2005). Because HIV integrase is crucial for virus replication, the search for integrase inhibitors has been ongoing (Fesen et al., 1993; Hazuda et al., 2000; Debyser et al., 2002; Deprez et al., 2004; Johnson et al., 2004; Pommier et al., 2005). Integrase inserts the proviral DNA into host chromosomes in two steps: 3' processing (3'-P) and strand transfer (ST). 3'-P is an endonucleolytic cleavage reaction removing the 3' ends of the viral LTR DNA (generally a dinucleotide pGpT for HIV-1) immediately 3' from the conserved sequence (CA for HIV-1) (Fig. 1A). ST is the insertion of the processed 3' ends of the viral DNA into the cell genome (Asante-Appiah and Skalka, 1997). The HIV-1 integrase catalytic site contains three essential amino acids: Asp64, Asp116, and Glu152 (D,D-35 E-motif) that coordinate at least one and probably two divalent cations (Mg2+ or Mn2+) between the enzyme and its DNA substrates (Engelman and Craigie, 1992; Chiu and Davies, 2004).
|
; Marchand et al., 2003; Pommier et al., 2005). 5-Chloroindolyltetrazolylpropenone has been cocrystallized in the catalytic domain of HIV integrase and shown to bind in the DDE motif (Goldgur et al., 1999). The diketo acid derivative L-731,988 was shown to block binding of target DNA in the integrase active site (Espeseth et al., 2000). The selective inhibition of the strand transfer reaction by diketo acids has been proposed to be due to their interfacial inhibition on preformed integrase-viral DNA complexes (Pommier and Marchand, 2005).
We previously reported one derivative (NSC 624404) containing the characteristic seven-membered tropolone ring as novel inhibitor of HIV-1 integrase in a four-point pharmacophore analysis of the National Cancer Institute drug database (Neamati et al., 1997). While screening the National Cancer Institute chemical library for HIV-1 integrase inhibitors, we recently found additional positive hits with tropolone derivatives. We report here the structure-activity relationship of tropolones available in the National Cancer Institute compound repository on HIV integrase activities. Tropolone derivatives are present in cupressaceous trees from genus Thuja and are probably responsible for resistance of fungal and insect attack on the heartwood (Baya et al., 2001; Diouf et al., 2002; Lim et al., 2005). Our experiments demonstrate the ability of the monomer 7-hydroxytropolones (-hydroxytropolones) to preferentially inhibit the ST reaction by interfering with the enzyme catalytic site. -Hydroxytropolone derivatives are new lead inhibitors for HIV-1 integrase.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Recombinant HIV Integrase and Oligonucleotide Substrates. Expression and purification of the recombinant HIV-1 integrase in Escherichia coli were performed according to Leh et al. (2000) and Marchand et al. (2001) with addition of 10% glycerol to all buffers. The preparation of the Q148C/SSS-mutant integrase is described in Johnson et al. (2006). The oligonucleotide substrates, except those used for the disulfide cross-linking (Fig. 5A), were purchased from Integrated DNA Technologies, Inc. (Coraville, IA) and purified by polyacrylamide gel. The sequences of DNA substrates are shown in Figs. 1A, 2A, 3A, and 6A. The single-stranded oligonucleotides were 5' end-labeled with [
-32P]ATP (PerkinElmer Life and Analytical Sciences, Boston, MA) and T4 polynucleotide kinase (New England BioLabs, Ipswich, MA). Unincorporated nucleotide was removed using mini Quick Spin Oligo columns (Roche Diagnostics, Indianapolis, IN). Substrates were obtained after annealing with complementary nonlabeled oligonucleotides. The thiol-modified substrate (Fig. 5A) for disulfide cross-linking assay was synthesized by W. Santos and G. Verdine (Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA) as described previously (He and Verdine, 2002).
|
|
|
|
-mercaptoethanol, and 7.5 mM divalent cations (MgCl2 or MnCl2 as indicated). Reactions were stopped by addition of 20 µl of loading dye (10 mM EDTA, 98% deionized formamide, 0.025% xylene cyanol, and 0.025% bromphenol blue). Reactions were heated at 95°C for 1 min before electrophoresis in 20% polyacrylamide-7 M urea gels. Gels were dried, and reaction products were visualized and quantitated with a PhosphorImager (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Densitometric analyses were performed using ImageQuant from the Molecular Dynamics software. The concentrations at which enzyme activity was reduced by 50% (IC50) was determined using Prism software (GraphPad Software Inc., San Diego, CA) for nonlinear regression to fit dose-response data to logistic curve models.
Integrase Binding to HIV DNA Using the Disulfide-Cross-Linking Assay. The disulfide cross-linking assay was described in detail previously (Johnson et al., 2006). In brief, 10 µM recombinant Q148C/SSS-mutant integrase was incubated with 10 µM DNA substrate (Fig. 5A) containing tethered thiols in the presence of 20 mM Tris, pH 7.4, 10% glycerol, and 7.5 mM divalent cations (MgCl2 or MnCl2 as indicated) for 20 min at 37°C. Reactions were stopped by the addition of 20 mM methylmethanethiosulfonate (capping reagent). Nonreducing gel loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, and 20% glycerol) was added, and samples were heated at 95°C before loading onto 16% Tricine gels (Invitrogen, Carlsbad, CA). Gels were stained with Microwave Blue according to manufacturer's recommendations (Protiga, Frederick, MD).
Otherwise, for dose-response experiments, 500 nM integrase was incubated with NSC 18806 as shown at Fig. 5C in the buffer described above for 20 min. DNA (20 nM) containing a 5' 32P label on one strand and a thiol-modified cytosine on the other strand was added, and reactions were capped with methylmethanethiosulfonate at 1 min. After capping, nonreducing gel loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, and 20% glycerol) was added, and samples were directly loaded on 16% Tricine gels (Invitrogen). Gels were dried, and reaction products were quantitated as described above.
Integrase Binding to HIV DNA Using the Schiff-Base Assay. The Schiff-base assay was performed as described previously (Mazumder and Pommier, 1995). In brief, 300 nM recombinant IN was incubated with inhibitors (at the indicated concentration) for 15 min at 37°C. Subsequently, 20 nM 5' end-labeled substrate containing the abasic oligonucleotide (Fig. 6A) was added for 10 min at room temperature in reaction buffer described above for integrase catalytic assays. A freshly prepared solution of sodium borohydride (0.1 M final concentration) was added for 5 min. An equal volume (10 µl) of 2x SDS-polyacrylamide gel electrophoresis buffer (Invitrogen) was added in each reaction. Reaction products were heated at 95°C for 1 min before analysis by electrophoresis in 12 to 20% polyacrylamide gels (Invitrogen). Gels were dried, and reaction products were quantitated using the same method as described above.
Fluorimetric HIV-1 Protease Assay. The fluorescent HIV-1 protease substrate [RE-(EDANS)-SQNYPIVQK-(DABCYL)-R] was obtained from Molecular Probes (Eugene, OR). Substrate and buffer were prewarmed at 37°C for at least 20 min before use. The protease (25 nM final concentration) was incubated in the manufacturer's recommended assay buffer (100 mM sodium acetate, 1 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% DMSO and 1 mg/ml bovine serum albumin, pH 4.7) at 37°C in the presence of 1 to 25 µM NSC 18806 for 10 min and then added to the warmed substrate solution (40 µM) containing the different treatments to initiate the reaction. Acetyl pepstatin (Sigma-Aldrich, St. Louis, MO) at 20 nM was used a positive HIV-1 protease inhibitor control. The total assay volume was 100 µl. Fluorescence was monitored for 30 min in a fluorescence microplate reader (FMAX; Molecular Devices, Sunnyvale, CA) with 355-nm excitation and 460-nm emission filters, and the rate of reactions was compared for the different conditions.
Inhibition of HIV-Induced Cytopathic Effect in Cell Culture. The MT-2 cells were grown in RPMI 1640 medium with GlutaMAX, supplemented with 10% (v/v) heat-inactivated fetal bovine serum (both from Invitrogen). The cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Every 4 to 5 days, cells were spun down and seeded at 2 x 105 cells/ml in new cell culture flasks. HIV (HTLV-IIIB isolate) was obtained from Advanced Biotechnology Incorporated (Columbia, MD). The virus stock (3.2 x 104 50% cell culture infective dose per milliliter as determined for MT-2 cells) was stored at -70°C until used. Stock solutions of compounds were diluted using medium directly into 96-well assay plate (Corning, Corning, NY).
MT-2 cells (5 x 105 cells/ml) were pretreated for 2 h with test compounds at various concentrations as indicated in Fig. 7. Cells were then infected with 100 50% cell culture infective dose or mock-infected. The cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Four days after infection, the viability of mock- and HIV-infected cells was examined spectrophotometrically by the CellTiter 96 nonradioactive cell proliferation assay (Promega, Madison, WI) and also confirmed microscopically in a hemacytometer by the trypan blue exclusion method. The percentage of cell viability in drug-treated uninfected and infected cells was determined based on the viability of the uninfected control drug-treated cells. The concentration of drug required to inhibit approximately 50% of the HIV-1-induced cytotoxicity was calculated from the plot of compound concentration versus the percentage of viable cells.
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-hydroxytropolone) resulted in inhibitory activity against HIV-1 integrase (NSC 18806 and NSC 310618). NSC 18806 was the most inhibitory against integrase in strand transfer reaction (IC50 = 4.8 ± 2.5 µM) compared with NSC 310618 (IC50 = 11.7 ± 5.2 µM) (Table 1).
|
The Tropolone NSC 18806 Inhibits Preferentially Strand Transfer in the Presence of Magnesium. For detailed characterization of NSC 18806, we compared its effect on the three reactions catalyzed by HIV integrase. 3' Processing, strand transfer, and disintegration can be independently measured in biochemical assays using specific oligonucleotides (Figs. 1A, 2A, and 3A) (Marchand et al., 2001). A divalent cation, either Mg2+ or Mn2+, is required for integrase activity in vitro (Engelman and Craigie, 1995). Mg2+, however, is the more likely cofactor in vivo. Because the integrase active site could be structurally different in the presence of Mg2+ or Mn2+ and inhibitors can act in different ways in Mg2+ or Mn2+ (Grobler et al., 2002; Neamati et al., 2002; Marchand et al., 2003), all assays were performed in the presence of either Mg2+ or Mn2+. Figures 1 to 3 show the results of representative experiments for the different assays, and Fig. 4 and Table 1 summarize the results of these three assays.
|
NSC 18806 exhibited greater potency against 3'-P and ST using the standard 21-bp oligonucleotide duplex in the presence of Mn2+ than in the presence Mg2+ (Figs. 1B and 4; Table 1). The IC50 values for 3'-P were approximately 5-fold higher than the IC50 values for ST in the presence of either Mg2+ or Mn2+. Therefore, NSC 18806 shows some selectivity for ST.
Because ST follows 3'-P in the reaction using the 21-bp DNA substrate shown in Fig. 1, independent measurement of ST was performed with a preprocessed substrate (Fig. 2A). This assay segregates the action of a compound against ST from a decrease of the integrase activity related to the 3'-P inhibition in the overall integration. Results from this assay show similar ST inhibition and comparable IC50 values for ST as were observed for overall integration (Figs. 1B, 2B, and 4; Table 1) with exception for NSC 310618.
Disintegration was suggested as a reverse reaction of ST (Chow et al., 1992) (Fig. 3A). Figure 3B shows the inability of NSC 18806 to inhibit disintegration in the presence of Mg2+. Disintegration was only inhibited in the presence of Mn2+ at high drug concentration. These results indicate that NSC 18806 is a more potent inhibitor of ST compared with disintegration (Fig. 4; Table 1).
NSC 18806 Affects the HIV-1 Integrase Catalytic Site without Inhibiting Overall DNA Binding. For determination of the possible binding site of NSC 18806 within the integrase catalytic site, we evaluated the ability of NSC 18806 to inhibit a cross-linking reaction between the cytosine in the 5'-AC dinucleotides overhang of the viral DNA and integrase glutamine 148 (Fig. 5A). A Q148C mutant form of HIV-1 integrase allows specific covalent interaction with a thiol-modified cytosine in the 5'-AC dinucleotide overhang without nonspecific interference of other integrase cysteine residues (Johnson et al., 2006).
The results of this assay show metal-dependent inhibition of integrase-DNA disulfide-cross-linking by NSC 18806 (Fig. 5B). The importance of the -hydroxy group in this inhibition is illustrated by the lack of inhibition observed for NSC 18804 (structural analog of NSC 18806 lacking the -hydroxy group) (Fig. 5B). For quantitative analysis of dose-dependent inhibition of integrase-DNA disulfide-cross-linking by NSC 18806, we performed the assay using 5' 32P-labeled DNA substrate (Johnson et al., 2006). The sensitivity of this method allows reactions to be performed with the same ratio of integrase-DNA as in the assays measuring ST. Inhibition of integrase cross-linking by NSC 18806 was observed at concentrations similar to those required to inhibit ST (Fig. 5C). Integrase-DNA complexes were represented by several bands (Fig. 5C, left), with the lowest corresponding to integrase-monomer-DNA (confirmed by Western blot experiments; data not shown). The slower migrating bands correspond to integrase-multimer-DNA complexes (dimer, trimer, tetramer, and so on) because of nondenaturing electrophoresis conditions. The IC50 for cross-linking inhibition (32 µM) is comparable with the IC50 for the ST inhibition in the presence of Mg2+ (21.6 ± 3.4 µM; Table 1).
To determine whether cross-linking inhibition could be because of inhibition of overall binding of HIV-1 integrase to the viral DNA end, we investigated the effect of tropolone derivatives using a Schiff-base assay (Mazumder and Pommier, 1995) measuring cross-linking between integrase and a DNA substrate mimicking the viral U5 LTR end and containing an abasic site corresponding to the adenine in the conserved CA-dinucleotide (Fig. 6A). Tropolone derivatives did not block the Schiff base IN-DNA interaction even at 1 mM concentration (Fig. 6B), indicating specific inhibition of disulfide cross-linking by NSC 18806.
NSC 18806 Exhibits Moderate Cytoprotective Activity against HIV-1 in Cell-Based Assay. The tropolone compounds shown in Table 1 were tested in an HIV infectivity assay (Pauwels et al., 1988). All compounds were inactive in this assay except for the NSC 18806, which showed moderate protection of infected cells from HIV-induced cytopathic effect with an estimated IC50 of approximately 12 µM (Fig. 7). The IC50 could only be estimated because of the presence of toxicity at concentrations at and above 12 µM. Note, that the cytoprotective concentration for this compound is comparable with the IC50 for integrase inhibition in vitro in the presence of Mg2+.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
), antibacterial (Morita et al., 2004), and insecticidal (Morita et al., 2003). They also exhibit antioxidant properties (Doulias et al., 2005). Specific inhibition of the RNaseH domain of HIV-1 reverse transcriptase by 7-hydroxytropolone derivatives (-hydroxytropolones) have been reported (Budihas et al., 2005). These various biological activities of tropolone derivatives have been linked with their ability to chelate metals (Matsumura et al., 2001; Budihas et al., 2005; Doulias et al., 2005). Tropolone interacts with Mg2+ by forming of a 1:1 complex (Andreu and Timasheff, 1982). The current study suggests that -hydroxytropolones may also inhibit HIV-1 integrase by chelation of one or two Mg2+ or Mn2+ in the enzyme active site (Fig. 8).
|
-hydroxytropolones interfere with the protruding 5' end of the LTR and the integrase amino group glutamine 148 in the integrase flexible loop (inhibition of disulfide cross-linking) without affecting the overall binding of integrase to the viral DNA end (no inhibition of Schiff-base cross-linking). It has been suggested that the interaction between the cytosine in the 5' overhang of the viral DNA and the Q148 occurs after a conformational change of the integrase-viral (donor) DNA complex, which is necessary for triggering ST (Johnson et al., 2006). Thus, the cross-linking results coupled with the ability of -hydroxytropolones to preferentially inhibit ST (full-length or preprocessed substrate) compared with 3'-P in the presence of Mg2+ suggest preferential binding of the -hydroxytropolones to the integrase-DNA complex after 3'-P.
The lack of inhibition of the disintegration reaction by -hydroxytropolones in the presence of Mg2+ compared with effective inhibition of the ST reaction is noticeable because disintegration corresponds to the reverse reaction of strand transfer (Chow et al., 1992). The same selectivity for strand transfer versus disintegration was shown for the diketo acid derivative L-731,988 and led to the interpretation that diketo acids bind to the target DNA site (Espeseth et al., 2000). Competition with target (acceptor) DNA could explain why NSC 18806 has a lower affinity for binding to the integrase catalytic active site if this site is already occupied by the donor and acceptor DNA, which would be the case for the disintegration substrate. Hence, -hydroxytropolones might act as interfacial inhibitors (Pommier and Cherfils, 2005; Pommier and Marchand, 2005) for HIV-1 integrase-divalent metal-DNA complexes and block the binding of the acceptor (genomic) DNA.
We find that -hydroxytropolones are more potent but less selective for ST in the presence of Mn2+ than in the presence of Mg2+. Such metal-dependent inhibition might be because of a different folding of the integrase active site in the presence of Mg2+ or Mn2+. Mn2+ is geometrically wider than Mg2+ (Huang et al., 1997; Bock et al., 1999); therefore, the catalytic site of integrase could be more "open" in the presence of Mn2+, which might allow the -hydroxytropolones to enter various conformations of this site. The lack of integrase inhibition using preprocessed substrate by NSC 310618 in the presence of Mg2+ might be indicative of different integrase configurations when ST proceeds from a precleaved (preprocessed) substrate versus a blunt end substrate.
NSC 18806 shows weak cytoprotective activity on HIV-infected cells, which seems limited by the cytotoxicity of the drug. The cytoprotection against virus could be because of drug action against other steps beside HIV replication. However, we can exclude HIV protease as NSC 18806 failed to inhibit HIV-1 protease at concentration up to 25 µM in a standard fluorescent-based HIV-1 protease assay (data not shown). It has been reported that the -hydroxytropolones inhibit the RNaseH domain of HIV-1 reverse transcriptase (Budihas et al., 2005). The topological similarity between the catalytic domain of HIV integrase and the HIV reverse transcriptase RNaseH domain could explain a common mechanism of metal chelation in the enzyme catalytic site(s) (Dyda et al., 1994; Yang and Steitz, 1995). While our study was under review, it was reported that 3,7-dihydroxytropolones also inhibit HIV integrase but at the same time block preferentially the polymerase activity of HIV reverse transcriptase over its RNaseH activity (Didierjean et al., 2005). The synthesis of more active compounds based on the 7-hydroxytropolone core is needed for developing therapeutically relevant compounds and for investigating which step of the retroviral infection is inhibited by -hydroxytropolones.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: HIV, human immunodeficiency virus; IN, integrase; 3'-P, 3'-processing; ST, strand transfer; LTR, long terminal repeat; DMSO, dimethyl sulfoxide; MOPS, 3-(N-morpholino)propanesulfonic acid; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; L-731,988, 4-(1-(4-fluoro-benzyl)-1H-pyrrol-2-yl)-2,4-dioxo-butyric acid; NSC 624404, 2-(4-{bis[2-hydroxy-5-(methylethyl)-3-oxocyclohepta-1,4,6-trienyl]methyl}phenoxy) acetic acid, sodium salt; NSC 310618, 1,2,3,4-tetrahydro-2-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one.
Address correspondence to: Dr. Yves Pommier, Laboratory of Molecular Pharmacology, Bldg. 37, Room 5068, National Institutes of Health, Bethesda, MD 20892. E-mail: pommier{at}nih.gov
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Asante-Appiah E and Skalka AM (1997) Molecular mechanisms in retrovirus DNA integration. Antiviral Res 36: 139-156.[CrossRef][Medline]
Barbaro G, Scozzafava A, Mastrolorenzo A, and Supuran CT (2005) Highly active antiretroviral therapy: current state of the art, new agents and their pharmacological interactions useful for improving therapeutic outcome. Curr Pharm Des 11: 1805-1843.[CrossRef][Medline]
Baya M, Soulounganga P, Gelhaye E, and Gerardin P (2001) Fungicidal activity of beta-thujaplicin analogues. Pest Manag Sci 57: 833-838.[CrossRef][Medline]
Bock CW, Katz AK, Markham GD, and Glusker JP (1999) Manganese as a replacement for magnesium and zinc: functional comparison of the divalent ions. J Am Chem Soc 121: 7360-7372.[CrossRef]
Brown PO (1990) Integration of retroviral DNA. Curr Top Microbiol Immunol 157: 19-48.[Medline]
Budihas SR, Gorshkova I, Gaidamakov S, Wamiru A, Bona MK, Parniak MA, Crouch RJ, McMahon JB, Beutler JA, and Le Grice SF (2005) Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones. Nucleic Acids Res 33: 1249-1256.
Chiu TK and Davies DR (2004) Structure and function of HIV-1 integrase. Curr Top Med Chem 4: 965-977.[CrossRef][Medline]
Chow SA, Vincent KA, Ellison V, and Brown PO (1992) Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus. Science (Wash DC) 255: 723-726.
Debyser Z, Cherepanov P, Van Maele B, De Clercq E, and Witvrouw M (2002) In search of authentic inhibitors of HIV-1 integration. Antivir Chem Chemother 13: 1-15.[Medline]
De Clercq E (2005) New approaches toward anti-HIV chemotherapy. J Med Chem 48: 1297-1313.[CrossRef][Medline]
Deprez E, Barbe S, Kolaski M, Leh H, Zouhiri F, Auclair C, Brochon JC, Le Bret M, and Mouscadet JF (2004) Mechanism of HIV-1 integrase inhibition by styrylquinoline derivatives in vitro. Mol Pharmacol 65: 85-98.
Didierjean J, Isel C, Querre F, Mouscadet JF, Aubertin AM, Valnot JY, Piettre SR, and Marquet R (2005) Inhibition of human immunodeficiency virus type 1 reverse transcriptase, RNase H and integrase activities by hydroxytropolones. Antimicrob Agents Chemother 49: 4884-4894.
Diouf PN, Delbarre N, Perrin D, Gerardin P, Rapin C, Jacquot JP, and Gelhaye E (2002) Influence of tropolone on Poria placenta wood degradation. Appl Environ Microbiol 68: 4377-4382.
Doulias PT, Nousis L, Zhu BZ, Frei B, and Galaris D (2005) Protection by tropolones against H2O2-induced DNA damage and apoptosis in cultured Jurkat cells. Free Radic Res 39: 125-135.[CrossRef][Medline]
Dyda F, Hickman AB, Jenkins TM, Engelman A, Craigie R, and Davies DR (1994) Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science (Wash DC) 266: 1981-1986.
Engelman A and Craigie R (1992) Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro. J Virol 66: 6361-6369.
Engelman A and Craigie R (1995) Efficient magnesium-dependent human immunodeficiency virus type 1 integrase activity. J Virol 69: 5908-5911.[Abstract]
Espeseth AS, Felock P, Wolfe A, Witmer M, Grobler J, Anthony N, Egbertson M, Melamed JY, Young S, Hamill T, et al. (2000) HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase. Proc Natl Acad Sci USA 97: 11244-11249.
Fesen MR, Kohn KW, Leteurtre F, and Pommier Y (1993) Inhibitors of human immunodeficiency virus integrase. Proc Natl Acad Sci USA 90: 2399-2403.
Goldgur Y, Craigie R, Cohen GH, Fujiwara T, Yoshinaga T, Fujishita T, Sugimoto H, Endo T, Murai H, and Davies DR (1999) Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: a platform for antiviral drug design. Proc Natl Acad Sci USA 96: 13040-13043.
Grobler JA, Stillmock K, Hu B, Witmer M, Felock P, Espeseth AS, Wolfe A, Egbertson M, Bourgeois M, Melamed J, et al. (2002) Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes. Proc Natl Acad Sci USA 99: 6661-6666.
Hazuda DJ, Felock P, Witmer M, Wolfe A, Stillmock K, Grobler JA, Espeseth A, Gabryelski L, Schleif W, Blau C, et al. (2000) Inhibitors of strand transfer that prevent integration and inhibit HIV-1 replication in cells. Science (Wash DC) 287: 646-650.
He C and Verdine GL (2002) Trapping distinct structural states of a protein/DNA interaction through disulfide crosslinking. Chem Biol 9: 1297-1303.[CrossRef][Medline]
Huang Y, Beaudry A, McSwiggen J, and Sousa R (1997) Determinants of ribose specificity in RNA polymerization: effects of Mn2+ and deoxynucleoside monophosphate incorporation into transcripts. Biochemistry 36: 13718-13728.[CrossRef][Medline]
Johnson A, Santos W, Pais GC, Marchand C, Amin R, Burke TR Jr, Verdine G, and Pommier Y (2006) Integration requires a specific interaction of the donor DNA terminal 5'-cytosine with glutamine 148 of the HIV-1 integrase flexible loop. J Biol Chem 281: 461-467.
Johnson AA, Marchand C, and Pommier Y (2004) HIV-1 integrase inhibitors: a decade of research and two drugs in clinical trial. Curr Top Med Chem 4: 1059-1077.[CrossRef][Medline]
Kellerman S, Begley E, Boyett B, Clark H, and Schulden J (2005) Changes in HIV and AIDS in the United States: entering the third decade. Curr Infect Dis Rep 7: 138-143.[Medline]
Leh H, Brodin P, Bischerour J, Deprez E, Tauc P, Brochon JC, LeCam E, Coulaud D, Auclair C, and Mouscadet JF (2000) Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase. Biochemistry 39: 9285-9294.[CrossRef][Medline]
Lim YW, Kim JJ, Chedgy R, Morris PI, and Breuil C (2005) Fungal diversity from western redcedar fences and their resistance to beta-thujaplicin. Antonie Van Leeuwenhoek 87: 109-117.[CrossRef][Medline]
Marchand C, Johnson AA, Karki RG, Pais GC, Zhang X, Cowansage K, Patel TA, Nicklaus MC, Burke TR Jr, and Pommier Y (2003) Metal-dependent inhibition of HIV-1 integrase by beta-diketo acids and resistance of the soluble double-mutant (F185K/C280S). Mol Pharmacol 64: 600-609.
Marchand C, Neamati N, and Pommier Y (2001) In vitro human immunodeficiency virus type 1 integrase assays. Methods Enzymol 340: 624-633.[Medline]
Matsumura E, Morita Y, Date T, Tsujibo H, Yasuda M, Okabe T, Ishida N, and Inamori Y (2001) Cytotoxicity of the hinokitiol-related compounds, gamma-thujaplicin and beta-dolabrin. Biol Pharm Bull 24: 299-302.[CrossRef][Medline]
Mazumder A and Pommier Y (1995) Processing of deoxyuridine mismatches and abasic sites by human immunodeficiency virus type-1 integrase. Nucleic Acids Res 23: 2865-2871.
Morita Y, Matsumura E, Okabe T, Fukui T, Ohe T, Ishida N, and Inamori Y (2004) Biological activity of beta-dolabrin, gamma-thujaplicin and 4-acetyltropolone, hinokitiol-related compounds. Biol Pharm Bull 27: 1666-1669.[CrossRef][Medline]
Morita Y, Matsumura E, Okabe T, Shibata M, Sugiura M, Ohe T, Tsujibo H, Ishida N, and Inamori Y (2003) Biological activity of tropolone. Biol Pharm Bull 26: 1487-1490.[CrossRef][Medline]
Neamati N, Hong H, Sunder S, Milne GW, and Pommier Y (1997) Potent inhibitors of human immunodeficiency virus type 1 integrase: identification of a novel four-point pharmacophore and tetracyclines as novel inhibitors. Mol Pharmacol 52: 1041-1055.
Neamati N, Lin Z, Karki RG, Orr A, Cowansage K, Strumberg D, Pais GC, Voigt JH, Nicklaus MC, Winslow HE, et al. (2002) Metal-dependent inhibition of HIV-1 integrase. J Med Chem 45: 5661-5670.[CrossRef][Medline]
Pauwels R, Balzarini J, Baba M, Snoeck R, Schols D, Herdewijn P, Desmyter J, and De Clercq E (1988) Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds. J Virol Methods 20: 309-321.[CrossRef][Medline]
Pommier Y and Cherfils J (2005) Interfacial inhibition of macromolecular interactions: nature's paradigm for drug discovery. Trends Pharmacol Sci 26: 138-145.[CrossRef][Medline]
Pommier Y, Johnson AA, and Marchand C (2005) Integrase inhibitors to treat HIV/AIDS. Nat Rev Drug Discov 4: 236-248.[CrossRef][Medline]
Pommier Y and Marchand C (2005) Interfacial inhibitors of protein-nucleic acid interactions. Curr Med Chem Anticancer Agents 5: 421-429.
Turner BG and Summers MF (1999) Structural biology of HIV. J Mol Biol 285: 1-32.[CrossRef][Medline]
Van Maele B and Debyser Z (2005) HIV-1 integration: an interplay between HIV-1 integrase, cellular and viral proteins. AIDS Rev 7: 26-43.[Medline]
Yang W and Steitz TA (1995) Recombining the structures of HIV integrase, RuvC and RNase H. Structure 3: 131-134.[Medline]
This article has been cited by other articles:
|
|
 |
|
  C. Marchand, J. A. Beutler, A. Wamiru, S. Budihas, U. Mollmann, L. Heinisch, J. W. Mellors, S. F. Le Grice, and Y. Pommier Madurahydroxylactone Derivatives as Dual Inhibitors of Human Immunodeficiency Virus Type 1 Integrase and RNase H Antimicrob. Agents Chemother., January 1, 2008; 52(1): 361 - 364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Johnson, C. Marchand, S. S. Patil, R. Costi, R. Di Santo, T. R. Burke Jr., and Y. Pommier Probing HIV-1 Integrase Inhibitor Binding Sites with Position-Specific Integrase-DNA Cross-Linking Assays Mol. Pharmacol., March 1, 2007; 71(3): 893 - 901. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
C; cysteines 56, 65, and 280 are mutated to serine to eliminate nonspecific cross-linking. Top right, modified oligonucleotide used for cross-linking (DNA X-1) with a thioalkyl modification (Johnson et al., 2006
) and mock-infected cells (
).
