Disruption of HIV-1 Integrase−DNA Complexes by Short 6-Oxocytosine-Containing Oligonucleotides<sup>,</sup>

Brodin, Priscille; Pinskaya, Marina; Buckle, Malcolm; Parsch, Uwe; Романова, Е. А.; Engels, Joachim W.; Gottikh, Marina; Mouscadet, Jean‐François

doi:10.1021/bi015732y

Cited by 19 publications

(19 citation statements)

References 39 publications

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“…As we showed previously that IN-DNA complexes form fairly slowly (38,39), this step might be differentially affected by DNA modifications, with effects on subsequent IN activity. We therefore investigated the effects of substrate modifications on the kinetics of DNA binding to IN (Table 4).…”

Section: Dna Duplexmentioning

confidence: 59%

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

Agapkina

Smolov

Barbe

et al. 2006

Journal of Biological Chemistry

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The specific activity of the human immunodeficiency virus, type 1 (HIV-1), integrase on the viral long terminal repeat requires the binding of the enzyme to certain sequences located in the U3 and U5 regions at the ends of viral DNA, but the determinants of this specific DNA-protein recognition are not yet completely understood. We synthesized DNA duplexes mimicking the U5 region and containing either 2-modified nucleosides or 1,3-propanediol insertions and studied their interactions with HIV-1 integrase, using Mn 2؉ or Mg 2؉ ions as integrase cofactors. These DNA modifications had no strong effect on integrase binding to the substrate analogs but significantly affected 3-end processing rate. The effects of nucleoside modifications at positions 5, 6, and especially 3 strongly depended on the cationic cofactor used. These effects were much more pronounced in the presence of Mg 2؉ than in the presence of Mn 2؉ . Modifications of base pairs 7-9 affected 3-end processing equally in the presence of both ions. Adenine from the 3rd bp is thought to form at least two hydrogen bonds with integrase that are crucial for specific DNA recognition. The complementary base, thymine, is not important for integrase activity. For other positions, our results suggest that integrase recognizes a fine structure of the sugar-phosphate backbone rather than heterocyclic bases. Integrase interactions with the unprocessed strand at positions 5-8 are more important than interactions with the processed strand for specific substrate recognition. Based on our results, we suggest a model for integrase interaction with the U5 substrate.Following reverse transcription, a DNA copy of the human immunodeficiency virus, type 1 (HIV-1), 2 RNA is integrated into the genome of infected cells. Integration is a prerequisite for viral replication and is catalyzed by the viral enzyme integrase (IN). IN binds to sequences located at the end of U3 and U5 parts of long terminal repeats (LTRs) of viral DNA and catalyzes the trimming, or 3Ј-end processing, of the terminal dinucleotide from the 3Ј-ends of both strands of the DNA. IN then mediates a strand transfer reaction that inserts the viral DNA into the host DNA. During this reaction, IN must bind simultaneously to viral and target DNA. However, IN interacts with these two DNA molecules in different ways as follows: binding to host DNA does not depend directly on host DNA sequence, whereas interaction with the viral DNA is a sequence-specific process. Nevertheless, the U5 and U3 sequences recognized by IN are not exactly identical.Strand transfer and 3Ј-end processing reactions may be carried out in vitro, using recombinant HIV IN, DNA duplexes mimicking U3 or U5 sequences of LTRs, and divalent metal ions, such as Mg 2ϩ or Mn 2ϩ . However, the Mn 2ϩ -and Mg 2ϩ -dependent activities of IN are not equivalent, with lower specificity reported for Mn 2ϩ -dependent IN (1, 2). Moreover, the inhibition of HIV-1 IN by compounds such as ␤-diketo acids, which interact with the active site of HIV-1 IN, is also metal-de...

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Section: Dna Duplexmentioning

confidence: 59%

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

Agapkina

Smolov

Barbe

et al. 2006

Journal of Biological Chemistry

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“…The sequence is identical to the sequence beginning seven bases from the 5’ end of the non-cleaved strand of the U5 LTR. Substitution of two cytosines with 6-oxocytosine residues results in a potent IN inhibitor even though the unmodified oligonucleotide has similar affinity for IN but fails to inhibit IN activity 163. Inhibition was dependent on sequence, presence of 6-oxocytosine, and positioning the 6-oxocytosine at the terminus of the oligonucleotide 162, 163.…”

Section: Update On Oligonucleotide Inhibitorsmentioning

confidence: 99%

HIV-1 IN Inhibitors: 2010 Update and Perspectives

Marchand¹,

Maddali²,

Métifiot³

et al. 2009

CTMC

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Integrase (IN) is the newest validated target against AIDS and retroviral infections. The remarkable activity of raltegravir (Isentress ® ) led to its rapid approval by the FDA in 2007 as the first IN inhibitor. Several other IN strand transfer inhibitors (STIs) are in development with the primary goal to overcome resistance due to the rapid occurrence of IN mutations in raltegravirtreated patients. Thus, many scientists and drug companies are actively pursuing clinically useful IN inhibitors. The objective of this review is to provide an update on the IN inhibitors reported in the last two years, including second generation strand transfer inhibitors (STI), recently developed hydroxylated aromatics, natural products, peptide, antibody and oligonucleotide inhibitors. Additionally, the targeting of IN cofactors such as LEDGF and Vpr will be discussed as novel strategies for the treatment of AIDS.

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“…Submicromolar inhibition was observed, with an IC 50 of 0.4 M. This value was lower than that determined above (1.2 M) because the concentration of IN was lower (50 nM compared with 120 nM). Second, IN/LTR complexes were preformed for 20 min at 25°C in the presence of Mg 2ϩ , because it has been shown previously that the amount of complexes is optimal at this time (Brodin et al, 2002). The drug was then added and the reaction was allowed to proceed at 37°C (Fig.…”

Section: Docking Of Khd161 To the Catalytic Core Domain Of Inmentioning

confidence: 99%

Mechanism of HIV-1 Integrase Inhibition by Styrylquinoline Derivatives in Vitro

Deprez¹,

Barbe²,

Kolaski³

et al. 2004

Mol Pharmacol

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Styrylquinoline derivatives (SQ) efficiently inhibit the 3Ј-processing activity of integrase (IN) with IC 50 values of between 0.5 and 5 M. We studied the mechanism of action of these compounds in vitro. First, we used steady-state fluorescence anisotropy to assay the effects of the SQ derivatives on the formation of IN-viral DNA complexes independently of the catalytic process. The IC 50 values obtained in activity and DNAbinding tests were similar, suggesting that the inhibition of 3Ј-processing can be fully explained by the prevention of IN-DNA recognition. SQ compounds act in a competitive manner, with K i values of between 400 and 900 nM. In contrast, SQs did not inhibit 3Ј-processing when IN-DNA complexes were preassembled. Computational docking followed or not by molecular dynamics using the catalytic core of HIV-1 IN suggested a competitive inhibition mechanism, which is consistent with our previous data obtained with the corresponding Rous sarcoma virus domain. Second, we used preassembled IN-preprocessed DNA complexes to assay the potency of SQs against the strand transfer reaction, independently of 3Ј-processing. Inhibition occurred even if the efficiency was decreased by about 5-to 10-fold. Our results suggest that two inhibitorbinding modes exist: the first one prevents the binding of the viral DNA and then the two subsequent reactions (i.e., 3Ј-processing and strand transfer), whereas the second one prevents the binding of target DNA, thus inhibiting strand transfer. SQ derivatives have a higher affinity for the first site, in contrast to that observed for the diketo acids, which preferentially bind to the second one.Integration of the HIV-1 DNA into the host genome ensures stable maintenance of the viral genome and perpetuation of the virus in the host organism. Therefore, this reaction, catalyzed by integrase (IN), is a key process in the life cycle of the virus. Inhibitors of therapeutic interest for AIDS exist. Inhibitors that act early in the replication cycle target reverse transcriptase and those that act later target protease. The high mutation rate of HIV means that drug resistance is emerging, making it necessary to develop new drugs with different targets (combined therapies). Thus, it is essential to find drugs that target alternative steps of the HIV-1 replication cycle. IN, the third enzyme of the Pol precursor protein, is then an attractive target for novel drugs because of its central role in the HIV-1 life cycle.IN is a 32-kDa protein (288 amino acids) that consists of three functional domains. The central domain (or core domain, residues 50 -210) contains the catalytic triad (DDE) that is essential for enzymatic activity. This domain is flanked by the N-terminal domain (which has a role in the assembly of an active multimeric form of the enzyme) and the C-terminal domain (which has a role in the nonspecific DNAbinding activity). These three domains, protein oligomerization, the integrity of the catalytic triad, and a cationic cofactor such as magnesium are all necessary fo...

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Disruption of HIV-1 Integrase−DNA Complexes by Short 6-Oxocytosine-Containing Oligonucleotides^,

Cited by 19 publications

References 39 publications

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

HIV-1 IN Inhibitors: 2010 Update and Perspectives

Mechanism of HIV-1 Integrase Inhibition by Styrylquinoline Derivatives in Vitro

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Disruption of HIV-1 Integrase−DNA Complexes by Short 6-Oxocytosine-Containing Oligonucleotides,

Cited by 19 publications

References 39 publications

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

Probing of HIV-1 Integrase/DNA Interactions Using Novel Analogs of Viral DNA

HIV-1 IN Inhibitors: 2010 Update and Perspectives

Mechanism of HIV-1 Integrase Inhibition by Styrylquinoline Derivatives in Vitro

Contact Info

Product

Resources

About

Disruption of HIV-1 Integrase−DNA Complexes by Short 6-Oxocytosine-Containing Oligonucleotides^,