Metal-Dependent Inhibition of HIV-1 Integrase

Neamati, Nouri; Lin, Zhaiwei; Karki, Rajeshri G.; Orr, Ann; Cowansage, Kiriana; Strumberg, Dirk; Pais, Godwin C. G.; Voigt, Johannes H.; Nicklaus, Marc C.; Winslow, Heather E.; Zhao, He; Turpin, Jim A.; Yi, Jizu; Skalka, Anna Marie; Burke, Terrence R.; Pommier, ﻿Yves

doi:10.1021/jm0201417

Cited by 77 publications

(67 citation statements)

References 45 publications

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“…3Ј Processing, strand transfer, and disintegration can be independently measured in biochemical assays using specific oligonucleotides (Figs. 1A, 2A, and 3A) (Marchand et al, 2001 , is required for integrase activity in vitro (Engelman and Craigie, 1995 (Grobler et al, 2002;Neamati et al, 2002;Marchand et al, 2003), all assays were performed in the presence of either Mg 2ϩ or Mn 2ϩ . .…”

Section: The 7-hydroxy Group Is Essential For Inhibition Of Hiv-1 Intmentioning

confidence: 99%

Preferential Inhibition of the Magnesium-Dependent Strand Transfer Reaction of HIV-1 Integrase by α-Hydroxytropolones

Семенова

Johnson²,

Marchand³

et al. 2006

Mol Pharmacol

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Section: The 7-hydroxy Group Is Essential For Inhibition Of Hiv-1 Intmentioning

confidence: 99%

Preferential Inhibition of the Magnesium-Dependent Strand Transfer Reaction of HIV-1 Integrase by α-Hydroxytropolones

Семенова

Johnson²,

Marchand³

et al. 2006

Mol Pharmacol

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“…[4][5][6][7] It has become apparent over recent years that inhibition of the HIV-IN ST reaction is the primary key to the effective suppression of viral replication in vivo. 8 Small molecules, on the other hand, which inhibit the in vitro activity of HIV-IN by interfering with DNA binding or the 3P reaction alone, generally lack antiviral activity.…”

Section: +mentioning

confidence: 99%

Homogeneous High-Throughput Screening Assays for HIV-1 Integrase 3'-Processing and Strand Transfer Activities

et al. 2005

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HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3′-processing and strand transfer activities. Both assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3′ processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs. (Journal of Biomolecular Screening 2005:456-462)

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“…Differences in the coordination properties of Mg 2ϩ and Mn 2ϩ ions may result in different conformational changes in the IN catalytic core. This may account for differences in the specificity of HIV-1 IN interactions with substrate DNA (1,2), inhibitors (3,12), and the nucleophiles used to carry out 3Ј-end processing (13). As IN is more sensitive to the structure of molecules interacting with its catalytic core in the presence of magnesium ions, it has been suggested that the catalytic core of this molecule is more rigid in the presence of magnesium ions, as reported for other magnesium-dependent enzymes (1).…”

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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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Metal-Dependent Inhibition of HIV-1 Integrase

Cited by 77 publications

References 45 publications

Preferential Inhibition of the Magnesium-Dependent Strand Transfer Reaction of HIV-1 Integrase by α-Hydroxytropolones

Preferential Inhibition of the Magnesium-Dependent Strand Transfer Reaction of HIV-1 Integrase by α-Hydroxytropolones

Homogeneous High-Throughput Screening Assays for HIV-1 Integrase 3'-Processing and Strand Transfer Activities

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

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