Use of Patient-Derived Human Immunodeficiency Virus Type 1 Integrases To Identify a Protein Residue That Affects Target Site Selection

Harper, Amy L.; Skinner, Lynn M.; Sudol, Malgorzata; Katzman, Michael

doi:10.1128/jvi.75.16.7756-7762.2001

Cited by 45 publications

(46 citation statements)

References 37 publications

(35 reference statements)

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“…Recently, Ser119 contribution to target site preferences was reported in the CCD of IN. 30 Our results corroborate with the literature reports in terms of substrate− enzyme interactions. Gratifyingly, 24 compounds were successfully synthesized using an operationally simple, direct approach.…”

supporting

confidence: 83%

Design, Synthesis, and Biological Evaluation of 1,2-Dihydroisoquinolines as HIV-1 Integrase Inhibitors

Tandon

Urvashi

Yadav

et al. 2015

ACS Med. Chem. Lett.

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6-Endo-dig-cyclization is an efficient method for the synthesis of 1,2-dihydroisoquinolines. We have synthesized few 1,2-dihydroisoquinolines having different functionality at the C-1, C-3, C-7, and N-2 positions for evaluation against HIV-1 integrase (HIV1-IN) inhibitory activity. A direct nitro-Mannich condensation of o-alkynylaldimines and dual activation of o-alkynyl aldehydes by inexpensive cobalt chloride yielded desired compounds. Out of 24 compounds, 4m and 6c came out as potent integrase inhibitors in in vitro strand transfer (ST) assay, with IC 50 value of 0.7 and 0.8 μM, respectively. Molecular docking of these compounds in integrase revealed strong interaction between metal and ligands, which stabilizes the enzyme−inhibitor complex. The ten most active compounds were subjected to antiviral assay. Out of those, 6c reduced the level of p24 viral antigen by 91%, which is comparable to RAL in antiviral assay. Interestingly, these compounds showed similar ST inhibitory activity in G140S mutant, suggesting they can act against resistant strains.

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supporting

confidence: 83%

Design, Synthesis, and Biological Evaluation of 1,2-Dihydroisoquinolines as HIV-1 Integrase Inhibitors

Tandon

Urvashi

Yadav

et al. 2015

ACS Med. Chem. Lett.

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“…The following color code was used: red, active-site residues (Asp-64, Asp-116, and Glu-152); blue, residues that cross-linked to viral DNA substrates (Tyr-143, Gln-148, and Lys-159; references 28 and 43) and/or when mutated yielded class I IN mutant viruses (see Fig. 1 and 2) (43); green, residues that when mutated yielded viruses displaying WT replication kinetics; yellow, Glu-138; magenta, residues that predictably interact with target DNA (38,67). The amphipathic alpha-helix is labeled helix 4. well conserved among HIV-1 strains and divergent retroviruses (Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…Peptides spanning residues 49 to 69 (40) and 51 to 64 (28) cross-linked to viral DNA and IN proteins altered at Gln-62 were sensitive to the concentration of salt in in vitro assays (26,33), suggesting that Gln-62 also binds HIV-1 DNA (13,28,33). Since IN enzymes carrying substitutions of Ser-119 (38) or Asn-120 (67) displayed altered patterns of DNA strand transfer, these residues likely contact target DNA during integration. Since Lys-159, Gln-148, and Tyr-143 interacted with 1-(5-chloroindol-3-yl)-3-hydroxy-(2 H-tetrazol-5-yl)-propenone (5CITEP) in an IN-inhibitor cocrystal structure, it was suggested that 5CITEP might inhibit IN activity by interfering with critical IN-DNA contacts that occur during integration (36).…”

mentioning

confidence: 99%

“…The CCD (residues 50 to 212 of the 288-residue protein) harbors a triad of invariant carboxylate amino acids (HIV-1 residues Asp-64, Asp-116, and Glu-152, which form the D,D-35-E motif) that comprises the enzyme active site (7,20,21,24,46,49,67). Results of 40,43), footprinting (19), and in vitro enzyme assays (4,26,33,38,43,67) revealed other conserved CCD residues that likely contact viral (28,33,40,41,43) and target (4,19,38,40,67) DNA during integration. Specifically, Lys-159, Tyr-143, and Gln-148 cross-linked to viral DNA substrates (28, 43) and IN enzymes with alterations at Lys-159 or Gln-148 were defective in in vitro integration assays (33,43,67,68).…”

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confidence: 99%

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Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase

Limón

Ghory

et al. 2005

J Virol

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The catalytic core domain (CCD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) harbors the enzyme active site and binds viral and chromosomal DNA during integration. Thirty-five CCD mutant viruses were constructed, paying particular attention to conserved residues in the Phe 139 -Gln 146 flexible loop and abutting Ser 147 -Val 165 amphipathic alpha helix that were implicated from previous in vitro work as important for DNA binding. Defective viruses were typed as class I mutants (specifically blocked at integration) or pleiotropic class II mutants (additional particle assembly and/or reverse transcription defects). Whereas HIV-1 P145A and HIV-1 Q146K grew like the wild type, HIV-1 N144K and HIV-1 Q148L were class I mutants, reinforcing previous results that Gln-148 is important for DNA binding and uncovering for the first time an important role for Asn-144 in integration. HIV-1 Q62K , HIV-1 H67E , HIV-1 N120K , and HIV-1 N155K were also class I mutants, supporting findings that Gln-62 and Asn-120 interact with viral and target DNA, respectively, and suggesting similar integration-specific roles for His-67 and Asn-155. Although results from complementation analyses established that IN functions as a multimer, the interplay between active-site and CCD DNA binding functions was unknown. By using Vpr-IN complementation, we determined that the CCD protomer that catalyzes integration also preferentially binds to viral and target DNA. We additionally characterized E138K as an intramolecular suppressor of Gln-62 mutant virus and IN. The results of these analyses highlight conserved CCD residues that are important for HIV-1 replication and integration and define the relationship between DNA binding and catalysis that occurs during integration in vivo.An essential step in the retroviral life cycle is the integration of the cDNA made by reverse transcription into a cell chromosome. Integration is mediated by the viral integrase (IN) protein, a specialized DNA recombinase that catalyzes two distinct endonucleolytic reactions during the early phase of infection. The first reaction, 3Ј processing, occurs shortly after reverse transcription is completed. During 3Ј processing, human immunodeficiency virus type 1 (HIV-1) IN removes a GT dinucleotide from each end of linear viral cDNA. IN performs its second reaction, DNA strand transfer, after finding a suitable chromosome target for integration. During DNA strand transfer, IN uses the oxygen moieties at the processed 3Ј ends to cut the target DNA at two 5Ј phosphates that are separated by 5 bp, which concomitantly joins the 3Ј ends to the chromosome. The resulting DNA recombination intermediate, with viral 5Ј ends unattached to the chromosome, is likely repaired by cellular enzymes (see references 16 and 34 for reviews).Partial proteolysis (24), deletion mutagenesis (9, 70), and functional complementation (23, 69) analyses revealed that HIV-1 IN is comprised of three domains, the N-terminal domain, catalytic core domain (CCD), and C-terminal domain (CTD) (reviewe...

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“…The CCD harbors additional conserved residues that contact viral DNA (86-94) and target DNA (90,(95)(96)(97)(98) during integration. The lysine residue that engages the phosphate backbone of the invariant CA dinucleotide in viral DNA (86,94) is conserved across retroviral integrase proteins and some bacterial insertion elements ( Fig.…”

Section: Integrase Protein Structures Domain Organization Of Integrasmentioning

confidence: 99%

Retroviral Integrase Structure and DNA Recombination Mechanism

Engelman

Cherepanov

2014

Microbiol Spectr

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SUMMARYDue to the importance of human immunodeficiency virus type 1 (HIV-1) integrase as a drug target, the biochemistry and structural aspects of retroviral DNA integration have been the focus of intensive research during the past three decades. The retroviral integrase enzyme acts on the linear double-stranded viral DNA product of reverse transcription. Integrase cleaves specific phosphodiester bonds near the viral DNA ends during the 3′ processing reaction. The enzyme then uses the resulting viral DNA 3′-OH groups during strand transfer to cut chromosomal target DNA, which simultaneously joins both viral DNA ends to target DNA 5′-phosphates. Both reactions proceed via direct transesterification of scissile phosphodiester bonds by attacking nucleophiles: a water molecule for 3′ processing, and the viral DNA 3′-OH for strand transfer. X-ray crystal structures of prototype foamy virus integrase-DNA complexes revealed the architectures of the key nucleoprotein complexes that form sequentially during the integration process and explained the roles of active site metal ions in catalysis. X-ray crystallography furthermore elucidated the mechanism of action of HIV-1 integrase strand transfer inhibitors, which are currently used to treat AIDS patients, and provided valuable insights into the mechanisms of viral drug resistance.

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Use of Patient-Derived Human Immunodeficiency Virus Type 1 Integrases To Identify a Protein Residue That Affects Target Site Selection

Cited by 45 publications

References 37 publications

Design, Synthesis, and Biological Evaluation of 1,2-Dihydroisoquinolines as HIV-1 Integrase Inhibitors

Design, Synthesis, and Biological Evaluation of 1,2-Dihydroisoquinolines as HIV-1 Integrase Inhibitors

Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase

Retroviral Integrase Structure and DNA Recombination Mechanism

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