Model of full-length HIV-1 integrase complexed with viral DNA as template for anti-HIV drug design

Karki, Rajeshri G.; Tang, Yun; Burke, Terrence R.; Nicklaus, Marc C.

doi:10.1007/s10822-005-0365-5

Cited by 56 publications

(38 citation statements)

References 77 publications

(142 reference statements)

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“…This may be the reason why the strand transfer activity is abolished, even though the ability to conduct 3Ј-processing is preserved, with, however, more resilience to coumarin inhibition. Indeed current models and experimental evidence on retroviral IN composition indicate that a dimeric species may be sufficient for 3Ј-processing, but a tetrameric arrangement, stabilized through target DNA binding at the dimerdimer interface, is necessary for the integration process and more relevant to the in vivo nucleo-protein complex (28,29). Results on avian sarcoma virus IN also suggest that tetramer formation is an obligatory step that occurs during strand transfer catalysis (28).…”

Section: Discussionmentioning

confidence: 99%

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site

Al‐Mawsawi

Fikkert

Dayam

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photocrosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide 128 AACWWAGIK 136 was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling͞MS analysis with site-directed mutagenesis͞molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors.drug design ͉ mass spectrometry ͉ photoaffinity labeling H IV-1 integrase (IN) mediates the insertion of viral DNA into the host genome. This process occurs through two separate events, both catalyzed by IN. In the 3Ј-processing reaction, IN cleaves a dinucleotide adjacent to a conserved CA on each terminus of the reverse-transcribed viral DNA. This cleavage results in two 3Ј hydroxyl groups that are used for a subsequent nucleophilic attack. IN then inserts this DNA product into the host genome in the second reaction, termed strand transfer (1, 2). IN reactions can be carried out in vitro by using purified protein, a DNA substrate with ends mimicking the U3 or U5 viral DNA termini, and Mg 2ϩ or Mn 2ϩ as a cofactor (3).Structural information detailing the association between IN and inhibitors under development is of enormous therapeutic importance. Knowledge of key amino acid residues involved in the binding of potential drugs, and therefore which residues are likely to mutate under therapeutic pressure, would inevitably help researchers stay one step ahead of drug-resistant viral strains. A co-crystal structure of one of our inhibitors was previously solved with the ASV -IN (4, 5). This complex was subsequently used as a surrogate structure to discover IN inhibitors through highthroughput docking studies (6). Thus far, only two examples of co-crystal structures of HIV-1 IN core in complex with inhibitors have been reported (7,8). Despite our own repeated attempts, solving co-crystal structures of IN with our potent inhibitors has failed. This pauc...

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Section: Discussionmentioning

confidence: 99%

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site

Al‐Mawsawi

Fikkert

Dayam

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…To date, there has been no description of the detailed conformational arrangement of the DNA at the terminus of the viral LTR that gives rise to the inhibitor-bound state. However, base substitutions, cross-linking studies, molecular models (17)(18)(19)(20)(21)(22), and resistance data converge on the idea that STIs bind near to the LTR terminus (reference 16 and references therein). A recent modeling paper places the integrase inhibitor within this structural context in detail (42).…”

Section: Discussionmentioning

confidence: 99%

The Terminal (Catalytic) Adenosine of the HIV LTR Controls the Kinetics of Binding and Dissociation of HIV Integrase Strand Transfer Inhibitors

et al. 2008

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Specific HIV integrase strand transfer inhibitors are thought to bind to the integrase active site, positioned to coordinate with two catalytic magnesium atoms in a pocket flanked by the end of the viral LTR. A structural role for the 3′ terminus of the viral LTR in the inhibitor-bound state has not previously been examined. This study describes the kinetics of binding of a specific strand transfer inhibitor to integrase variants assembled with systematic changes to the terminal 3′ adenosine. Kinetic experiments are consistent with a two-step binding model in which there are different functions for the terminal adenine base and the terminal deoxyribose sugar. Adenine seems to act as a "shield" which retards the rate of inhibitor association with the integrase active site, possibly by acting as an internal competitive inhibitor. The terminal deoxyribose is responsible for retarding the rate of inhibitor dissociation, either by sterically blocking inhibitor egress or by a direct interaction with the bound inhibitor. These findings further our understanding of the details of the inhibitor binding site of specific strand transfer inhibitors.

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“…These biochemical experiments together with crystallographic determination of the two-domain structures prompted molecular modeling research. However, the IN-DNA models obtained by different groups vary significantly, indicating that the available experimental data comprises an insufficient number of constraints for formulating a common outcome (28,(37)(38)(39)(40)(41)(42)(43).…”

Section: Hiv-1 Integrase (In)mentioning

confidence: 99%

Subunit-specific Protein Footprinting Reveals Significant Structural Rearrangements and a Role for N-terminal Lys-14 of HIV-1 Integrase during Viral DNA Binding

Zhao

McKee

Kessl

et al. 2008

Journal of Biological Chemistry

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To identify functional contacts between HIV-1 integrase (IN)and its viral DNA substrate, we devised a new experimental strategy combining the following two methodologies. First, disulfide-mediated cross-linking was used to site-specifically link select core and C-terminal domain amino acids to respective positions in viral DNA. Next, surface topologies of free IN and IN-DNA complexes were compared using Lys-and Arg-selective small chemical modifiers and mass spectrometric analysis. This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. The footprinting studies for the first time revealed the importance of a specific N-terminal domain residue, Lys-14, in viral DNA binding. In addition, a DNA-induced conformational change involving the connection between the core and C-terminal domains was observed. Site-directed mutagenesis experiments confirmed the importance of the identified contacts for recombinant IN activities and virus infection. These new findings provided major constraints, enabling us to identify the viral DNA binding channel in the active full-length IN multimer. The experimental approach described here has general application to mapping interactions within functional nucleoprotein complexes. HIV-1 integrase (IN)4 is commonly viewed as an important therapeutic target for the following reasons: its catalytic activities are required for viral replication, there is no closely related cellular equivalent of IN, and specific IN inhibitors are likely to be effective against viral strains resistant to currently available therapies targeting reverse transcriptase (RT), protease, and virus-cell fusion. Detailed structural information on functional IN-DNA complexes could aid drug design efforts. For example, the promising diketo acid class of inhibitors preferentially bind to the assembled IN-viral DNA complex rather than the free protein (1-4).The two chemical reactions catalyzed by HIV-1 IN, 3Ј processing and DNA strand transfer, have been characterized in detail (reviewed in Ref. 5). First, IN removes two nucleotides from each 3Ј-end of the viral DNA synthesized by reverse transcriptase. In the following step, concerted transesterification reactions covalently join the viral DNA ends into the host genome (6). In vivo, the enzyme acts in the context of a large nucleoprotein complex with a number of viral and host proteins contributing to the integration process (7-21).HIV-1 IN is composed of three distinct structural and functional domains: the N-terminal domain (NTD) (residues 1-50) that contains an HHCC zinc binding motif, the catalytic core domain (CCD) (residues 51-212) containing the DDE motif essential for coordinating catalytic divalent metals, and the C-terminal domain (CTD) (residues 213-288) that is thought to provide a platform for DNA binding. Crystallographic or NMR structural data are available for each of the individual domains (22-26). In addition, two-domain CCD/ CTD (27) and NTD/CCD (28) crystal structures have been determined. However,...

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Model of full-length HIV-1 integrase complexed with viral DNA as template for anti-HIV drug design

Cited by 56 publications

References 77 publications

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site

The Terminal (Catalytic) Adenosine of the HIV LTR Controls the Kinetics of Binding and Dissociation of HIV Integrase Strand Transfer Inhibitors

Subunit-specific Protein Footprinting Reveals Significant Structural Rearrangements and a Role for N-terminal Lys-14 of HIV-1 Integrase during Viral DNA Binding

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