Development of Resistance against Diketo Derivatives of Human Immunodeficiency Virus Type 1 by Progressive Accumulation of Integrase Mutations

Fikkert, Valery; Maele, Bénédicte Van; Vercammen, Jo; Hantson, Anke; Remoortel, Barbara Van; Michiels, Martine; Gurnari, Cristina; Pannecouque, Christophe; Maeyer, Marc De; Engelborghs, Yves; Clercq, Erik De; Debyser, Zeger; Witvrouw, Myriam

doi:10.1128/jvi.77.21.11459-11470.2003

Cited by 95 publications

(116 citation statements)

References 34 publications

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“…The N155S virus has been reported to be less fit than WT (42,43), and the N155E, N155K, and N155L variants were reported to have 6% of WT strand transfer activity (44), similar to the N155H results reported here. T66I had a relative efficiency of 36%, indicating that this enzyme is not much altered catalytically, as compared with WT, similar to what has been reported previously (38). Certain double variants were particularly defective.…”

Section: Discussionsupporting

confidence: 71%

“…Selection for resistance to the action of strand transfer inhibitors has been shown to map to the integrase gene and to encode changes near the catalytic triad responsible for catalytic activity (16,38). Such changes have also been observed in the clinic to raltegravir, the first HIV integrase approved for treatment of HIV-infected patients (15).…”

Section: Discussionmentioning

confidence: 94%

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Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Dicker

Terry

Lin

et al. 2008

Journal of Biological Chemistry

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In this study, eight different HIV-1 integrase proteins containing mutations observed in strand transfer inhibitor-resistant viruses were expressed, purified, and used for detailed enzymatic analyses. All the variants examined were impaired for strand transfer activity compared with the wild type enzyme, with relative catalytic efficiencies (k p /K m ) ranging from 0.6 to 50% of wild type. The origin of the reduced strand transfer efficiencies of the variant enzymes was predominantly because of poorer catalytic turnover (k p ) values. However, smaller secondorder effects were caused by up to 4-fold increases in K m values for target DNA utilization in some of the variants. All the variants were less efficient than the wild type enzyme in assembling on the viral long terminal repeat, as each variant required more protein than wild type to attain maximal activity. In addition, the variant integrases displayed up to 8-fold reductions in their catalytic efficiencies for 3-processing. The Q148R variant was the most defective enzyme. The molecular basis for resistance of these enzymes was shown to be due to lower affinity binding of the strand transfer inhibitor to the integrase complex, a consequence of faster dissociation rates. In the case of the Q148R variant, the origin of reduced compound affinity lies in alterations to the active site that reduce the binding of a catalytically essential magnesium ion. Finally, except for T66I, variant viruses harboring the resistance-inducing substitutions were defective for viral integration.

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Section: Discussionsupporting

confidence: 71%

Section: Discussionmentioning

confidence: 94%

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Dicker

Terry

Lin

et al. 2008

Journal of Biological Chemistry

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“…A prior study examined HIV-1 viral resistance to L-708,906 and S-1360, which are derivatives from the diketo acid class of IN inhibitors. Although a 10-fold viral resistance to these IN inhibitors was the observed phenotype, the corresponding triple mutant (T66I͞L74M͞S230R) protein exhibited only 2-to 3-fold resistance in vitro to the same compounds (30). The coumarins interacted directly with the peptide backbone in the region 128 AACWWAGIK 136 .…”

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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“…Published models of HIV IN (19,22) suggest that these specific STIs bind to the IN active site in a pocket flanked by the end of the LTR such that the diketoacid moiety, or its mimic, is positioned to coordinate with both of the catalytic magnesium atoms (23)(24)(25) (14,19,22,(26)(27)(28)(29). Amino acid substitutions which confer resistance to STIs cluster around the IN active site (30)(31)(32)(33)(34)(35)(36)(37), and changes to the 5′ end of the LTR reduce the affinity of a specific STI for its binding site (16). Published models do not address the issue of the precise placement of the 3′ terminus of the viral LTR in the inhibitor-bound state.…”

mentioning

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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Development of Resistance against Diketo Derivatives of Human Immunodeficiency Virus Type 1 by Progressive Accumulation of Integrase Mutations

Cited by 95 publications

References 34 publications

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

Biochemical Analysis of HIV-1 Integrase Variants Resistant to Strand Transfer Inhibitors

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

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