Probing Resistance Mutations in Retroviral Integrases by Direct Measurement of Dolutegravir Fluorescence (original) (raw)
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Journal of Virology, 2012
link the reverse-transcribed viral DNA to the host genome. INSTIs have proven their high efficiency in inhibiting viral replication in vitro and in patients. However, first-generation INSTIs have only a modest genetic barrier to resistance, allowing the virus to escape these powerful drugs through several resistance pathways. Second-generation INSTIs, such as dolutegravir (DTG, S/GSK1349572), have been reported to have a higher resistance barrier, and no novel drug resistance mutation has yet been described for this drug. Therefore, we performed in vitro selection experiments with DTG using viruses of subtypes B, C, and A/G and showed that the most common mutation to emerge was R263K. Further analysis by site-directed mutagenesis showed that R263K does confer low-level resistance to DTG and decreased integration in cell culture without altering reverse transcription. Biochemical cell-free assays performed with purified IN enzyme containing R263K confirmed the absence of major resistance against DTG and showed a slight decrease in 3= processing and strand transfer activities compared to the wild type. Structural modeling suggested and in vitro IN-DNA binding assays show that the R263K mutation affects IN-DNA interactions.
Antimicrobial Agents and Chemotherapy, 2011
ABSTRACTThe integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10−6s−1and a dissociative half-life (t1/2) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t1/2, at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the singl...
Antimicrobial Agents and Chemotherapy, 2013
ABSTRACTDrug resistance mutations (DRMs) have been reported for all currently approved anti-HIV drugs, including the latest integrase strand transfer inhibitors (INSTIs). We previously used the new INSTI dolutegravir (DTG) to select a G118R integrase resistance substitution in tissue culture and also showed that secondary substitutions emerged at positions H51Y and E138K. Now, we have characterized the impact of the G118R substitution, alone or in combination with either H51Y or E138K, on 3′ processing and integrase strand transfer activity. The results show that G118R primarily impacted the strand transfer step of integration by diminishing the ability of integrase-long terminal repeat (LTR) complexes to bind target DNA. The addition of H51Y and E138K to G118R partially restored strand transfer activity by modulating the formation of integrase-LTR complexes through increasing LTR DNA affinity and total DNA binding, respectively. This unique mechanism, in which one function of HIV i...
Three Integrase (IN) strand transfer inhibitors are in intensive clinical use, raltegravir, elvitegravir anddolutegravir. However, the onset of IN resistance mutations limits their therapeutic efficiency. As put forth earlier, the drug affinity for the intasome could be improved by targeting preferentially the retroviralnucleobases, which are little, if at all, mutation-prone. We report experimental results of anisotropy fluorescence titrations of viral DNA by these three drugs . These show that the ranking of their inhibitory activities of the intasome corresponds to that of their free energies of binding, D Gs,to retroviral DNA, and that such a ranking is only governed by the binding enthalpies, D H, the entropy undergoing marginal variations.This ranking can therefore be directly correlated to that of model Quantum Chemistry (QC) calculations of intermolecular interaction energies of the sole halobenzene ring with the highly conserved retroviral nucleobases G4 and C14, using Dens...
Molecular Pharmacology, 2011
and suppress viremia in patients. These small molecules bind to the IN active site, causing it to disengage from the deoxyadenosine at the 3Ј end of viral DNA. The emergence of viral strains that are highly resistant to RAL underscores the pressing need to develop INSTIs with improved resistance profiles. Herein, we show that the candidate second-generation drug dolutegravir (DTG, S/GSK1349572) effectively inhibits a panel of HIV-1 IN variants resistant to first-generation INSTIs. To elucidate the structural basis for the increased potency of DTG against RAL-resistant INs, we determined crystal structures of wild-type and mutant prototype foamy virus intasomes bound to this compound. The overall IN binding mode of DTG is strikingly similar to that of the tricyclic hydroxypyrrole MK-2048. Both second-generation INSTIs occupy almost the same physical space within the IN active site and make contacts with the 4 -␣2 loop of the catalytic core domain. The extended linker region connecting the metal chelating core and the halobenzyl group of DTG allows it to enter farther into the pocket vacated by the displaced viral DNA base and to make more intimate contacts with viral DNA, compared with those made by RAL and other INSTIs. In addition, our structures suggest that DTG has the ability to subtly readjust its position and conformation in response to structural changes in the active sites of RAL-resistant INs.
Biochemical and Biophysical Research Communications
The Human Immunodeficiency Virus-1 integrase is responsible for the covalent insertion of a newly synthesized double-stranded viral DNA into the host cells, and is an emerging target for antivirus drug design. Raltegravir (RAL) and elvitegravir (EVG) are the first two integrase strand transfer inhibitors used in therapy. However, treated patients eventually develop detrimental resistance mutations. By contrast, a recently approved drug, dolutegravir (DTG), presents a high barrier to resistance. This study aims to understand the increased efficiency of DTG upon focusing on its interaction properties with viral DNA. The results showed DTG to be involved in more extended interactions with viral DNA than EVG. Such interactions involve the halobenzene and scaffold of DTG and EVG and bases 5'G-4 3', 3'A 3 5'and 3'C 4 5'.
We recently reported that viral DNA could be the primary target of raltegravir (RAL), an efficient anti-HIV-1 drug, which acts by inhibiting integrase. To elucidate this mechanism, we conducted a comparative analysis of RAL and TB11, a diketoacid abandoned as an anti-HIV-1 drug for its weak efficiency and marked toxicity, and tested the effects of the catalytic cofactor Mg 2+ (5 mM) on drug-binding properties. We used circular dichroism and fluorescence to determine drug affinities for viral DNA long terminal repeats (LTRs) and peptides derived from the integrase active site and DNA retardation assays to assess drug intercalation into DNA base pairs. We found that RAL bound more tightly to LTR ends than did TB11 (a diketo acid bearing an azido group) and that Mg 2+ significantly increased the affinity of both RAL and TB11. We also observed a good relationship between drug binding with processed LTR and strand transfer inhibition. This unusual type of inhibition was caused by Mg 2+ -assisted binding of drugs to DNA substrate, rather than to enzyme. Notably, while RAL bound exclusively to the cleavable/cleaved site, TB11 further intercalated into DNA base pairs and interacted with the integrase-derived peptides. These unwanted binding sites explain the weaker bioavailability and higher toxicity of TB11 compared with the more effective RAL.
Unprocessed Viral DNA Could Be the Primary Target of the HIV-1 Integrase Inhibitor Raltegravir
PLoS ONE, 2012
Integration of HIV DNA into host chromosome requires a 39-processing (39-P) and a strand transfer (ST) reactions catalyzed by virus integrase (IN). Raltegravir (RAL), commonly used in AIDS therapy, belongs to the family of IN ST inhibitors (INSTIs) acting on IN-viral DNA complexes (intasomes). However, studies show that RAL fails to bind IN alone, but nothing has been reported on the behaviour of RAL toward free viral DNA. Here, we assessed whether free viral DNA could be a primary target for RAL, assuming that the DNA molecule is a receptor for a huge number of pharmacological agents. Optical spectroscopy, molecular dynamics and free energy calculations, showed that RAL is a tight binder of both processed and unprocessed LTR (long terminal repeat) ends. Complex formation involved mainly van der Waals forces and was enthalpy driven. Dissociation constants (Kds) revealed that RAL affinity for unbound LTRs was stronger than for bound LTRs. Moreover, Kd value for binding of RAL to LTRs and IC50 value (half concentration for inhibition) were in same range, suggesting that RAL binding to DNA and ST inhibition are correlated events. Accommodation of RAL into terminal base-pairs of unprocessed LTR is facilitated by an extensive end fraying that lowers the RAL binding energy barrier. The RAL binding entails a weak damping of fraying and correlatively of 39-P inhibition. Noteworthy, present calculated RAL structures bound to free viral DNA resemble those found in RAL-intasome crystals, especially concerning the contacts between the fluorobenzyl group and the conserved 59C 4 pA 3 39 step. We propose that RAL inhibits IN, in binding first unprocessed DNA. Similarly to anticancer drug poisons acting on topoisomerases, its interaction with DNA does not alter the cut, but blocks the subsequent joining reaction. We also speculate that INSTIs having viral DNA rather IN as main target could induce less resistance.
Proceedings of the National Academy of Sciences, 2000
Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3 end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731,988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.
Journal of Biological Chemistry, 2007
Human immunodeficiency virus (HIV) integrase enzyme is required for the integration of viral DNA into the host cell chromosome. Integrase complex assembly and subsequent strand transfer catalysis are mediated by specific interactions between integrase and bases at the end of the viral long terminal repeat (LTR). The strand transfer reaction can be blocked by the action of small molecule inhibitors, thought to bind in the vicinity of the viral LTR termini. This study examines the contributions of the terminal four bases of the nonprocessed strand (G 2 T 1 C ؊1 A ؊2) of the HIV LTR on complex assembly, specific strand transfer activity, and inhibitor binding. Base substitutions and abasic replacements at the LTR terminus provided a means to probe the importance of each nucleotide on the different functions. An approach is described wherein the specific strand transfer activity for each integrase/LTR variant is derived by normalizing strand transfer activity to the concentration of active sites. The key findings of this study are as follows. 1) The G 2 :C 2 base pair is necessary for efficient assembly of the complex and for maintenance of an active site architecture, which has high affinity for strand transfer inhibitors. 2) Inhibitor-resistant enzymes exhibit greatly increased sensitivity to LTR changes. 3) The strand transfer and inhibitor binding defects of a Q148R mutant are due to a decreased affinity of the complex for magnesium. 4) Gln 148 interacts with G 2 , T 1 , and C ؊1 at the 5 end of the viral LTR, with these four determinants playing important and overlapping roles in assembly, strand transfer catalysis and high affinity inhibitor binding.