Heterocycle amide isosteres: An approach to overcoming resistance for HIV-1 integrase strand transfer inhibitors

A series of heterocyclic pyrimidinedione-based HIV-1 integrase inhibitors was prepared and screened for activity against purified integrase enzyme and/or viruses modified with the following mutations within integrase: Q148R, Q148H/G140S and N155H. These are mutations that result in resistance to the first generation integrase inhibitors raltegravir and elvitegravir. Based on consideration of drug-target interactions, an approach was undertaken to replace the amide moiety of the first generation pyrimidinedione inhibitor with azole heterocycles that could retain potency against these key resistance mutations. An imidazole moiety was found to be the optimal amide substitute and the observed activity was rationalized with the use of calculated properties and modeling. Rat pharmacokinetic (PK) studies of the lead imidazole compounds demonstrated moderate clearance and moderate exposure.     

Comparison of raltegravir and elvitegravir on HIV-1 integrase catalytic reactions and on a series of drug-resistant integrase mutants

HIV-1 integrase (IN) is the molecular target of the newly approved anti-AIDS drug raltegravir (MK-0518, Isentress) while elvitegravir (GS-9137, JTK-303) is in clinical trials. The aims of the present study were (1) to investigate and compare the effects of raltegravir and elvitegravir on the three IN-mediated reactions, 3'-processing (3'-P), strand transfer (ST), and disintegration, (2) to determine the biochemical activities of seven IN mutants (T66I, L74M, E92Q, F121Y, Q148K, S153Y, and N155H) previously selected from drug-resistant patients and isolates, and (3) to determine the resistance profile for raltegravir and elvitegravir in those IN mutants. Our findings demonstrate that both raltegravir and elvitegravir are potent IN inhibitors and are highly selective for the ST reaction of IN. Elvitegravir was more potent than raltegravir, but neither drug could block disintegration. All resistance mutations were at least partially impaired for ST. Q148K was also markedly impaired for 3'-P. Both drugs exhibited a parallel resistance profile, although resistance was generally greater for elvitegravir. Q148K and T66I conferred the highest resistance to both drugs while S153Y conferred relatively greater resistance to elvitegravir than raltegravir. Drug resistance could not be overcome by preincubating the drugs with IN, consistent with the binding of raltegravir and elvitegravir at the IN-DNA interface. Finally, we found an inverse correlation between resistance and catalytic activity of the IN mutants.     

Substitutions at amino acid positions 143, 148, and 155 of HIV-1 integrase define distinct genetic barriers to raltegravir resistance in vivo

Mutations at amino acids 143, 148, and 155 in HIV-1 integrase (IN) define primary resistance pathways in subjects failing raltegravir (RAL)-containing treatments. Although each pathway appears to be genetically distinct, shifts in the predominant resistant virus population have been reported under continued drug pressure. To better understand this dynamic, we characterized the RAL susceptibility of 200 resistant viruses, and we performed sequential clonal analysis for selected cases. Patient viruses containing Y143R, Q148R, or Q148H mutations consistently exhibited larger reductions in RAL susceptibility than patient viruses containing N155H mutations. Sequential analyses of virus populations from three subjects revealed temporal shifts in subpopulations representing N155H, Y143R, or Q148H escape pathways. Evaluation of molecular clones isolated from different time points demonstrated that Y143R and Q148H variants exhibited larger reductions in RAL susceptibility and higher IN-mediated replication capacity (RC) than N155H variants within the same subject. Furthermore, shifts from the N155H pathway to either the Q148R or H pathway or the Y143R pathway were dependent on the amino acid substitution at position 148 and the secondary mutations in Y143R- or Q148R- or H-containing variants and correlated with reductions in RAL susceptibility and restorations in RC. Our observations in patient viruses were confirmed by analyzing site-directed mutations. In summary, viruses that acquire mutations defining the 143 or 148 escape pathways are less susceptible to RAL and exhibit greater RC than viruses containing 155 pathway mutations. These selective pressures result in the displacement of N155H variants by 143 or 148 variants under continued drug exposure.     

Resistance mutations in human immunodeficiency virus type 1 integrase selected with elvitegravir confer reduced susceptibility to a wide range of integrase inhibitors

Integration of viral DNA into the host chromosome is an essential step in the life cycle of retroviruses and is facilitated by the viral integrase enzyme. The first generation of integrase inhibitors recently approved or currently in late-stage clinical trials shows great promise for the treatment of human immunodeficiency virus (HIV) infection, but virus is expected to develop resistance to these drugs. Therefore, we used a novel resistance selection protocol to follow the emergence of resistant HIV in the presence of the integrase inhibitor elvitegravir (GS-9137). We find the primary resistance-conferring mutations to be Q148R, E92Q, and T66I and demonstrate that they confer a reduction in susceptibility not only to elvitegravir but also to raltegravir (MK-0518) and other integrase inhibitors. The locations of the mutations are highlighted in the catalytic sites of integrase, and we correlate the mutations with expected drug-protein contacts. In addition, mutations that do not confer reduced susceptibility when present alone (H114Y, L74M, R20K, A128T, E138K, and S230R) are also discussed in relation to their position in the catalytic core domain and their proximity to known structural features of integrase. These data broaden the understanding of antiviral resistance against integrase inhibitors and may give insight facilitating the discovery of second-generation compounds.     

Design,synthesis and SAR study of bridged tricyclic pyrimidinone carboxamides as HIV-1 integrase inhibitors

The design, synthesis and structure-activity relationships associated with a series of bridged tricyclic pyrimidinone carboxamides as potent inhibitors of HIV-1 integrase strand transfer are described. Structural modifications to these molecules were made in order to examine the effect on potency towards wild-type and clinically-relevant resistant viruses. The [3.2.2]-bridged tricyclic system was identified as an advantageous chemotype, with representatives exhibiting excellent antiviral activity against both wild-type viruses and the G140S/Q148H resistant virus that arises in response to therapy with raltegravir and elvitegravir.     

HIV‐1 IN alternative molecular recognition of DNA induced by raltegravir resistance mutations

Virologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified Ω‐shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir‐resistant viruses. Copyright © 2009 John Wiley & Sons, Ltd.     

HIV-2 Integrase Variation in Integrase Inhibitor-Na?ve Adults in Senegal,West Africa

Background

Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs) raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance.

Methods

We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2–infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1.

Results

No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155). Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein.

Conclusion

Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at “secondary” HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2–infected patients.     

Understanding the effect of drug-resistant mutations of HIV-1 intasome on raltegravir action through molecular modeling study

Raltegravir is the first FDA-approved drug targeting the strand transfer step of HIV-1 integration. However, the rapid emergence of viral strains that are highly resistant to raltegravir has become a critical problem. Unfortunately, the detailed molecular mechanism of how HIV-1 integrase (IN) mutations actually confer drug resistance is not well understood. In the present study, starting from our previously constructed complex of HIV-1 IN and viral DNA, we employed molecular dynamics (MD) simulation and molecular mechanics generalized Born surface area (MM-GBSA) calculation, to uncover the molecular mechanism behind the resistant mechanism of HIV-1 IN to raltegravir. The values of the calculated binding free energy follow consistently the experimentally observed ranking of resistance levels. A detailed analysis of the results of MD simulation suggests that the Tyr143 located in the 140s loop (e.g., residues from Gly140 to Gly149) is a key anchoring residue that leads to stable raltegravir binding. The decrease in the interaction at this residue is one of the key reasons responsible for the resistance of HIV-1 IN to raltegravir. Additionally, the calculation results also proved that the 3' adenosine flip in different conformations in the wild-type and mutant HIV-1 IN-viral DNA complexes play an important role in raltegravir binding. Our results could provide a structural and energetic understanding of the raltegravir-resistant mechanism at the atomic level and provide some new clues on how to design new drugs that may circumvent the known resistance mutations.     

Synthesis,docking, and biological studies of phenanthrene β-diketo acids as novel HIV-1 integrase inhibitors

In the present study we report the synthesis of halogen-substituted phenanthrene β-diketo acids as new HIV-1 integrase inhibitors. The target phenanthrenes were obtained using both standard thermal- and microwave-assisted synthesis. 4-(6-Chlorophenanthren-2-yl)-2,4-dioxobutanoic acid (18) was the most active compound of the series, inhibiting both 3′-end processing (3′-P) and strand transfer (ST) with IC₅₀ values of 5 and 1.3 μM, respectively. Docking studies revealed two predominant binding modes that were distinct from the binding modes of raltegravir and elvitegravir, and suggest a novel binding region in the IN active site. Moreover, these compounds are predicted not to interact significantly with some of the key amino acids (Q148 and N155) implicated in viral resistance. Therefore, this series of compounds can further be investigated for a possible chemotype to circumvent resistance to clinical HIV-1 IN inhibitors.     

Drug resistant integrase mutants cause aberrant HIV integrations

Background

HIV-1 integrase is the target for three FDA-approved drugs, raltegravir, elvitegravir, and dolutegravir. All three drugs bind at the active site of integrase and block the strand transfer step of integration. We previously showed that sub-optimal doses of the anti-HIV drug raltegravir can cause aberrant HIV integrations that are accompanied by a variety of deletions, duplications, insertions and inversions of the adjacent host sequences.

Results

We show here that a second drug, elvitegravir, also causes similar aberrant integrations. More importantly, we show that at least two of the three clinically relevant drug resistant integrase mutants we tested, N155H and G140S/Q148H, which reduce the enzymatic activity of integrase, can cause the same sorts of aberrant integrations, even in the absence of drugs. In addition, these drug resistant mutants have an elevated IC₅₀ for anti-integrase drugs, and concentrations of the drugs that would be optimal against the WT virus are suboptimal for the mutants.

Conclusions

We previously showed that suboptimal doses of a drug that binds to the HIV enzyme integrase and blocks the integration of a DNA copy of the viral genome into host DNA can cause aberrant integrations that involve rearrangements of the host DNA. We show here that suboptimal doses of a second anti-integrase drug can cause similar aberrant integrations. We also show that drug-resistance mutations in HIV integrase can also cause aberrant integrations, even in the absence of an anti-integrase drug. HIV DNA integrations in the oncogenes BACH2 and MKL2 that do not involve rearrangements of the viral or host DNA can stimulate the proliferation of infected cells. Based on what is known about the association of DNA rearrangements and the activation of oncogenes in human tumors, it is possible that some of the deletions, duplications, insertions, and inversions of the host DNA that accompany aberrant HIV DNA integrations could increase the chances that HIV integrations could lead to the development of a tumor.

     

Dolutegravir Resistance Mutation R263K Cannot Coexist in Combination with Many Classical Integrase Inhibitor Resistance Substitutions

The new integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) displays limited cross-resistance with older drugs of this class and selects for the R263K substitution in treatment-experienced patients. We performed tissue culture selections with DTG, using viruses resistant to older INSTIs and infectivity and resistance assays, and showed that the presence of the E92Q or N155H substitution was compatible with the emergence of R263K, whereas the G140S Q148R, E92Q N155H, G140S, Y143R, and Q148R substitutions were not.     

The G140S mutation in HIV integrases from raltegravir-resistant patients rescues catalytic defect due to the resistance Q148H mutation

Raltegravir (MK-0518) is the first integrase (IN) inhibitor to be approved by the US FDA and is currently used in clinical treatment of viruses resistant to other antiretroviral compounds. Virological failure of Raltegravir treatment is associated with mutations in the IN gene following two main distinct genetic pathways involving either the N155 or Q148 residue. Importantly, in most cases, an additional mutation at the position G140 is associated with the Q148 pathway. Here, we investigated the viral DNA kinetics for mutants identified in Raltegravir-resistant patients. We found that (i) integration is impaired for Q148H when compared with the wild-type, G140S and G140S/Q148H mutants; and (ii) the N155H and G140S mutations confer lower levels of resistance than the Q148H mutation. We also characterized the corresponding recombinant INs properties. Enzymatic performances closely parallel ex vivo studies. The Q148H mutation ‘freezes’ IN into a catalytically inactive state. By contrast, the conformational transition converting the inactive form into an active form is rescued by the G140S/Q148H double mutation. In conclusion, the Q148H mutation is responsible for resistance to Raltegravir whereas the G140S mutation increases viral fitness in the G140S/Q148H context. Altogether, these results account for the predominance of G140S/Q148H mutants in clinical trials using Raltegravir.     

Development of an AlphaScreen-based HIV-1 integrase dimerization assay for discovery of novel allosteric inhibitors

In recent years, HIV-1 integrase (IN) has become an established target in the field of antiretroviral drug discovery. However, its sole clinically approved inhibitor, the integrase strand transfer inhibitor (INSTI) raltegravir, has a surprisingly low genetic barrier for resistance. Furthermore, the only two other integrase inhibitors currently in advanced clinical trials, elvitegravir and dolutegravir, share its mechanism of action and certain resistance pathways. To maintain a range of treatment options, drug discovery efforts are now turning toward allosteric IN inhibitors, which should be devoid of cross-resistance with INSTIs. As IN requires a precise and dynamic equilibrium between several oligomeric species for its activities, the modulation of this equilibrium presents an interesting allosteric target. We report on the development, characterization, and validation of an AlphaScreen-based assay for high-throughput screening for modulators of HIV-1 IN dimerization. Compounds identified as hits in this assay proved to act as allosteric IN inhibitors. Additionally, the assay offers a flexible platform to study IN dimerization.     

Raltegravir and elvitegravir-resistance mutation E92Q affects HLA-B*40:02-restricted HIV-1-specific CTL recognition

Interplay between drug-resistance mutations in CTL epitopes and HIV-1-specific CTLs may influence the control of HIV-1 viremia. However, the effect of integrase inhibitor (INI)-resistance mutations on the CTL recognition has not been reported. We here investigated the effect of a raltegravir and elvitegravir-resistance mutation (E92Q) on HLA-B*40:02-restricted Int92-102 (EL11: ETGQETAYFLL)-specific CTLs. EL11-specific CTLs recognized E92Q peptide-pulsed and E92Q mutant virus-infected cells less effectively than EL11 peptide-pulsed and wild-type virus-infected cells, respectively. Ex vivo ELISpot analysis showed no induction of E92Q-specific T cells in chronically HIV-1-infected individuals. Thus, we demonstrated that EL11-specific CTL recognition was affected by the INI-resistance mutation.