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.     

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.     

Dolutegravir Interactions with HIV-1 Integrase-DNA: Structural Rationale for Drug Resistance and Dissociation Kinetics

Signature HIV-1 integrase mutations associated with clinical raltegravir resistance involve 1 of 3 primary genetic pathways, Y143C/R, Q148H/K/R and N155H, the latter 2 of which confer cross-resistance to elvitegravir. In accord with clinical findings, in vitro drug resistance profiling studies with wild-type and site-directed integrase mutant viruses have shown significant fold increases in raltegravir and elvitegravir resistance for the specified viral mutants relative to wild-type HIV-1. Dolutegravir, in contrast, has demonstrated clinical efficacy in subjects failing raltegravir therapy due to integrase mutations at Y143, Q148 or N155, which is consistent with its distinct in vitro resistance profile as dolutegravir’s antiviral activity against these viral mutants is equivalent to its activity against wild-type HIV-1. Kinetic studies of inhibitor dissociation from wild-type and mutant integrase-viral DNA complexes have shown that dolutegravir also has a distinct off-rate profile with dissociative half-lives substantially longer than those of raltegravir and elvitegravir, suggesting that dolutegravir’s prolonged binding may be an important contributing factor to its distinct resistance profile. To provide a structural rationale for these observations, we constructed several molecular models of wild-type and clinically relevant mutant HIV-1 integrase enzymes in complex with viral DNA and dolutegravir, raltegravir or elvitegravir. Here, we discuss our structural models and the posited effects that the integrase mutations and the structural and electronic properties of the integrase inhibitors may have on the catalytic pocket and inhibitor binding and, consequently, on antiviral potency in vitro and in the clinic.     

Discovery of potent HIV integrase inhibitors active against raltegravir resistant viruses

A series of novel HIV integrase inhibitors active against rategravir resistant strains are reported. Initial SAR studies revealed that activities against wild-type virus were successfully maintained at single digit nanomolar level with a wide range of substitutions. However, inclusion of nitrogen-based cyclic substitutions was crucial for achieving potency against mutant viruses. Several compounds with excellent activities against wild-type virus as well as against the viruses with the mutations Q148H/G140S or N155H/E92Q were reported.     

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.     

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.     

Design, synthesis, and biological evaluation of novel tricyclic HIV-1 integrase inhibitors by modification of its pyridine ring

This communication details both the syntheses and biological evaluation of a novel class of HIV-1 integrase inhibitors. When the quinoline moiety is replaced with the quinoxoline moiety, the antiviral activity is significantly compromised. Similarly, introduction of imidazole to replace the pyridine ring is deleterious to the potency of the compound against the enzyme. Substitution at the 3-position of the pyridine has been investigated. The presence of the pyridine ring in the tricyclic core is preferred for antiviral activity against HIV integrase.     

Selection and analysis of HIV-1 integrase strand transfer inhibitor resistant mutant viruses

This report describes methods for the selection and analysis of antiretroviral resistance to HIV integrase strand transfer inhibitors (InSTIs) in cell culture. The method involves the serial passage of HIV-1 in the presence of increasing concentrations of test inhibitors, followed by the cloning and sequencing of the integrase coding region from the selected viruses. The identified mutations are subsequently re-engineered into a reference wild-type molecular clone, and the resulting replication capacity and level of drug resistance are determined relative to the wild-type virus. Here we describe examples of selection and analysis of InSTI-resistant viruses using four integrase inhibitors from three structurally distinct chemical classes; a diketo acid, two naphthyridines, and a pyrimidinecarboxamide. Each inhibitor selected an independent route to resistance. Interestingly, the shift in the IC₅₀ required to suppress the re-engineered resistant mutant viruses closely matched the concentration of compound used during the selection of drug resistance.     

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.     

Naphthyridinone (NTD) integrase inhibitors 4. Investigating N1 acetamide substituent effects with C3 amide groups

A series of N1 acetamide substituted naphthyridinone HIV-1 integrase inhibitors have been explored to understand structure–activity relationships (SAR) with various C3 amide groups. Investigations were evaluated using integrase enzyme inhibition, antiviral activity and protein binding effects to optimize the sub-structures. Lipophilicity was also incorporated to understand ligand lipophilic efficiency as a function of the structural modifications. Three representative analogs were further examined in a peripheral blood mononuclear cell (PBMC) antiviral assay as well as in vitro and in vivo drug metabolism and pharmacokinetic studies.     

E92A是HIV-1整合酶耐药突变N155S的活性回复突变

HIV-1整合酶是目前抗艾滋病药物研发的重要靶点之一,整合酶的耐药突变是导致整合酶抑制剂类药物治疗失败的主要原因,但突变产生耐药性的机理仍不清楚.本工作通过人工构建突变型整合酶,测试其活性和耐药性,对整合酶的耐药机理进行初步探索.构建整合酶的突变型包括E92A、N155S两种单突变及E92A/N155S双突变.通过基因工程操作引入突变、构建质粒、表达纯化得到整合酶蛋白.用基于磁珠的整合酶链转移ELISA测试整合酶的链转移活性,用S-1360和Raltegravir两种抑制剂测试整合酶的耐药性.另外,用Autodock软件做了S-1360和整合酶核心区(包括野生型和突变型)的分子对接.结果表明,N155S突变使整合酶链转移活性下降约80%,而E92A/N155S双突变仅使活性下降约42%,这表明N155S突变基础上的E92A突变可使整合酶的活性大幅回复.E92A和E92A/N155S对不同的抑制剂可产生不同的耐药性,它们对Raltegravir的耐药性强于对S-1360.突变对整合酶活性和耐药性的影响主要是通过改变整合酶活性中心结构实现的,E92A突变可能导致其与周围残基静电相互作用减弱,间接影响到D64和D116残基,产生活性回复作用.     

Role of the 207-218 peptide region of Moloney murine leukemia virus integrase in enzyme catalysis

X-ray diffraction data on a few retroviral integrases show a flexible loop near the active site. By sequence alignment, the peptide region 207-218 of Mo-MLV IN appears to correspond to this flexible loop. In this study, residues H208, Y211, R212, Q214, S215 and S216 of Mo-MLV IN were mutated to determine their role on enzyme activity. We found that Y211A, R212A, R212K and Q214A decreased integration activity, while disintegration and 3′-processing were not significantly affected. By contrast H208A was completely inactive in all the assays. The core domain of Mo-MLV integrase was modeled and the flexibility of the region 207-216 was analyzed. Substitutions with low integration activity showed a lower flexibility than wild type integrase. We propose that the peptide region 207-216 is a flexible loop and that H208, Y211, R212 and Q214 of this loop are involved in the correct assembly of the DNA-integrase complex during integration.     

Combining symmetry elements results in potent naphthyridinone (NTD) HIV-1 integrase inhibitors

A series of naphthyridinone HIV-1 integrase strand-transfer inhibitors have been designed based on a psdeudo-C2 symmetry element present in the two-metal chelation pharmacophore. A combination of two distinct inhibitor binding modes resulted in potent inhibition of the integrase strand-transfer reaction in the low nM range. Effects of aryl and N1 substitutions are disclosed including the impact on protein binding adjusted antiviral activity.     

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.