RESUMO
The recently approved HIV-1 integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) (S/GSK1349572) has overall advantageous activity when tested in vitro against HIV-1 with raltegravir (RAL) and elvitegravir (EVG) resistance signature mutations. We conducted an in vitro resistance selection study using wild-type HIV-1 and mutants with the E92Q, Y143C, Y143R, Q148H, Q148K, Q148R, and N155H substitutions to assess the DTG in vitro barrier to resistance. No viral replication was observed at concentrations of ≥ 32 nM DTG, whereas viral replication was observed at 160 nM RAL or EVG in the mutants. In the Q148H, Q148K, or Q148R mutants, G140S/Q148H, E138K/Q148K, E138K/Q148R, and G140S/Q148R secondary mutations were identified with each INSTI and showed high resistance to RAL or EVG but limited resistance to DTG. E138K and G140S, as secondary substitutions to Q148H, Q148K, or Q148R, were associated with partial recovery in viral infectivity and/or INSTI resistance. In the E92Q, Y143C, Y143R, and N155H mutants, no secondary substitutions were associated with DTG. These in vitro results suggest that DTG has a high barrier to the development of resistance in the presence of RAL or EVG signature mutations other than Q148. One explanation for this high barrier to resistance is that no additional secondary substitution of E92Q, Y143C, Y143R, or N155H simultaneously increased the fold change in 50% effective concentration (EC50) to DTG and infectivity. Although increased DTG resistance via the Q148 pathway and secondary substitutions occurs at low concentrations, a higher starting concentration may reduce or eliminate the development of DTG resistance in this pathway in vitro.
Assuntos
Farmacorresistência Viral/genética , Quinolonas/farmacologia , Raltegravir Potássico/farmacologia , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Mutação/genética , Oxazinas , Piperazinas , PiridonasRESUMO
GSK1265744 is a new HIV integrase strand transfer inhibitor (INSTI) engineered to deliver efficient antiviral activity with a once-daily, low-milligram dose that does not require a pharmacokinetic booster. The in vitro antiviral profile and mechanism of action of GSK1265744 were established through integrase enzyme assays, resistance passage experiments, and cellular assays with site-directed molecular (SDM) HIV clones resistant to other classes of anti-HIV-1 agents and earlier INSTIs. GSK1265744 inhibited HIV replication with low or subnanomolar efficacy and with a selectivity index of at least 22,000 under the same culture conditions. The protein-adjusted half-maximal inhibitory concentration (PA-EC50) extrapolated to 100% human serum was 102 nM. When the virus was passaged in the presence of GSK1265744, highly resistant mutants with more than a 10-fold change (FC) in EC50 relative to that of the wild-type were not observed for up to 112 days of culture. GSK1265744 demonstrated activity against SDM clones containing the raltegravir (RAL)-resistant Y143R, Q148K, N155H, and G140S/Q148H signature variants (FC less than 6.1), while these mutants had a high FC in the EC50 for RAL (11 to >130). Either additive or synergistic effects were observed when GSK1265744 was tested in combination with representative anti-HIV agents, and no antagonistic effects were seen. These findings demonstrate that, similar to dolutegravir, GSK1265744 is differentiated as a new INSTI, having a markedly distinct resistance profile compared with earlier INSTIs, RAL, and elvitegravir (EVG). The collective data set supports further clinical development of GSK1265744.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , Piridonas/uso terapêutico , Linhagem Celular , Farmacorresistência Viral/genética , Infecções por HIV/virologia , Integrase de HIV/efeitos dos fármacos , HIV-1/genética , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Testes de Sensibilidade Microbiana , Oxazinas , Piperazinas , Quinolonas/uso terapêutico , Raltegravir Potássico/uso terapêutico , Replicação Viral/efeitos dos fármacosRESUMO
Human immunodeficiency virus type-1 (HIV-1) protease is essential for viral propagation, and its inhibitors are key anti-HIV-1 drug candidates. In this study, we discovered a novel HIV-1 protease inhibitor (compound 16) with potent antiviral activity and oral bioavailability using a structure-based drug design approach via X-ray crystal structure analysis and improved metabolic stability, starting from hit macrocyclic peptides identified by mRNA display against HIV-1 protease. We found that the improvement of the proteolytic stability of macrocyclic peptides by introducing a methyl group to the α-position of amino acid is crucial to exhibit strong antiviral activity. In addition, macrocyclic peptides, which have moderate metabolic stability and solubility in solutions containing taurocholic acid, exhibited desirable plasma total clearance and oral bioavailability. These approaches may contribute to the successful discovery and development of orally bioavailable peptide drugs.
RESUMO
A novel strategy for lead identification that we have dubbed the "Pocket-to-Lead" strategy is demonstrated using HIV-1 protease as a model target. Sometimes, it is difficult to obtain hit compounds because of the difficulties in satisfying the complex pharmacophoric features. In this study, a virtual fragment hit which does not match all of the pharmacophore features but has key interactions and vectors that could grow into remaining pharmacophore features was optimized in silico. The designed compound 9 demonstrated weak but evident inhibitory activity (IC50 = 54 µM), and the design concept was proven by the co-crystal structure. Then, structure-based drug design promptly gave compound 14 (IC50 = 0.0071 µM, EC50 = 0.86 µM), an almost 10,000-fold improvement in activity from 9. The structure of the designed molecules proved to be novel with high synthetic feasibility, indicating the usefulness of this strategy to tackle tough targets with complex pharmacophore.
Assuntos
Inibidores da Protease de HIV , HIV-1 , Desenho de Fármacos , Protease de HIV/metabolismo , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , HIV-1/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Inibidores de ProteasesRESUMO
S/GSK1349572 is a next-generation HIV integrase (IN) inhibitor designed to deliver potent antiviral activity with a low-milligram once-daily dose requiring no pharmacokinetic (PK) booster. In addition, S/GSK1349572 demonstrates activity against clinically relevant IN mutant viruses and has potential for a high genetic barrier to resistance. S/GSK1349572 is a two-metal-binding HIV integrase strand transfer inhibitor whose mechanism of action was established through in vitro integrase enzyme assays, resistance passage experiments, activity against viral strains resistant to other classes of anti-HIV agents, and mechanistic cellular assays. In a variety of cellular antiviral assays, S/GSK1349572 inhibited HIV replication with low-nanomolar or subnanomolar potency and with a selectivity index of 9,400. The protein-adjusted half-maximal effective concentration (PA-EC(50)) extrapolated to 100% human serum was 38 nM. When virus was passaged in the presence of S/GSK1349572, highly resistant mutants were not selected, but mutations that effected a low fold change (FC) in the EC(50) (up to 4.1 fold) were identified in the vicinity of the integrase active site. S/GSK1349572 demonstrated activity against site-directed molecular clones containing the raltegravir-resistant signature mutations Y143R, Q148K, N155H, and G140S/Q148H (FCs, 1.4, 1.1, 1.2, and 2.6, respectively), while these mutants led to a high FC in the EC(50) of raltegravir (11- to >130-fold). Either additive or synergistic effects were observed when S/GSK1349572 was tested in combination with representative approved antiretroviral agents; no antagonistic effects were seen. These findings demonstrate that S/GSK1349572 would be classified as a next-generation drug in the integrase inhibitor class, with a resistance profile markedly different from that of first-generation integrase inhibitors.
Assuntos
Fármacos Anti-HIV/farmacologia , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Linhagem Celular , Linhagem Celular Transformada , Farmacorresistência Viral , Inibidores de Integrase de HIV/síntese química , Inibidores de Integrase de HIV/química , HIV-1/enzimologia , HIV-1/fisiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Naftiridinas/síntese química , Naftiridinas/química , Naftiridinas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/virologia , Integração Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
SKG mice, a newly established model of rheumatoid arthritis (RA), spontaneously develop autoimmune arthritis accompanying extra-articular manifestations, such as interstitial pneumonitis. To examine possible roles of T cells for mediating this systemic autoimmunity, we generated T cell clones from arthritic joints of SKG mice. Two distinct CD8(+) clones were established and both showed in vitro autoreactivity by killing syngeneic synovial cells and a variety of MHC-matched cell lines. Transfer of each clone to histocompatible athymic nude mice elicited joint swelling and histologically evident synovitis accompanying the destruction of adjacent cartilage and bone. Notably, the transfer also produced diffuse severe interstitial pneumonitis. Clone-specific TCR gene messages in the inflamed joints and lungs of the recipients gradually diminished, becoming hardly detectable in 6-11 months; yet, arthritis and pneumonitis continued to progress. Thus, the same CD8(+) T cell clones from arthritic lesions of SKG mice can elicit both synovitis and pneumonitis, which chronically progress and apparently become less T cell dependent in a later phase. The results provide clues to our understanding of how self-reactive T cells cause both articular and extra-articular lesions in RA as a systemic autoimmune disease.
Assuntos
Artrite Experimental/imunologia , Linfócitos T CD8-Positivos/imunologia , Pneumonia/imunologia , Transferência Adotiva , Animais , Artrite Experimental/patologia , Artrite Experimental/fisiopatologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Autoantígenos/imunologia , Autoimunidade/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Células Clonais/imunologia , Células Clonais/metabolismo , Células Clonais/patologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Progressão da Doença , Humanos , Articulações/imunologia , Articulações/patologia , Camundongos , Pneumonia/patologia , Pneumonia/fisiopatologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologiaRESUMO
Specificities of cytotoxic T lymphocyte (CTL) effector cells induced in BALB/c mouse by immunization with the single modified CTL epitope peptide derived from NS3 of dengue virus types 1 and 3, or that of dengue virus types 2 and 4 were examined. The effector cells included CTLs specific for the epitope peptide used for immunization and those cross-reactive to epitope peptides of other flaviviruses. A CTL clone, 2F7, was established from the effector cells. The clone 2F7 was specific for the epitope peptide used for immunization. Recognition by the effector cells was remarkably impaired by amino acid substitutions at positions 3, 5, and 6 of the epitope peptides. These results indicate that immunization with a single CTL epitope peptide of dengue viruses induces serotype-specific CTLs as well as CTLs cross-reactive to the other flaviviruses, and that the a.a. residues at positions 3, 5, and 6 are critical for cross-reaction.
Assuntos
Vírus da Dengue/imunologia , Epitopos de Linfócito T/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas não Estruturais Virais/imunologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Reações Cruzadas , Epitopos de Linfócito T/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , RNA Helicases/genética , RNA Helicases/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Proteínas não Estruturais Virais/genéticaRESUMO
Passage of HIV-1 in the presence of integrase inhibitors (INIs) generates resistant viruses that have mutations in the integrase region. Integrase-resistant mutations Q148K and Q148R were identified as primary mutations with the passage of HIV-1 IIIB in the presence of INIs S-1360 or S/GSK-364735, respectively. Secondary amino acid substitutions E138K or G140S were observed when passage with INI was continued. The role of these mutations was investigated with molecular clones. Relative to Q148K alone, Q148K/E138K had 2- and >6-fold increases in resistance to S-1360 and S/GSK-364735, respectively, and the double mutant had slightly better infectivity and replication kinetics. In contrast, Q148K/G140S and Q148R/E138K had nearly equivalent or slightly reduced fold resistance to the INI compared with their respective Q148 primary mutants, and had increases in infectivity and replication kinetics. Recovery of these surrogates of viral fitness coincided with the recovery of integration efficiency of viral DNA into the host cell chromosome for these double mutants. These data show that recovery of viral integration efficiency can be an important factor for the emergence and maintenance of INI-resistant mutations.
Assuntos
Fármacos Anti-HIV/farmacologia , Integrase de HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Mutação de Sentido Incorreto , Replicação Viral/efeitos dos fármacos , Furanos/farmacologia , Integrase de HIV/genética , HIV-1/genética , Humanos , Células Jurkat , Triazóis/farmacologiaRESUMO
Resistance passage studies were conducted with five INIs (integrase inhibitors) that have been tested in clinical trials to date: a new naphthyridinone-type INI S/GSK-364735, raltegravir, elvitegravir, L-870,810 and S-1360. In establishing the passage system and starting from concentrations several fold above the EC(50) value, resistance mutations against S-1360 and related diketoacid-type compounds could be isolated from infected MT-2 cell cultures from day 14 to 28. Q148R and F121Y were the two main pathways of resistance to S/GSK-364735. Q148R/K and N155H, which were found in patients failing raltegravir treatment in Phase IIb studies, were observed during passage with raltegravir with this method. The fold resistance of 40 mutant molecular clones versus wild type virus was compared with these five INIs. The overall resistance pattern of S/GSK-364735 was similar to that of raltegravir and other INIs. However, different fold resistances of particular mutations were noted among different INIs, reflecting a potential to develop INIs with distinctly different resistant profiles.
Assuntos
Farmacorresistência Viral , Infecções por HIV/virologia , Inibidores de Integrase de HIV/farmacologia , HIV-1/genética , Mutação , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Infecções por HIV/tratamento farmacológico , Integrase de HIV/genética , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/isolamento & purificação , Células HeLa , Humanos , Linfócitos T/virologiaRESUMO
The naphthyridinone GSK364735 potently inhibited recombinant human immunodeficiency virus type 1 (HIV-1) integrase in a strand transfer assay (mean 50% inhibitory concentration +/- standard deviation, 8 +/- 2 nM). As expected based on the structure of the drug, it bound competitively with another two-metal binding inhibitor (Kd [binding constant], 6 +/- 4 nM). In a number of different cellular assays, GSK364735 inhibited HIV replication with potency at nanomolar concentrations (e.g., in peripheral blood mononuclear cells and MT-4 cells, 50% effective concentrations were 1.2 +/- 0.4 and 5 +/- 1 nM, respectively), with selectivity indexes of antiviral activity versus in-assay cytotoxicity of at least 2,200. When human serum was added, the antiviral potency decreased (e.g., a 35-fold decrease in the presence of 100% human serum was calculated by extrapolation from the results of the MT-4 cell assay). In cellular assays, GSK364735 blocked viral DNA integration, with a concomitant increase in two-long-terminal-repeat circles. As expected, this integrase inhibitor was equally active against wild-type viruses and mutant viruses resistant to approved drugs targeting either reverse transcriptase or protease. In contrast, some but not all viruses resistant to other integrase inhibitors were resistant to GSK364735. When virus was passaged in the presence of the inhibitor, we identified resistance mutations within the integrase active site that were the same as or similar to mutations arising in response to other two-metal binding inhibitors. Finally, either additive or synergistic effects were observed when GSK364735 was tested in combination with approved antiretrovirals (i.e., no antagonistic effects were seen). Thus, based on all the data, GSK364735 exerted potent antiviral activity through the inhibition of viral DNA integration by interacting at the two-metal binding site within the catalytic center of HIV integrase.