RESUMO
Novel cardiac troponin activators were identified using a high throughput cardiac myofibril ATPase assay and confirmed using a series of biochemical and biophysical assays. HTS hit 2 increased rat cardiomyocyte fractional shortening without increasing intracellular calcium concentrations, and the biological target of 1 and 2 was determined to be the cardiac thin filament. Subsequent optimization to increase solubility and remove PDE-3 inhibition led to the discovery of CK-963 and enabled pharmacological evaluation of cardiac troponin activation without the competing effects of PDE-3 inhibition. Rat echocardiography studies using CK-963 demonstrated concentration-dependent increases in cardiac fractional shortening up to 95%. Isothermal calorimetry studies confirmed a direct interaction between CK-963 and a cardiac troponin chimera with a dissociation constant of 11.5 ± 3.2 µM. These results provide evidence that direct activation of cardiac troponin without the confounding effects of PDE-3 inhibition may provide benefit for patients with cardiovascular conditions where contractility is reduced.
RESUMO
Replacement of a secondary amide with a piperidine or azetidine moiety in a series of CCR5 antagonists led to the discovery of compounds with increased intrinsic permeability. This effort led to the identification of a potent CCR5 antagonist which exhibited an improved in vivo pharmacokinetic profile.
Assuntos
Amidas/química , Compostos Aza/farmacologia , Antagonistas dos Receptores CCR5 , Compostos Aza/química , Compostos Aza/farmacocinética , Relação Estrutura-AtividadeRESUMO
The introduction of N-substituted pyrazoles in a new series of CCR5 antagonists was shown to substantially increase antiviral activity.
Assuntos
Fármacos Anti-HIV/química , Antagonistas dos Receptores CCR5 , Pirazóis/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Humanos , Pirazóis/síntese química , Pirazóis/farmacologia , Receptores CCR5/metabolismo , Relação Estrutura-AtividadeRESUMO
Elaboration of our previously disclosed spiropiperidine template led to the development of a series of novel CCR5 antagonists. Results of SAR exploration and preliminary lead characterization are described.
Assuntos
Antagonistas dos Receptores CCR5 , Imidazolidinas/síntese química , Imidazolidinas/farmacologia , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Imidazolidinas/química , Imidazolidinas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Starting with a high-throughput screening lead, a novel series of CCR5 antagonists was developed utilizing an information-based approach. Improvement of pharmacokinetic properties for the series was pursued by SAR exploration of the lead template. The synthesis, SAR and biological profiles of the series are described.
Assuntos
Fármacos Anti-HIV/química , Benzamidas/química , Antagonistas dos Receptores CCR5 , Inibidores da Fusão de HIV/química , Pirróis/química , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacocinética , Benzamidas/síntese química , Benzamidas/farmacologia , Inibidores da Fusão de HIV/síntese química , Inibidores da Fusão de HIV/farmacocinética , Humanos , Microssomos Hepáticos/metabolismo , Pirróis/síntese química , Pirróis/farmacocinética , Ratos , Receptores CCR5/metabolismo , Relação Estrutura-AtividadeRESUMO
The bicyclic 5-amino-3-azabicyclo[3.3.0]octanes were shown to be effective replacements for the 3-amino-8-azabicyclo[3.2.1]octane found in the CCR5 antagonist maraviroc.
Assuntos
Fármacos Anti-HIV/química , Compostos Azabicíclicos/química , Antagonistas dos Receptores CCR5 , Cicloexanos/química , Inibidores da Fusão de HIV/química , Triazóis/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Linhagem Celular Tumoral , Cicloexanos/síntese química , Cicloexanos/farmacologia , Inibidores da Fusão de HIV/síntese química , Inibidores da Fusão de HIV/farmacologia , Humanos , Maraviroc , Modelos Químicos , Receptores CCR5/metabolismo , Triazóis/síntese química , Triazóis/farmacologiaRESUMO
The bicyclic 5-amino-3-azabicyclo[3.3.0]octanes were shown to be effective replacements for the conformationally restricted 4-aminopiperidine ring found in several series of CCR5 antagonists.
Assuntos
Antagonistas dos Receptores CCR5 , Avaliação Pré-Clínica de Medicamentos , Piperidinas/química , Modelos MolecularesRESUMO
Replacement of a secondary amide with an N-acyl or N-sulfonyl gem-disubstituted azacyle in a series of CCR5 antagonists led to the identification of compounds with excellent in vitro HIV antiviral activity and increased intrinsic membrane permeability.
Assuntos
Amidas/química , Fármacos Anti-HIV/química , Compostos Aza/química , Antagonistas dos Receptores CCR5 , Inibidores da Fusão de HIV/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Compostos Aza/síntese química , Compostos Aza/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Inibidores da Fusão de HIV/síntese química , Inibidores da Fusão de HIV/farmacologia , Humanos , Receptores CCR5/metabolismoRESUMO
A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.
Assuntos
Antagonistas dos Receptores CCR5 , Piperidinas/química , Piperidinas/farmacologia , Receptores CCR5/metabolismo , Animais , Células CACO-2 , Cães , Haplorrinos , Humanos , Piperidinas/farmacocinética , Ratos , Compostos de Espiro/química , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
Replacement of the cyclic carbamate in our previously disclosed 1-oxa-3,9-diazaspiro[5.5]undecan-2-one template led to the discovery of two novel series of 3,9-diazaspiro[5.5]undecane and undeca-2-one CCR5 antagonists. The synthesis, SAR, and antiviral activities of these two series are described. One compound (32) was found to have attractive combination of antiviral potency, selectivity, and pharmacokinetic profile. The asymmetric synthesis of 32 was also accomplished and both enantiomers were equally potent.
Assuntos
Alcanos/síntese química , Antivirais/síntese química , Antagonistas dos Receptores CCR5 , Compostos de Espiro/síntese química , Administração Oral , Alcanos/farmacologia , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Disponibilidade Biológica , Descoberta de Drogas , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
A panel of four CCR5 monoclonal antibodies (mAbs) recognizing different epitopes on CCR5 was examined in CCR5-mediated cell-cell fusion assay, alone or in combination with a variety of small molecule CCR5 antagonists. Although no antagonism was observed between any of the CCR5 inhibitors, surprisingly potent synergy was observed between CCR5 mAbs and antagonists, and the synergistic activity was confirmed in other antiviral assays. Strong synergy was also observed between CCR5 inhibitors and the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide. There was no synergy observed between small molecule CCR5 inhibitors; however, potent synergy was observed between mAbs recognizing different parts of CCR5. In all synergistic combinations, greater synergy was achieved at higher percent inhibition levels. A negative correlation was found between the degree of synergy between the two classes of CCR5 inhibitors and the ability to compete each other for binding to the receptor. For example, the greatest synergy, observed between the mAb ROAb13 and the small molecule inhibitor maraviroc, did not interfere with binding to CCR5 for either inhibitor, whereas no synergy was found between mAb 45523 and maraviroc, which do compete for binding to CCR5. In addition, in contrast to a recent report, the CCR5 inhibitors tested here were found to inhibit the same stage of HIV entry. Based on the data presented here, we hypothesize that CCR5 inhibitors exert synergistic antiviral actions through a cobinding mechanism.
Assuntos
Fármacos Anti-HIV/farmacologia , Anticorpos Monoclonais/farmacologia , Antagonistas dos Receptores CCR5 , Animais , Fármacos Anti-HIV/metabolismo , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Sinergismo Farmacológico , Enfuvirtida , Proteína gp41 do Envelope de HIV/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores CCR5/metabolismoRESUMO
Six mouse anti-human CCR5 monoclonal antibodies (mAbs) that showed potent antiviral activities were identified from over 26,000 mouse hybridomas. The epitopes for these mAbs were determined by using various CCR5 mutants, including CCR5/CCR2B chimeras. One mAb, ROAb13, was found to bind to a linear epitope in the N terminus of CCR5. Strikingly, the other five mAbs bind to epitopes derived from extracellular loop 2 (ECL2). The three most potent mAbs, ROAb12, ROAb14, and ROAb18, require residues from both the N-terminal (Lys171 and Glu172) and C-terminal (Trp190) halves of ECL2 for binding; two other mAbs, ROAb10 and ROAb51, which also showed potent antiviral activities, require Lys171 and Glu172 but not Trp190 for binding. Binding of the control mAb 2D7 completely relies on Lys171 and Glu172. Unlike 2D7, the novel mAbs ROAb12, ROAb14, and ROAb18 do not bind to the linear peptide 2D7-2SK. In addition, all three mAbs bind to monkey CCR5 (with Arg at position 171 instead of Lys); however, 2D7 does not. Since five of the six most potent CCR5 mAbs derived from the same pool of immunized mice require ECL2 as epitopes, we hypothesize that CCR5 ECL2 contains the dominant epitopes for mAbs with potent antiviral activities. These dominant epitopes were found in CCR5 from multiple species and were detected in large proportions of the total cell surface CCR5. mAbs recognizing these epitopes also showed high binding affinity. A homology model of CCR5 was generated to aid in the interpretation of these dominant epitopes in ECL2.
Assuntos
Anticorpos Monoclonais/imunologia , Antagonistas dos Receptores CCR5 , Epitopos/química , HIV-1/imunologia , Receptores CCR5/imunologia , Mapeamento de Epitopos , Epitopos/genética , Anticorpos Anti-HIV , HIV-1/patogenicidade , Humanos , Receptores CCR5/química , Receptores CCR5/genética , Relação Estrutura-AtividadeRESUMO
Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.