RESUMO
Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Biblioteca de Peptídeos , Alanina/genética , Alanina/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Técnicas de Visualização da Superfície Celular/economia , Análise Custo-Benefício , Citometria de Fluxo/economia , Citometria de Fluxo/métodos , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Mutação , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular activity and in vivo efficacy. This structure-activity relationship study focused on the P4 oxamides and warhead moieties. Primarily on the basis of in vitro data, inhibitors were selected for study in a murine model of alpha-Fas-induced liver injury. IDN-6556 (1) was further profiled in additional in vivo models and pharmacokinetic studies. This first-in-class caspase inhibitor is now the subject of two Phase II clinical trials, evaluating its safety and efficacy for use in liver disease.
Assuntos
Inibidores de Caspase , Hepatopatias/tratamento farmacológico , Ácidos Pentanoicos/síntese química , Adulto , Alanina Transaminase/sangue , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/sangue , Disponibilidade Biológica , Caspase 3 , Colestase/tratamento farmacológico , Colestase/patologia , Ensaios Clínicos Fase I como Assunto , Meia-Vida , Hepatite C Crônica/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Células Jurkat , Fígado/efeitos dos fármacos , Fígado/patologia , Hepatopatias/enzimologia , Hepatopatias/etiologia , Camundongos , Ácidos Pentanoicos/química , Ácidos Pentanoicos/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Various heterocyclic hetero-methyl ketones of the 1-naphthyloxyacetyl-Val-Asp backbone have been prepared. A study of their structure-activity relationship (SAR) related to caspase-1, -3, -6, and -8 is reported. Their efficacy in a cellular model of cell death is also discussed. Potent broad-spectrum caspase inhibitors have been identified.
Assuntos
Inibidores de Caspase , Morte Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Compostos Heterocíclicos/farmacologia , Cetonas/farmacologia , Animais , Ácido Aspártico/química , Células Cultivadas , Compostos Heterocíclicos/síntese química , Cetonas/síntese química , Camundongos , Modelos Biológicos , Naftóis/química , Relação Estrutura-Atividade , Valina/químicaRESUMO
Various aryloxy methyl ketones of the 1-naphthyloxyacetyl-Val-Asp backbone have been prepared. A systematic study of their structure-activity relationship (SAR) related to caspases 1, 3, 6, and 8 is reported. Highly potent irreversible broad-spectrum caspase inhibitors have been identified. Their efficacy in cellular models of cell death and inflammation are also discussed.
Assuntos
Inibidores de Caspase , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Relação Estrutura-AtividadeRESUMO
Structural modifications were made to a previously described acyl dipeptide caspase inhibitor, leading to the oxamyl dipeptide series. Subsequent SAR studies directed toward the warhead, P2, and P4 regions of this novel peptidomimetic are described herein.