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1.
Anal Biochem ; 497: 8-17, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26743718

RESUMO

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is a central apoptosis regulator that blocks cell death by inhibiting caspase-3, caspase-7, and caspase-9 via binding interactions with the XIAP BIR2 and BIR3 domains (where BIR is baculovirus IAP repeat). Smac protein, in its dimeric form, effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Here we describe the development of highly sensitive homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assays to measure binding affinities of potent bivalent peptidomimetic inhibitors of XIAP. Our results indicate that these assays can differentiate Smac-mimetic inhibitors with a wide range of binding affinities down to the picomolar range. Furthermore, we demonstrate the utility of these fluorescent tools for characterization of inhibitor off-rates, which as a crucial determinant of target engagement and cellular potency is another important parameter to guide optimization in a structure-based drug discovery effort. Our study also explores how increased inhibitor valency can lead to enhanced potency at multimeric proteins such as IAP.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Peptidomiméticos/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Caspase 3/metabolismo , Linhagem Celular , Humanos , Camundongos Endogâmicos BALB C , Peptidomiméticos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química
2.
Bioanalysis ; 8(4): 265-74, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26807991

RESUMO

BACKGROUND: A target protein-based affinity extraction LC-MS/MS method was developed to enable plasma level determination following ultralow dosing (0.1-3 µg/kg) of an inhibitor of apoptosis proteins molecule. Methodology & results: Affinity extraction (AE) utilizing immobilized target protein BIR2/BIR3 was used to selectively capture the inhibitor of apoptosis proteins molecule from dog plasma and enable removal of background matrix components. Pretreatment of plasma samples using protein precipitation was found to provide an additional sensitivity gain. A LLOQ of 7.8 pM was achieved by combining protein precipitation with AE. The method was used to support an ultralow dose dog toxicity study. CONCLUSION: AE-LC-MS/MS, utilizing target protein, is a highly sensitive methodology for small molecule quantification with potential for broader applicability.


Assuntos
Análise Química do Sangue/métodos , Fracionamento Químico/métodos , Cromatografia Líquida/métodos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Isoquinolinas/análise , Limite de Detecção , Oligopeptídeos/análise , Bibliotecas de Moléculas Pequenas/análise , Espectrometria de Massas em Tandem/métodos , Animais , Cães , Feminino , Humanos , Proteínas Imobilizadas/antagonistas & inibidores , Proteínas Imobilizadas/química , Proteínas Inibidoras de Apoptose/química , Isoquinolinas/química , Isoquinolinas/farmacologia , Masculino , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
3.
Exp Cell Res ; 338(2): 251-60, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26302264

RESUMO

Cellular levels of inhibitor of apoptosis (IAP) proteins are elevated in multiple human cancers and their activities often play a part in promoting cancer cell survival by blocking apoptotic pathways, controlling signal transduction pathways and contributing to resistance. These proteins function through interactions of their BIR (baculoviral IAP repeat) protein domains with pathway components and these interactions are endogenously antagonized by Smac/Diablo (second mitochondrial activator of caspases/direct IAP binding protein with low isoelectric point). This report describes development of synthetic smac mimetics (SM) and compares their binding, antiproliferative and anti-tumor activities. All dimeric antagonists inhibit in vitro smac tetrapeptide binding to recombinant IAP proteins, rescue IAP-bound caspase-3 activity and show anti-proliferative activity against human A875 melanoma cells. One heterodimeric SM, SM3, binds tightly to IAP proteins in vitro and slowly dissociates (greater than two hours) from these protein complexes compared to the other antagonists. In addition, in vitro SM anti-proliferation potency is influenced by ABCB1 transporter (ATP-binding cassette, sub-family B; MDR1, P-gp) activities and one antagonist, SM5, does not appear to be an ABCB1 efflux pump substrate. All dimeric smac mimetics inhibit the growth of human melanoma A875 tumors implanted in athymic mice at well-tolerated doses. One antagonist, SM4, shows broad spectrum in vivo anti-tumor activity and modulates known pharmacodynamic markers of IAP antagonism. These data taken together demonstrate the range of diverse dimeric IAP antagonist activities and supports their potential as anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Caspase 3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Mitocondriais/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Biomimética/métodos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HCT116 , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos
4.
ACS Med Chem Lett ; 6(7): 770-5, 2015 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-26191364

RESUMO

A series of dimeric macrocyclic compounds were prepared and evaluated as antagonists for inhibitor of apoptosis proteins. The most potent analogue 11, which binds to XIAP and c-IAP proteins with high affinity and induces caspase-3 activation and ultimately cell apoptosis, inhibits growth of human melanoma and colorectal cell lines at low nanomolar concentrations. Furthermore, compound 11 demonstrated significant antitumor activity in the A875 human melanoma xenograft model at doses as low as 2 mg/kg on a q3d schedule.

5.
J Med Chem ; 58(3): 1556-62, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25584393

RESUMO

The prominent role of IAPs in controlling cell death and their overexpression in a variety of cancers has prompted the development of IAP antagonists as potential antitumor therapies. We describe the identification of a series of heterodimeric antagonists with highly potent antiproliferative activities in cIAP- and XIAP-dependent cell lines. Compounds 15 and 17 further demonstrate curative efficacy in human melanoma and lung cancer xenograft models and are promising candidates for advanced studies.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Prolina/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Estrutura Molecular , Neoplasias Experimentais/patologia , Prolina/síntese química , Prolina/química , Relação Estrutura-Atividade
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