RESUMO
Proline-based trypsin inhibitors occupying the S1-S2-S1' region were identified by an HTS screening campaign. It was discovered that truncation of the P1' moiety and appropriate extension into the S4 region led to highly potent trypsin inhibitors with excellent selectivity against related serine proteases and a favorable hERG profile.
Assuntos
Pancreatite/tratamento farmacológico , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/uso terapêutico , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologiaRESUMO
Fluorescence lifetime is an intrinsic parameter describing the fluorescence process. Changes in the fluorophore's physicochemical environment can lead to changes in the fluorescence lifetime. When used as the readout in biological assays, it is thought to deliver superior results to conventional optical readouts. Hence it has the potential to replace readout technologies currently established in drug discovery such as absorption, luminescence or fluorescence intensity. Here we report the development of an activity assay for human kallikrein 7, a serine protease involved in skin diseases. As a probe, we have selected a blue-fluorescent acridone dye, featuring a remarkably long lifetime that can be quenched by either of the 2 natural amino acids, tyrosine and tryptophan. Incorporating this probe and 1 of the quenching amino acids on either side of the scissile bond of the substrate peptide enables us to monitor the enzymatic activity by quantifying the increase in the fluorescence lifetime signal. A systematic investigation of substrate structures has led to a homogenous, microplate-based, compound profiling assay that yields inhibitory constants down into the single-digit nanomolar range. This type of assay has now been added to our standard portfolio of screening techniques, and is routinely used for compound profiling.
Assuntos
Fluorescência , Calicreínas/antagonistas & inibidores , Medições Luminescentes/métodos , Inibidores de Proteases/análise , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Humanos , Calicreínas/metabolismo , Dados de Sequência Molecular , Especificidade por Substrato , Fatores de TempoRESUMO
Fluorescence lifetime (FLT)-based assays have developed to become highly attractive tools in drug discovery. All recently published examples of FLT-based assays essentially describe their use for monitoring enzyme-mediated peptide modifications, such as proteolytic cleavage or phosphorylation/dephosphorylation. Here we report the development of competitive binding assays as novel, inhibitor-centric assays, principally employing the FLT of the acridone dye Puretime 14 (PT14) as the readout parameter. Exemplified with two case studies on human serine proteases, the details of the rationale for both the design and synthesis of probes (i.e., active site-directed low-molecular-weight inhibitors conjugated to PT14) are provided. Data obtained from testing inhibitors with the novel assay format match those obtained with alternative formats such as FLT-based protease activity and time-resolved fluorescence resonance energy transfer-based competitive binding assays.
Assuntos
Descoberta de Drogas/métodos , Inibidores de Proteases/química , Espectrometria de Fluorescência/métodos , Acridonas/química , Ligação Competitiva , Soluções Tampão , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Pulmão/enzimologia , Conformação Molecular , Peso Molecular , Peptídeos/química , Ligação Proteica , Proteínas Recombinantes/química , Serina Proteases/química , Triptases/químicaRESUMO
BACKGROUND AND AIMS: Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis. METHODS: Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent "smart" probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L-lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging. RESULTS: We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence-based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio. CONCLUSIONS: We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition.
Assuntos
Corantes Fluorescentes , Imagem Óptica , Pancreatite/diagnóstico , Doença Aguda , Animais , Carbocianinas , Modelos Animais de Doenças , Endopeptidases/metabolismo , Ativação Enzimática , Feminino , Pancreatite/tratamento farmacológico , Pancreatite/enzimologia , Inibidores de Proteases/farmacologia , Ratos , Tripsina/metabolismo , Inibidores da Tripsina/administração & dosagem , Inibidores da Tripsina/farmacologiaRESUMO
INTRODUCTION: Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. AREAS COVERED: To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. EXPERT OPINION: The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.
RESUMO
We present a novel homogeneous in vitro assay format and apply it to the quantitative determination of the enzymatic activity of a tyrosine kinase. The assay employs a short peptidic substrate containing a single tyrosine and a single probe attached via a cysteine side chain. The structural flexibility of the peptide allows for the dynamic quenching of the probe by the nonphosphorylated tyrosine side chain. The probe responds with changes in its fluorescence lifetime depending on the phosphorylation state of the tyrosine. We use this effect to directly follow the enzymatic phosphorylation of the substrate, without having to resort to additional assay components such as an antibody against the phosphotyrosine. As an example for the application of this assay principle, we present results from the development of an assay for Abelson kinase (c-Abl) used for compound profiling. Adjustments in the peptide sequence would make this assay format suitable to a wide variety of other tyrosine kinases.
Assuntos
Corantes Fluorescentes/química , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/metabolismo , Espectrometria de Fluorescência/métodos , Sequência de Aminoácidos , Bioensaio , Humanos , Concentração Inibidora 50 , Peptídeos/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Especificidade por Substrato , Tirosina/metabolismoRESUMO
Fragment-based screening (FBS) has gained acceptance in the pharmaceutical industry as an attractive approach for the identification of new chemical starting points for drug discovery programs in addition to classical strategies such as high-throughput screening. There is the concern that screening of fragments at high µM concentrations in biochemical assays results in increased false-positive and false-negative rates. Here the authors systematically compare the data quality of FBS obtained by enzyme activity-based fluorescence intensity, fluorescence lifetime, and mobility shift assays with the data quality from surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) methods. The serine protease trypsin and the matrix metalloprotease MMP12 were selected as model systems. For both studies, 352 fragments were selected each. From the data generated, all 3 biochemical protease assay methods can be used for screening of fragments with low false-negative and low false-positive rates, comparable to those achieved with the SPR-based assays. It can also be concluded that only fragments with a solubility higher than the screening concentration determined by means of NMR should be used for FBS purposes. Extrapolated to 10,000 fragments, the biochemical assays speed up the primary FBS process by approximately a factor of 10 and reduce the protease consumption by approximately 10,000-fold compared to NMR protein observation experiments.