Trypsin inhibitors for the treatment of pancreatitis.

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.

Fluorescence Lifetime-Based Competitive Binding Assays for Measuring the Binding Potency of Protease Inhibitors In Vitro.

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.

In vivo imaging with fluorescent smart probes to assess treatment strategies for acute pancreatitis.

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.

Fluorescence lifetime assays: current advances and applications in drug discovery.

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.

A fluorescence lifetime-based assay for abelson kinase.

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.

Fragment-based screening by biochemical assays: Systematic feasibility studies with trypsin and MMP12.

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.

A fluorescence lifetime-based assay for protease inhibitor profiling on human kallikrein 7.

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.