A Fluorescence Lifetime-Based Assay for Abelson Kinase

Abstract

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.

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

“…To illustrate the efficiency of the proposed FLT detection technology for HTS, we demonstrate the feasibility of an enzymatic activity assay relying on FLT detection. The assay (15,18,19) is designed to evidence the activity of the protease human pancreatic trypsin (from autolyzed human pancreas, Elastin Product Company, Inc., Owensville, MO, USA, catalogue number TR127) on a peptide substrate tagged with the fluorescent probe PT14. Due to the structural flexibility of the peptide the probe undergoes dynamic quenching resulting in a FLT of 7 ns.…”

“…The enzyme activity assay was developed and validated previously (15,18,19). It is based on the increase in the FLT of the PT14 probe which is initially quenched by collisions with the flexible tryrosine residue in the substrate.…”

“…This well-known advantage of FLT detection is for instance at the origin of the very successful development of Fluorescence Lifetime Imaging Microscopy (FLIM). (11)(12)(13)(14) In the context of HTS, the same argument has motivated the development of innovative biochemical constructs taking advantage of FLT detection for the efficient sensing of molecular interactions (15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”

Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes

“…For many years, a number of laboratories developed elegant optical sensors to evaluate the activities of these enzymes. In some of them, substrate peptide was conjugated (or fused) to a probe molecule (e.g., Tb(III) complexes [ 35 – 40 ], Mg(II) complexes [ 41 – 47 ], Ca(II) complex [ 48 ], Zn(II) complex [ 49 ], Cd(II) complex [ 50 ], peptide derivatives [ 51 , 52 ], and others [ 53 , 54 ]). The other sensors involve noncovalent interactions between a substrate and a probe (e.g., Tb(III) ion [ 55 – 62 ], Eu(III) complex [ 63 , 64 ], platinum(II) complex [ 65 ], and Tb(III) complexes [ 66 – 69 ]).…”

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

“…2 Assays based on monitoring enzyme-mediated peptide modifications that directly affect the FLT of a reporter dye have been successfully introduced for routine inhibitor profiling in automated processes within the hit-to-lead and lead optimization phases for enzyme classes such as proteases, kinases, and phosphatases. [3][4][5][6][7] The assay principle presented here employs changes in FLT, enabling determination of binding potency of compounds to the active site of an enzyme through the displacement of a conjugated binding probe. Using such an approach, compound screening in cases in which enzymes exhibit only low activity with known substrates is possible.…”