A FRET-Based Assay Platform for Ultra-High Density Drug Screening of Protein Kinases and Phosphatases

Abstract

Protein phosphorylation is one of the major regulatory mechanisms involved in signal-induced cellular events, including cell proliferation, apoptosis, and metabolism. Because many facets of biology are regulated by protein phosphorylation, aberrant kinase and/or phosphatase activity forms the basis for many different types of pathology. The disease relevance of protein kinases and phosphatases has led many pharmaceutical and biotechnology companies to expend significant resources in lead discovery programs for these two target classes. The existence of >500 kinases and phosphatases encoded by the human genome necessitates development of methodologies for the rapid screening for novel and specific compound inhibitors. We describe here a fluorescence-based, molecular assay platform that is compatible with robotic, ultra-high throughput screening systems and can be applied to virtually all tyrosine and serine/threonine protein kinases and phosphatases. The assay has a coupled-enzyme format, utilizing the differential protease sensitivity of phosphorylated versus nonphosphorylated peptide substrates. In addition to screening individual kinases, the assay can be formatted such that kinase pathways are re-created in vitro to identify compounds that specifically interact with inactive kinases. Miniaturization of this assay format to the 1-microl scale allows for the rapid and accurate compound screening of a host of kinase and phosphatase targets, thereby facilitating the hunt for new leads for these target classes.

Continuous assay of protein tyrosine phosphatases based on fluorescence resonance energy transfer

“…In fact, they only presented a discontinuous assay method and the rhodamine 110 fluorescence was read 90 min after the addition of aminopeptidase. To our surprise, Rodems et al [25], without citing our work [7], presented an assay method which is based on a principle almost identical to that of our original discontinuous assay method [7]. Their peptide substrates contained coumarin as a fluorescence donor and fluorescein as an acceptor instead of Mca and DNP in our peptide substrates.…”

A FRET-based microplate assay for human protein kinase CK2, a target in neoplastic disease

“…In the phosphorylated state, the protease recognition site is altered, the cleavage efficiency is lowered, and the EDANS fluorescence is quenched by DABCYL. Although in the present study the molecular basis of the differential protease sensitivity was not further analyzed, it can be assumed that either steric or electrostatic parameters might be involved in the reduction of protease activity 27 .…”

“…A similar approach using a tyrosine kinase and chymotrypsin has been described before 27 . The common CK2 substrate peptide RRRDDDSDDD 23 was labeled with a fluorophore (EDANS) at the C-terminus and a quencher (DABCYL) at the N-terminus ("D/E-peptide").…”

Multiplexed Random Peptide Library and Phospho-Specific Antibodies Facilitate Human Polo-Like Kinase 1 Inhibitor Screen

“…To select a specifi c antibody for phosphorylated form of biotinylated FKD peptide (biotin-DELMEFpSFKDQEAKV; I, Vertex) 25 and a 384-tip pintool (V&P Scientifi c, San Diego, CA) equipped on a CyBi-DISK (CyBio AG, Jena, Germany) to deliver the assay reagents and compounds, respectively. After adding EDTA/TR-FRET, we further incubated the plate for 45 min, and measured RFU665 and RFU615 under the excitation at 337 nm with the Victor 2 -V.…”