Binding assay for characterization of protein kinase inhibitors possessing sub-picomolar to sub-millimolar affinity

Hedi Sinijarv; Shanshan Wu; Taavi Ivan; Tonis Laasfeld; Kaido Viht; Asko Uri

doi:10.1016/j.ab.2017.05.017

Binding assay for characterization of protein kinase inhibitors possessing sub-picomolar to sub-millimolar affinity

Anal Biochem. 2017 Aug 15:531:67-77. doi: 10.1016/j.ab.2017.05.017. Epub 2017 May 18.

Authors

Hedi Sinijarv¹, Shanshan Wu¹, Taavi Ivan¹, Tonis Laasfeld², Kaido Viht¹, Asko Uri³

Affiliations

¹ Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia.
² Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia. Electronic address: tonis.laasfeld@gmail.com.
³ Institute of Chemistry, University of Tartu, 14A Ravila St., 50411 Tartu, Estonia. Electronic address: asko.uri@ut.ee.

PMID: 28527909
DOI: 10.1016/j.ab.2017.05.017

Abstract

High demand for inhibitors regulating the activity of protein kinases has stimulated the quest for high throughput and reliable compound screening assays. Here we introduce a method applying a non-metal photoluminescent probe ARC-Lum(Fluo) for determination of dissociation constants of competitive inhibitors of protein kinases. Employing a single probe instead of a combination of antibody and fluorescent tracer makes the assay simpler, cheaper, and more accurate than several other inhibitor-screening technologies. High affinity (20 pM) and low background signal of the free probe supports the determination of dissociation constants of tight-binding as well as low affinity inhibitors. The calculated lowest K_d value that can be accurately determined with the method is 60 fM. We also introduce graphical presentation of the linearized Cheng-Prusoff equation and demonstrate multiple possibilities for its application (deciding upon the assay formats, calculation of the limits of K_d determination, etc.). The open toolbox (http://www.ut.ee/medchem/toolbox-fluorescence-probes) is available for creating the map of resolvable affinities if applying the competitive probes at defined assay conditions.

Keywords: Affinity; Cheng-Prusoff equation; Control chart; Photoluminescence; Protein kinase inhibitor; Time-gated measurement.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biosensing Techniques / methods*
Cyclic AMP-Dependent Protein Kinases / chemistry*
Fluorescence*
Humans
Protein Kinase Inhibitors / analysis*
Protein Kinase Inhibitors / chemistry

Substances

Protein Kinase Inhibitors
Cyclic AMP-Dependent Protein Kinases