Fluorescent biosensors for high throughput screening of protein kinase inhibitors

Abstract

High throughput screening assays aim to identify small molecules that interfere with protein function, activity, or conformation, which can serve as effective tools for chemical biology studies of targets involved in physiological processes or pathways of interest or disease models, as well as templates for development of therapeutics in medicinal chemistry. Fluorescent biosensors constitute attractive and powerful tools for drug discovery programs, from high throughput screening assays, to postscreen characterization of hits, optimization of lead compounds, and preclinical evaluation of candidate drugs. They provide a means of screening for inhibitors that selectively target enzymatic activity, conformation, and/or function in vitro. Moreover, fluorescent biosensors constitute useful tools for cell- and image-based, multiplex and multiparametric, high-content screening. Application of fluorescence-based sensors to screen large and complex libraries of compounds in vitro, in cell-based formats or whole organisms requires several levels of optimization to establish robust and reproducible assays. In this review, we describe the different fluorescent biosensor technologies which have been applied to high throughput screens, and discuss the prerequisite criteria underlying their successful application. Special emphasis is placed on protein kinase biosensors, since these enzymes constitute one of the most important classes of therapeutic targets in drug discovery.

Chemical Probes for Human UDP‐Glucuronosyltransferases: A Comprehensive Review

“…Although many drugs and endogenous substances have been identified as probe substrates for a target UGT enzyme, the glucuronidation rates of these UGT probe substrates were routinely detected by LC‐UV or LC‐MS detectors, requiring relative long time for sample preparation and analysis. By contrast, fluorescence‐based biochemical assays have attracted great attention in biological and medical related fields, due to their inherent advantages including non‐destructive, highly sensitive, and applicable to HTS assays . Over the past decade, a wide range of fluorescence probes for serine hydrolases and other phase I metabolizing enzymes have been developed and widely used for evaluating enzyme activities and screening of enzyme modulators .…”

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

“…As such, fluorescent biosensors are extremely useful in the laboratory for fundamental purposes, allowing scientists to tackle questions concerning PK function and dynamics which could not be addressed previously, both in vitro and in living cells in a qualitative and quantitative fashion. Beyond fundamental studies, fluorescent biosensors have also become part of the essential toolbox in drug discovery programs, as they constitute useful probes for HTS and HCS as well as for postscreen characterization of candidate drugs and assessment of their therapeutic efficacy [22]. They are equally a source of inspiration for biomedical applications, including diagnostic assays, enabling development of complementary assays or serving as alternatives to the more traditional antigenic and/or genomic approaches [23].…”

“…The development of imaging systems that allow the acquisition of multispectral data sets has allowed devising assays in which the behavior of several targets can be monitored by combining the simultaneous use of several biosensors [21,22,23]. Although multiparametric imaging remains challenging, it is particularly informative in drug discovery programs, as it allows gaining information on the specificity of pharmacological inhibitors, to characterize differences in potency and inhibitory kinetics, and to identify off-target effects.…”

Editorial: Fluorescent biosensors

“…This article is followed by four reviews which describe the use of FRET-based genetically encoded biosensors for imaging kinase activities: to image neuronal cAMP/PKA signalling [17], early signalling events in T lymphocytes [18], entry and progression through mitosis [19] and necroptosis [20]. Another review article describes lanthanide-based luminescent peptide probes for the development of non-genetic biosensors monitoring kinase and phosphatase activities [21], which is followed by a review describing different classes of genetic and non-genetic fluorescent biosensors applied to high-throughput screening of protein kinase inhibitors [22]. The next two reviews describe fluorescent biosensors for probing protease activities [23] and for detection of ROS and redox metabolites [24].…”