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Fluorescent peptide biosensor for probing CDK5 kinase activity in glioblastoma and its applications for diagnostics and drug discovery in vitro and by fluorescence Imaging
Author(s) -
MORRIS May C.,
Peyressatre Marion,
Soussi Ines,
Boukhaddaoui Hassan
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.657.1
Subject(s) - cyclin dependent kinase 5 , biosensor , fluorescence , kinase , biophysics , chemistry , in vitro , cancer research , microbiology and biotechnology , biology , biochemistry , computational biology , protein kinase a , cyclin dependent kinase 2 , physics , quantum mechanics
Glioblastoma is an aggressive malignant primary brain tumour, for which chemo‐ and radiotherapy currently only offer palliation. Identification of relevant biomarkers and tools for their detection would greatly enable implementation of early‐stage diagnostics, patient stratification and development of targeted therapies to reduce mortality. The neurospecific kinase CDK5 constitutes a renowned biomarker in several neurodegenerative pathologies and has more recently been identified as a valuable predictive marker of tumorigenesis and progression in glioma ( Catania A. et al. 2001; Liu et al. 2008; Xie et al. 2015; Yushan et al. 2015) . In order to probe and quantify the relative activity of protein kinases in complex biological samples, we have developed a fluorescent biosensor technology through conjugation of environmentally‐sensitive probes to synthetic modular polypeptides ( Van et al. 2014; Prével et al. 2016 ). In order to develop a CDK5‐specific biosensor, we engineered a peptide library derived from CDK5 substrates, and screened for unbiased and selective response to CDK5 activity in vitro. This fluorescent biosensor responds to CDK5 activation and to its inhibition in a dose‐dependent fashion through sensitive changes in fluorescence intensity. It further enables comparative quantification of CDK5 activity between different neuronal and glioblastoma cell lines and can be implemented to probe this biomarker in tumour biopsies. Moreover, we designed a self‐cell‐penetrating variant which readily penetrates into living cells, and enables imaging of CDK5 activation dynamics in space and in time, revealing the kinetics of CDK5 activation initiated the cell membrane, then progressively diffusing into the cytoplasm. This original fluorescent biosensor constitutes a potent tool for studying the dynamic behaviour of CDK5 in neuronal cells and quantifying the extent of its activation in physiological and in pathological conditions, thereby offering attractive opportunities to develop a diagnostic assay for glioblastoma. This chemical probe has also proven useful to monitor the efficacy and and mechanism of action of small molecule inhibitors identified by high throughput screening. We therefore anticipate it holds promises for development of diagnostic and companion assays to evaluate the response to therapies targeting CDK5 kinase in neuronal pathologies. Support or Funding Information Grant to MCM from the Canceropole GSO (France) for Innovative Project for Health TechnologiesCDK5 kinase‐specific fluorescent peptide biosensor reporterThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .