Nanobiosensor Reports on CDK1 Kinase Activity in Tumor Xenografts in Mice

Abstract

Probing the dynamics and quantifying the activities of intracellular protein kinases that coordinate cell growth and division and constitute biomarkers and pharmacological targets in hyperproliferative and pathological disorders remain a challenging task. Here engineering and characterization of a nanobiosensor of the mitotic kinase CDK1, through multifunctionalization of carbon nanotubes with a CDK1‐specific fluorescent peptide reporter, are described. This original reporter of CDK1 activity combines the sensitivity of a fluorescent biosensor with the unique physico‐chemical and biological properties of nanotubes for multifunctionalization and efficient intracellular penetration. The functional versatility of this nanobiosensor enables implementation to quantify CDK1 activity in a sensitive and dose‐dependent fashion in complex biological environments in vitro, to monitor endogenous kinase in living cells and directly within tumor xenografts in mice by fluorescence imaging, thanks to a ratiometric quantification strategy accounting for response relative to concentration in space and in time.

A Toolbox of Fluorescent Peptide Biosensors to Highlight Protein Kinases in Complex Samples: Focus on Cyclin‐Dependent Kinases

“…More recently we engineered a hybrid CDKACT1 nanobiosensor by immobilizing CDK1-specific peptide biosensor onto the surface of multiwall carbon nanotubes (MWCNTs). [79] This hybrid biosensor responds to recombinant CDK1 but not to CDK2 and displays much greater sensitivity to kinase activity in vitro than CDKACT1 peptide alone. Moreover MWCNT-CDKACT1 penetrates readily into living cells and was successfully implemented to quantify differences in cultured cells treated with CDK1 inhibitor, as well as to in vivo imaging in mouse models.…”

“…[63][64][65][66][67][68][69][70][71][72] In order to monitor CDK kinase activities in complex biological samples, such as cell extracts, tissue or tumor biopsies, we have developed a toolbox of fluorescent biosensors through conjugation of environmentallysensitive probes to synthetic modular peptide scaffolds/bioreceptors (Figure 6). [73][74][75][76][77][78][79] CDKACT biosensors are modular bipartite peptides comprising a CDK-specific substrate moiety onto which an environmentally-sensitive dye is conjugated, and a PAABD that folds onto the substrate sequence phosphorylated by an active CDK. This intramolecular conformational change alters the local environment of the fluorescent probe, consequently promoting fluorescence enhancement.…”

“…More recently we engineered a hybrid CDKACT1 nanobiosensor by immobilizing CDK1‐specific peptide biosensor onto the surface of multiwall carbon nanotubes (MWCNTs) [79] . This hybrid biosensor responds to recombinant CDK1 but not to CDK2 and displays much greater sensitivity to kinase activity in vitro than CDKACT1 peptide alone.…”

Recent Advances in Fluorescent Chemosensors for Protein Kinases

“…In addition, the authors used carbon nanotubes (CNTs) to deliver a protein-based biosensor for detecting CDK1 kinase in living cells and mice. [122] Irudayaraj and coworkers added a cell-penetrating peptide (TAT) to a FAK peptide biosensor, enabling cellular internalization of biosensors. [123] Through time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM), the authors monitored the activity of FAK.…”

Insights on functionalized carbon nanotubes for cancer theranostics

“…CDK1 is responsible for the progression through mitosis and the coordination of G2/M transition, however, diagnostic methods for the detection and measurement of relative activities of CDK1 possess great difficulties due to the lack of sensitivity and specificity [ 179 ]. Tilmaciu et al designed functionalized MWNTs that were modified with a kind of fluorescent peptide reporter which was specific to CDK1 for the sensitive and fluorescence-based quantification of CDK1’s activity through fluorescence imaging [ 155 ]. This nanobiosensor could not only achieve the simple detection of CDK1, but report the enzymatic activity of CDK1 as well.…”