Nanobiosensor Reports on CDK1 Kinase Activity in Tumor Xenografts in Mice.

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.

Fluorescent Peptide Biosensor for Probing CDK6 Kinase Activity in Lung Cancer Cell Extracts.

CDK6 kinase regulates cell-cycle progression in G1, together with CDK4, but has cell-, tissue- and developmentally distinct functions associated with transcription, angiogenesis and metabolism. Although CDK6 makes an attractive cancer biomarker and target, there are no means of assessing its activity in a complex environment. In this study, we describe the design, engineering and characterisation of a fluorescent peptide biosensor derived from 6-phosphofructokinase that reports on CDK6 kinase activity through sensitive changes in fluorescence intensity. This biosensor can report on CDK6 activity in a dose-dependent fashion, thereby enabling quantification of differences in kinase activity in complex and physiologically relevant environments. Further implementation of this biosensor in different lung and melanoma cell lines, as well as in mesothelioma cell lines derived from patients together with a CDK4 biosensor highlighted differences in kinase activity between CDK6 and CDK4 kinase. This work demonstrates the utility of these selective tools for monitoring two closely related kinases comparatively and simultaneously in the same samples, thereby offering attractive perspectives for diagnostic and therapeutic purposes.

Fluorescent Biosensor for Detection of the R248Q Aggregation-Prone Mutant of p53.

The p53 tumour suppressor and guardian of the genome undergoes missense mutations that lead to functional inactivation in 50 % of human cancers. These mutations occur mostly in the DNA-binding domain of the protein, and several of these result in conformational changes that lead to amyloid-like protein aggregation. Herein, we describe a fluorescent biosensor that reports on the R248Q mutant of p53 in vitro and in living cells, engineered through conjugation of an environmentally sensitive probe onto a peptide derived from the primary aggregation segment of p53. This biosensor was characterised both in vitro and by means of fluorescence microscopy following facilitated delivery into cultured cells. It is shown that this biosensor preferentially reports on the p53 R248Q mutant in the PC9 lung cancer cell line compared with other lung cancer cell lines harbouring either wild-type or no p53.

Microneedle Array-Based Platforms for Future Theranostic Applications.

Theranostics involves finding the biomarkers of a disease, fighting them through site specific drug delivery and following them for prognosis of the disease. Microneedle array technology has been used for drug delivery and extended for continuous monitoring of analytes present in the skin compartment. We envisage the use of microneedle arrays for future theranostic applications. The potential of combining microneedle array-based drug delivery and diagnostics as part of closed-loop control system for the management of diseases and delivery of precision drugs in individual patients is reported in this paper.

Rationally Designed Peptides as Efficient Inhibitors of Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein.

Due to its essential roles in the viral replication cycle and to its highly conserved sequence, the nucleocapsid protein (NCp7) of the human immunodeficiency virus type 1 is a target of choice for inhibiting replication of the virus. Most NCp7 inhibitors identified so far are small molecules. A small number of short peptides also act as NCp7 inhibitors by competing with its nucleic acid (NA) binding and chaperone activities but exhibit antiviral activity only at relatively high concentrations. In this work, in order to obtain more potent NCp7 competitors, we designed a library of longer peptides (10-17 amino acids) whose sequences include most of the NCp7 structural determinants responsible for its specific NA binding and destabilizing activities. Using an in vitro assay, the most active peptide (pE) was found to inhibit the NCp7 destabilizing activity, with a 50% inhibitory concentration in the nanomolar range, by competing with NCp7 for binding to its NA substrates. Formulated with a cell-penetrating peptide (CPP), pE was found to accumulate into HeLa cells, with low cytotoxicity. However, either formulated with a CPP or overexpressed in cells, pE did not show any antiviral activity. In vitro competition experiments revealed that its poor antiviral activity may be partly due to its sequestration by cellular RNAs. The selected peptide pE therefore appears to be a useful tool for investigating NCp7 properties and functions in vitro, but further work will be needed to design pE-derived peptides with antiviral activity.

Rational Design of Nanobody80 Loop Peptidomimetics: Towards Biased ß2 Adrenergic Receptor Ligands.

G protein-coupled receptors (GPCRs) play an important role in many cellular responses; as such, their mechanism of action is of utmost interest. To gain insight into the active conformation of GPCRs, the X-ray crystal structures of nanobody (Nb)-stabilized ß2 -adrenergic receptor (ß2 AR) have been reported. Nb80, in particular, is able to bind the intracellular G protein binding site of ß2 AR and stabilize the receptor in an active conformation. Within Nb80, the complementarity-determining region 3 (CDR3) is responsible for most of the binding interactions. Hence, we hypothesized that peptidomimetics of the CDR3 loop might be sufficient for binding to the receptor, inhibiting the interaction of ß2 AR with intracellular GPCR interacting proteins (e.g., G proteins). Based on previous crystallographic data, a set of peptidomimetics were synthesized that, similar to the Nb80 CDR3 loop, adopt a ß-hairpin conformation. Syntheses, conformational analysis, binding and functional in vitro assays, as well as internalization experiments, were performed. We demonstrate that peptidomimetics can structurally mimic the CDR3 loop of a nanobody and its function by inhibiting G protein coupling as measured by partial inhibition of cAMP production.

Conformational Equilibrium of CDK/Cyclin Complexes by Molecular Dynamics with Excited Normal Modes.

Cyclin-dependent kinases (CDKs) and their associated regulatory cyclins are central for timely regulation of cell-cycle progression. They constitute attractive pharmacological targets for development of anticancer therapeutics, since they are frequently deregulated in human cancers and contribute to sustained, uncontrolled tumor proliferation. Characterization of their structural/dynamic features is essential to gain in-depth insight into structure-activity relationships. In addition, the identification of druggable pockets or key intermediate conformations yields potential targets for the development of novel classes of inhibitors. Structural studies of CDK2/cyclin A have provided a wealth of information concerning monomeric/heterodimeric forms of this kinase. There is, however, much less structural information for other CDK/cyclin complexes, including CDK4/cyclin D1, which displays an alternative (open) position of the cyclin partner relative to CDK, contrasting with the closed CDK2/cyclin A conformation. In this study, we carried out normal-mode analysis and enhanced sampling simulations with our recently developed method, molecular dynamics with excited normal modes, to understand the conformational equilibrium on these complexes. Interestingly, the lowest-frequency normal mode computed for each complex described the transition between the open and closed conformations. Exploration of these motions with an explicit-solvent representation using molecular dynamics with excited normal modes confirmed that the closed conformation is the most stable for the CDK2/cyclin A complex, in agreement with their experimentally available structures. On the other hand, we clearly show that an open↔closed equilibrium may exist in CDK4/cyclin D1, with closed conformations resembling that captured for CDK2/cyclin A. Such conformational preferences may result from the distinct distributions of frustrated contacts in each complex. Using the same approach, the putative roles of the Thr(160) phosphoryl group and the T-loop conformation were investigated. These results provide a dynamic view of CDKs revealing intermediate conformations not yet characterized for CDK members other than CDK2, which will be useful for the design of inhibitors targeting critical conformational transitions.

Tampering with cell division by using small-molecule inhibitors of CDK-CKS protein interactions.

Cyclin-dependent kinases (CDKs) control many cellular processes and are considered important therapeutic targets. Large collections of inhibitors targeting CDK active sites have been discovered, but their use in chemical biology or drug development has been often hampered by their general lack of specificity. An alternative approach to develop more specific inhibitors is targeting protein interactions involving CDKs. CKS proteins interact with some CDKs and play important roles in cell division. We discovered two small-molecule inhibitors of CDK-CKS interactions. They bind to CDK2, do not inhibit its enzymatic activity, inhibit the proliferation of tumor cell lines, induce an increase in G1 and/or S-phase cell populations, and cause a decrease in CDK2, cyclin A, and p27(Kip1) levels. These molecules should help decipher the complex contributions of CDK-CKS complexes in the regulation of cell division, and they might present an interesting therapeutic potential.

Fluorescent biosensors - probing protein kinase function in cancer and drug discovery.

One of the challenges of modern biology and medicine is to visualize biomolecules in their natural environment, in real-time and in a non-invasive fashion, so as to gain insight into their physiological behavior and highlight alterations in pathological settings, which will enable to devise appropriate therapeutic strategies. Fluorescent biosensors constitute a class of imaging agents which have provided major insights into the function and regulation of enzymes in their cellular context. GFP-based reporters and genetically-encoded FRET biosensors, have been successfully applied to study protein kinases in living cells with high spatial and temporal resolution. In parallel, combined efforts in fluorescence chemistry and in chemical biology have enabled the design of non-genetic, polypeptide biosensors coupled to small synthetic fluorescent probes, which have been applied to monitor protein kinase activities in vitro and in more complex biological samples, with an equally successful outcome. From a biomedical perspective, fluorescent biosensor technology is well suited to development of diagnostic approaches, for monitoring disease progression and for evaluating response to therapeutics. Moreover it constitutes an attractive technology for drug discovery programs, for high content, high throughput screening assays, to assess the potency of new hits and optimize lead compounds, whilst also serving to characterize drugs developed through rational design. This review describes the utility and versatility of fluorescence biosensor technology to probe protein kinases with a specific focus on CDK/cyclin biosensors we have developed to probe abundance, activity and conformation. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Fluorescent protein biosensor for probing CDK/cyclin activity in vitro and in living cells.

Cyclin-dependent kinases (CDKs) play an essential role in the coordination of cell cycle progression and transcriptional regulation; hyperactivation is associated with cancer. However there are few means of measuring their activity in a physiological context or their inhibition in response to therapeutics. To this aim we engineered a modular fluorescent protein biosensor that reports on phosphorylation by CDK/cyclins through real-time changes in fluorescence intensity. This allowed a comparison of enzymatic activity of recombinant kinases, monitoring inhibition by small molecules, and probing endogenous activities in lysates from healthy and cancer cell lines in a sensitive and quantitative fashion. This versatile tool was further implemented to probe the oscillatory activity of these kinases throughout the cell cycle by time-lapse imaging and ratiometric fluorescence quantification, following delivery of a red fluorescent protein fusion mediated by cell-penetrating peptides.

Ethaverine and Papaverine Target Cyclin-Dependent Kinase 5 and Inhibit Lung Cancer Cell Proliferation and Migration.

CDK5 kinase plays a central role in the regulation of neuronal functions, and its hyperactivation has been associated with neurodegenerative pathologies and more recently with several human cancers, in particular lung cancer. However, ATP-competitive inhibitors targeting CDK5 are poorly selective and suffer limitations, calling for new classes of inhibitors. In a screen for allosteric modulators of CDK5, we identified ethaverine and closely related derivative papaverine and showed that they inhibit cell proliferation and migration of non small cell lung cancer cell lines. Moreover the efficacy of these compounds is significantly enhanced when combined with the ATP-competitive inhibitor roscovitine, suggesting an additive dual mechanism of inhibition targeting CDK5. These compounds do not affect CDK5 stability, but thermodenaturation studies performed with A549 cell extracts infer that they interact with CDK5 in cellulo. Furthermore, the inhibitory potentials of ethaverine and papaverine are reduced in A549 cells treated with siRNA directed against CDK5. Taken together, our results provide unexpected and novel evidence that ethaverine and papaverine constitute promising leads that can be repurposed for targeting CDK5 in lung cancer.

Multiplexed Profiling of CDK Kinase Activities in Tumor Biopsies with Fluorescent Peptide Biosensors.

Detection of disease biomarkers constitutes a major challenge for the development of personalized and predictive diagnostics as well as companion assays. Protein kinases (PKs) involved in the coordination of cell cycle progression and proliferation that are hyperactivated in human cancers constitute attractive pharmacological targets and relevant biomarkers. Although it is relatively straightforward to assess the relative abundance of PKs in a biological sample, there is not always a direct correlation with enzymatic activity, which is regulated by several posttranslational mechanisms. Studies of relative abundance therefore convey limited information, and the lack of selective, sensitive, and standardized tools together with the inherent complexity of biological samples makes it difficult to quantify PK activities in physio-pathological tissues. To address this challenge, we have developed a toolbox of fluorescent biosensors that report on CDK activities in a sensitive, selective, dose-dependent, and quantitative fashion, which we have implemented to profile CDK activity signatures in cancer cell lines and biopsies from human tumors. In this study, we report on a standardized and calibrated biosensing approach to quantify CDK1,2,4, and 6 activities simultaneously through a combination of four different biosensors in a panel of 40 lung adenocarcinoma and 40 follicular lymphoma samples. CDK activity profiling highlighted two major patterns which were further correlated with age, sex of patients, tumor size, grade, and genetic and immunohistochemical features of the biopsies. Multiplex CDKACT biosensing technology provides new and complementary information relative to current genetic and immunohistochemical characterization of tumor biopsies, which will be useful for diagnostic purposes, potentially guiding therapeutic decision. These fluorescent peptide biosensors offer promise for personalized diagnostics based on kinase activity profiling.

CDK6 is activated by the atypical cyclin I to promote E2F-mediated gene expression and cancer cell proliferation.

Cyclin-dependent kinases (CDKs), together with their cyclin partners, are the master cell cycle regulators. Remarkably, the cyclin family was extended to include atypical cyclins, characterized by distinctive structural features, but their partner CDKs remain elusive. Here, we conducted a yeast two-hybrid screen to identify new atypical cyclin-CDK complexes. We identified 10 new complexes, including a complex between CDK6 and cyclin I (CCNI), which was found to be active against retinoblastoma protein. CCNI upregulation increased the proliferation of breast cancer cells in vitro and in vivo, with a magnitude similar to that seen upon cyclin D upregulation, an effect that was abrogated by CDK6 silencing or palbociclib treatment. In line with these findings, CCNI downregulation led to a decrease in cell number and a reduction in the percentage of cells reaching S phase. Finally, CCNI upregulation correlated with the high expression of E2F target genes in large panels of cancer cell lines and tissue samples from breast cancer patients. In conclusion, we unveil CCNI as a new player in the pathways that activate CDK6, enriching the wiring of cell cycle control.

Shining Light on Protein Kinase Biomarkers with Fluorescent Peptide Biosensors.

Protein kinases (PKs) are established gameplayers in biological signalling pathways, and a large body of evidence points to their dysregulation in diseases, in particular cancer, where rewiring of PK networks occurs frequently. Fluorescent biosensors constitute attractive tools for probing biomolecules and monitoring dynamic processes in complex samples. A wide variety of genetically encoded and synthetic biosensors have been tailored to report on PK activities over the last decade, enabling interrogation of their function and insight into their behaviour in physiopathological settings. These optical tools can further be used to highlight enzymatic alterations associated with the disease, thereby providing precious functional information which cannot be obtained through conventional genetic, transcriptomic or proteomic approaches. This review focuses on fluorescent peptide biosensors, recent developments and strategies that make them attractive tools to profile PK activities for biomedical and diagnostic purposes, as well as insights into the challenges and opportunities brought by this unique toolbox of chemical probes.

PEP and CADY-mediated delivery of fluorescent peptides and proteins into living cells.

Cell-penetrating peptides (CPPs) constitute a family of peptides with the characteristic ability to cross biological membranes and deliver cargo into the intracellular milieu. Several CPPs have been proposed for delivery of polypeptides and proteins into cells through either of two strategies: covalent or complexed in a non-covalent fashion. Members of the PEP family are primary amphipathic peptides which have been shown to deliver peptides and proteins into a wide variety of cells through formation of non-covalent complexes. CADY is a secondary amphipathic peptide which has been demonstrated to deliver short nucleic acids, in particular siRNA with high efficiency. Here we review the characteristics of the PEP and CADY carriers and describe a novel derivative of CADY termed CADY2, which also presents sequence similarities to Pep1. We have compared Pep1, CADY and CADY2 in their efficiency to interact with and internalize short fluorogenic peptides and proteins into cultured cells, and provide evidence that CADY2 can interact with proteins and peptides and deliver them efficiently into living cells, similar to Pep1, but in contrast to CADY which is unable to deliver any peptide, even short negatively charged peptides. This is the first study to investigate the influence of the cargo on the interactions between PEP and CADY carriers, thereby providing novel insights into the physicochemical parameters underlying interactions and cellular uptake of peptides and proteins by these non-covalent CPPs.

Fluorescent Peptide Biosensors for Probing CDK Kinase Activity in Cell Extracts.

Fluorescent biosensors can report on the relative abundance, activity, or conformation of biomolecules and analytes through changes in fluorescence emission. A wide variety of genetically-encoded and synthetic biosensors have been developed to monitor protein kinase activity. We have focused on the design, engineering and characterization of fluorescent peptide biosensors of cyclin-dependent kinases (CDKs) that constitute attractive cancer biomarkers and pharmacological targets. In this chapter, we describe the CDKACT fluorescent peptide biosensor technology and its application to assess the relative kinase activity of CDKs in vitro, either using recombinant proteins or cell extracts as a more complex source of kinase. This technology offers a straightforward means of comparing CDK activity in different cell lines and evaluating the specific impact of treatments intended to target kinase activity in a physiologically relevant environment.

Cell-cycle markers and biosensors.

Since the first schematic illustrations of dividing cells, we have come a long way in characterising eukaryotic cells and defining their cell-cycle status thanks to a number of complementary approaches. Although most of these approaches rely on cell-fixation procedures to identify molecular components in cell lysates, cultured cells or tissues, the development of GFP technology has enabled visualisation of virtually any fusion protein in cellulo and in vivo, and the exploitation of functional elements with well-defined spatiotemporal characteristics has enabled the development of genetically encoded fluorescent markers of cell-cycle phases, thus providing novel means of characterising the status of living cells in real time with high resolution. Together with technological advances in fluorescence chemistry and imaging approaches, the more recent development of fluorescent biosensors has provided direct means of probing cell-cycle regulators and of studying their dynamics with high spatial and temporal resolution. Here we review classical approaches that rely on cell fixation to characterise the cell-cycle status and its regulatory enzymes, and we describe the more recent development of cell-cycle markers based on genetically encoded fusions of fluorescent proteins with characteristic cell-cycle features, and of fluorescent biosensor technology to probe cell-cycle regulators in living cells. Biosensors not only provide a means of characterising the behaviour of cell-cycle regulators in their natural environment, they are also very useful for comparative studies of biological processes in healthy and pathological conditions, and can be further applied to diagnostic approaches to assess the status of a specific target, and to monitor response to therapeutic intervention.

Cks1-dependent proteasome recruitment and activation of CDC20 transcription in budding yeast.

Cks proteins are small evolutionarily conserved proteins that interact genetically and physically with cyclin-dependent kinases. However, in spite of a large body of genetic, biochemical and structural research, no compelling unifying model of their functions has emerged. Here we show, by investigating the essential role of Cks1 in Saccharomyces cerevisiae, that the protein is primarily involved in promoting mitosis by modulating the transcriptional activation of the APC/C protein-ubiquitin ligase activator Cdc20. Cks1 is required for both the periodic dissociation of Cdc28 kinase from the CDC20 promoter and the periodic association of the proteasome with the promoter. We propose that the essential role of Cks1 is to recruit the proteasome to, and/or dissociate the Cdc28 kinase from, the CDC20 promoter, thus facilitating transcription by remodelling transcriptional complexes or chromatin associated with the CDC20 gene.

Fluorescent Biosensor of CDK5 Kinase Activity in Glioblastoma Cell Extracts and Living Cells.

CDK5 plays a major role in neuronal functions, and is hyperactivated in neurodegenerative pathologies as well as in glioblastoma and neuroblastoma. Although this kinase constitutes an established biomarker and pharmacological target, there are few means of probing its activity in cell extracts or in living cells. To this aim a fluorescent peptide reporter of CDK5 kinase activity, derived from a library of CDK5-specific substrates, is engineered and its ability to respond to recombinant CDK5/p25 is established and CDK5 activity in glioblastoma cell extracts is reported on through sensitive changes in fluorescence intensity. A cell-penetrating variant of this biosensor which can be implemented to image CDK5 activation dynamics in space and in time is further implemented. This original biosensor constitutes a potent tool for quantifying differences in CDK5 activity following treatment with selective inhibitors and for monitoring CDK5 activation, following inhibition or stimulation, in a physiologically relevant environment. As such it offers attractive opportunities to develop a diagnostic assay for neuronal pathologies associated with hyperactivated CDK5, as well as a companion assay to evaluate response to new therapies targeting this kinase.