Search

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., (© 2020 Wiley-VCH GmbH.)

CDK4/cyclin D kinase constitutes an attractive pharmacological target for development of anticancer therapeutics, in particular in KRAS -mutant lung cancer patients, who have a poor prognosis and no targeted therapy available yet. Although several ATP-competitive inhibitors of CDK4 have been developed for anticancer therapeutics, they suffer from limited specificity and efficacy. Methods : As an alternative to ATP-competitive inhibitors we have designed a stapled peptide to target the main interface between CDK4 and cyclin D, and have characterized its physico-chemical properties and affinity to bind cyclin D1. Results : We have validated a positive correlation between CDK4/cyclin D level and KRAS mutation in lung cancer patients. The stapled peptide enters cells rapidly and efficiently, and inhibits CDK4 kinase activity and proliferation in lung cancer cells. Its intrapulmonary administration in mice enables its retention in orthotopic lung tumours and complete inhibition of their growth when co-administered with Abemaciclib. Conclusion : The stapled peptide targeting the main interface between CDK4 and cyclin D provides promising therapeutic perspectives for patients with lung cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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.

We describe a lanthanide biosensor that responds to CDK4 kinase activity in melanoma cell extracts through a significant and dose dependent increase in luminescence, thanks to sensitization of a DOTA[Tb 3+ ] complex incorporated into a CDK4 substrate peptide by a unique tryptophan residue in an adjacent phosphoaminoacid binding moiety.

Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma., (Copyright © 2016. Published by Elsevier B.V.)

Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancer cells, thereby constituting attractive pharmacological targets. However, there are no direct means of assessing their relative abundance in living cells, current approaches being limited to antigenic and proteomic analysis of fixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developed a fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognition of its targets. CDKSENS was introduced into living cells, through complexation with the cell-penetrating carrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detection of differences between different healthy and cancer cell lines, thereby enabling to distinguish cells that express high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extraction procedures. As such it provides promising perspectives for monitoring the response to therapeutics that affect CDK/Cyclin abundance, for cell-based drug discovery strategies and fluorescence-based cancer diagnostics.

The dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide-nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy based on short amphiphatic peptides (MPG8/PEP3) that have been successfully applied ex vivo and in vivo for the delivery of therapeutic siRNA and PNA molecules. PEP3 and MPG8 form stable nanoparticles with PNA analogues and siRNA, respectively, and promote their efficient cellular uptake, independently of the endosomal pathway, into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. This chapter describes easy-to-handle protocols for the use of MPG-8 or PEP-3-nanoparticle technologies for PNA and siRNA delivery into adherent and suspension cell lines as well as in vivo into cancer mouse models.

With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors, which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems, peptide and protein biosensors and describe biosensor applications in a wide variety of fields.

The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made the delivery of molecules a keystone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including CPPs (cell-penetrating peptides), which represent a new and innovative concept to bypass the problem of bioavailability of drugs. CPPs constitute very promising tools and have been successfully applied for in vivo. Two CPP strategies have been described to date; the first one requires chemical linkage between the drug and the carrier for cellular drug internalization, and the second is based on the formation of stable complexes with drugs, depending on their chemical nature. The Pep and MPG families are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG- and Pep-based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes, in a fully biologically active form, into a large variety of cell lines, as well as in animal models. This review focuses on the structure-function relationship of non-covalent MPG and Pep-1 strategies, and their requirement for cellular uptake of biomolecules and applications in cultured cells and animal models.

Cell-penetrating peptides (CPPs) constitute a family of peptides whose unique characteristic is their ability to insert into and cross biological membranes. Cell-penetrating peptide carriers of the Pep family are amphipathic peptides which have been shown to deliver peptides and proteins into a wide variety of cells through formation of non-covalent complexes with their cargo. In this study, we have investigated the morphological features of different Pep-1/cargo complexes by scanning electron microscopy and light scattering measurements. We provide first-time evidence that biologically efficient complexes of Pep-1/p27Kip tumour suppressor physically exist in the form of discrete nanoparticles. Moreover, we have characterized the relationship between the Pep-1/cargo ratio, the size and homogeneity of the nanoparticles formed, and their efficiency in delivering the cargo into cells, and report that particle size and homogeneity is both directly dependent on the ratio of Pep-1/cargo formulations, and responsible for their biological efficiency.

Control of gene expression using RNA interference (RNAi) technology constitutes a method of choice for investigating gene function in mammalian cells. However, like most oligonucleotide-based strategies, the major limitation of interfering RNA is their poor cellular uptake due to low permeability of the cell membrane to nucleic acids. Several strategies have been developed to improve delivery of oligonucleotides both in cultured cells and in vivo. So far, there is no universal method for their delivery, as they all present several limitations. Peptide-based strategies have been demonstrated to improve the cellular uptake of nucleic acids both in cultured cell and in vivo. This chapter describes a new peptide-based gene delivery system, MPG, which forms stable noncovalent complexes with oligonucleotides and promotes their delivery into a large panel of cell lines without the need for prior chemical covalent coupling. Protocols are described for both adherent and suspension cell lines.

The dramatic acceleration in identification of new nucleic-acid-based therapeutic molecules has provided new perspectives in pharmaceutical research. However, their development is limited by their poor cellular uptake and inefficient trafficking. Here we describe a short amphipathic peptide, Pep-3, that combines a tryptophan/phenylalanine domain with a lysine/arginine-rich hydrophilic motif. Pep-3 forms stable nano-size complexes with peptide-nucleic acid analogues and promotes their efficient delivery into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. We demonstrate that Pep-3-mediated delivery of antisense-cyclin B1-charged-PNA blocks tumour growth in vivo upon intratumoral and intravenous injection. Moreover, we show that PEGylation of Pep-3 significantly improves complex stability in vivo and consequently the efficiency of antisense cyclin B1 administered intravenously. Given the biological characteristics of these vectors, we believe that peptide-based delivery technologies hold a true promise for therapeutic applications of DNA mimics.

To identify rules for the design of efficient CPPs that can deliver therapeutic agents such as nucleic acids (DNAs, siRNAs) or proteins and PNAs into subcellular compartments, we compared the properties of several primary and secondary amphipathic CPPs. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration and compression experiments reveal that both peptides interact strongly with phospholipids, and observations on Langmuir-Blodgett transfers indicate that they can modify the lipid organization. Conformational investigations indicate that the lipid-peptide interactions govern the conformational state(s) of the peptides. On the basis of the ability of both peptides to promote ion permeation through both natural and artificial membranes, models illustrating the translocation processes have been proposed. One is based on the formation of a beta-barrel pore-like structure while another is based on the association of helices.

We have investigated the interactions between two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration experiments reveal that both peptides interact strongly with phospholipids. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG, it is based on the formation of a beta-barrel pore-like structure, while for Pep-1, it is based on association of helices.

The development of therapeutic peptides and proteins is limited by the poor permeability and the selectivity of the cell membrane. The discovery of protein transduction domains has given a new hope for administration of large proteins and peptides in vivo. We have developed a non-covalent strategy for protein transduction based on an amphipathic peptide, Pep-1, that consists of a hydrophobic domain and a hydrophilic lysine-rich domain. Pep-1 efficiently delivers a variety of fully biologically active peptides and proteins into cells, without the need for prior chemical cross-linking or chemical modifications. The mechanism through which Pep-1 delivers active macromolecules does not involve the endosomal pathway and the dissociation of the Pep-1/macromolecule particle occurs immediately after it crosses the cell membrane. Pep-1 has been successfully applied to the screening of therapeutic peptides in vivo and presents several advantages: stability in physiological buffer, lack of toxicity and of sensitivity to serum. In conclusion, Pep-1 technology could contribute significantly to the development of fundamental and therapeutic applications and be an alternative to covalent protein transduction domain-based technologies.

Cyclin dependent kinases (CDKs) are key regulators of the cell cycle progression and therefore constitute excellent targets for the design of anticancer agents. Most of the inhibitors identified to date inhibit kinase activity by interfering with the ATP-binding site of CDKs. We recently proposed that the protein/protein interface and conformational changes required in the molecular mechanism of CDK2-cyclin A activation were potential targets for the design of specific inhibitors of cell cycle progression. To this aim, we have designed and characterized a small peptide, termed C4, derived from amino acids 285-306 in the alpha5 helix of cyclin A. We demonstrate that this peptide does not interfere with complex formation but forms stable complexes with CDK2-cyclin A. The C4 peptide significantly inhibits kinase activity of complexes harboring CDK2 in a competitive fashion with respect to substrates but does not behave as an ATP antagonist. Moreover, when coupled with the protein transduction domain of Tat, the C4 peptide blocks the proliferation of tumor cell lines, thereby constituting a potent lead for the development of specific CDK-cyclin inhibitors.

This review focuses on two peptides, MPG and Pep-1, which facilitate the transfer of nucleic acids and proteins, respectively, into subcellular compartments. We have investigated the interactions between these two carrier peptides and model membrane systems as well as the conformational consequences of these interactions. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration and compression experiments reveal that both peptides interact strongly with phospholipids, and observations on Langmuir-Blodgett transfers indicate that they can modify the lipid organization. Conformational investigations indicate that the lipid-peptide interaction govern the conformational state of the peptides. Based on the ability of both peptides to promote ion permeabilization of both natural and artificial membranes, we propose a model illustrating the translocation process. For MPG it is based on the formation of a beta-barrel pore-like structure while for Pep-1 it is based on association of helices.

Recently, we described a new strategy for the delivery of nucleic acids into mammalian cells, based on an amphipathic peptide of 27 residues called MPG, which was designed on the basis of a hydrophobic domain derived from a fusion sequence associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of nucleic acids in cultured cells, without requiring any covalent coupling. We have examined the conformational states of MPG in its free form and complexed with a cargo, as well as its ability to interact with phospholipids, and have investigated the structural consequences of these interactions. In spite of its similarity to the similarly designed cell-penetrating peptide Pep-1, MPG behaves significantly differently from the conformational point of view. Circular dichroism (CD) analysis reveals a transition from a nonstructured to a beta-sheet conformation upon interaction with phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Partial conformational change of MPG is associated with formation of a complex with its cargo, and an increase in sheet content occurs upon association with the cell membrane.

Recently, we described a new strategy for the delivery of proteins and peptides into mammalian cells, based on an amphipathic peptide of 21 residues, Pep-1, which was designed on the basis of a protein-interacting domain associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of active proteins or peptides both in cultured cells and in vivo, without requiring any covalent coupling. We have examined the conformational states of Pep-1 in its free form and complexed with a cargo peptide and have investigated their ability to interact with phospholipids and the structural consequences of these interactions. From the conformational point of view, Pep-1 behaves significantly differently from other similarly designed cell-penetrating peptides. CD analysis revealed a transition from a nonstructured to a helical conformation upon increase of the concentration. Determination of the structure by NMR showed that in water, its alpha-helical domain extends from residues 4-13. CD and FTIR indicate that Pep-1 adopts a helical conformation in the presence of phospholipids. Adsorption measurements performed at the air-water interface are consistent with the helical form. Pep-1 does not undergo conformational changes upon formation of a particle with a cargo peptide. In contrast, we observe a partial conformational transition when the complex encounters phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Conformational change of Pep-1 is not associated with complexation with its cargo but is induced upon association with the cell membrane.

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. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases (CDK/Cyclins) form a family of heterodimeric kinases that play central roles in regulation of cell cycle progression, transcription and other major biological processes including neuronal differentiation and metabolism. Constitutive or deregulated hyperactivity of these kinases due to amplification, overexpression or mutation of cyclins or CDK, contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. The structural features of several of these kinases have been elucidated and their molecular mechanisms of regulation characterized in depth, providing clues for development of drugs and inhibitors to disrupt their function. However, like most other kinases, they constitute a challenging class of therapeutic targets due to their highly conserved structural features and ATP-binding pocket. Notwithstanding, several classes of inhibitors have been discovered from natural sources, and small molecule derivatives have been synthesized through rational, structure-guided approaches or identified in high throughput screens. The larger part of these inhibitors target ATP pockets, but a growing number of peptides targeting protein/protein interfaces are being proposed, and a small number of compounds targeting allosteric sites have been reported. [ABSTRACT FROM AUTHOR]

Examines the role of the thumb domain of p51 in HIV reverse transcriptase (RT) activation for the design of antiviral compounds. Effect of peptides on stability and dimerization; Mechanism of inhibition; Effect of peptide A on RT affinity for primer/template and RT transfer RNA and DNA binding; Conformational changes involved in maturation.

[Display omitted] • 9-Amino-quinolimide based fluorescent labelling reagents have been prepared. • 9-Amino-quinolimide-labelled peptides derived from CDK5 have been obtained. • CDK5 labelled peptides behave as p25 fluorescence sensors in vitro and in cellulo. • In cellulo imaging FRET response to a CDK5 labelled peptide correlates with p25 level. CDK5 kinase is activated through interactions with different partners including the p35/p25 regulators. Active CDK5 plays a key role in several neuronal functions and its hyperactivity contributes to a variety of neurodegenerative processes and several human cancers, in particular glioblastomas and neuroblastomas. In order to probe partners that interact with CDK5, we designed and synthesized fluorescent quinolimide-labelled peptides derived from the C-helix of CDK5, which were implemented to probe CDK5 partners in vitro and by in cellulo imaging in U87 glioblastoma and N2a neuroblastoma cells. Ectopic expression of a NIR fluorescent protein miRFP670 fusion with p25 (p25-miRFP670) in N2a cells induced FRET between a quinolimide-labelled peptide biosensor and p25-miRFP670, and fluorescence intensity was proportional to the expression level of p25, thereby demonstrating the potential of the quinolimide-labelled peptide biosensor to report on p25 relative abundance. [ABSTRACT FROM AUTHOR]