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Molecular Mechanism of Regulation of CDK/Cyclins Schematic representation of the general mechanism of assembly and regulation of Cyclin-dependent kinases. Following interaction of a CDK with a cyclin, heterodimeric CDK/Cyclin complexes, are subject to inhibitory phosphorylation on threonine 14 (Thr14) and tyrosine 15 (Tyr15) residues, for CDK1 and CDK2, by Wee1 and Myt1 kinases, then activated through dephosphorylation of these residues by members of the Cdc25 phosphatase family, together with activating phosphorylation of threonine 161 (Thr161 in CDK1) by CDK-activating Kinase CAK.
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Cyclin-dependent kinases play central roles in regulation of cell cycle progression, transcriptional regulation and other major biological processes such as neuronal differentiation and metabolism. These kinases are hyperactivated in most human cancers and constitute attractive pharmacological targets. A large number of ATP-competitive inhibitors o...
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... inactive and acquire basal kinase activity through binding of a cyclin partner. Although there are variations on the theme, following their assembly into hetero- dimeric complexes, cyclin-dependent kinases are generally subject to reversible activating and inhibitory phosphorylations which coordinate the yield of a fully active complex [1,16] (Fig. 2). CDK1/ cyclin B for instance is subject to inhibitory phosphorylation of threonine 14 (Thr14) and tyrosine 15 (Tyr15) residues are phos- phorylated by Wee1 and Myt1 kinases, which prevent ATP binding and thereby hold the complex in check [17]. These residues are subsequently dephosphorylated by members of the Cdc25 phos- phatase family ...
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... [1][2][3][4][5][6][7] A somewhat less popular yet growing class of synthetic biosensors, engineered through combination of peptides and synthetic fluorophores provides further means of detecting biomarkers ex vivo in complex samples with exquisite sensitivity and strong potential for diagnostic and biomedical applications. [8][9][10][11][12][13][14][15][16][17][18] Indeed, plasmids encoding genetically-encoded biosensors are routinely transfected into cultured cells through lipid-based or viral strategies, thereby enabling imaging studies following ectopic expression of the fluorescent protein biosensor. Synthetic biosensors require facilitated intracellular delivery, which can be mediated by a wide panel of available cell-penetrating peptides, or following conjugation to nanomaterial-based vectors, such as carbon nanotubes that readily penetrate mammalian cells. ...
... Synthetic biosensors require facilitated intracellular delivery, which can be mediated by a wide panel of available cell-penetrating peptides, or following conjugation to nanomaterial-based vectors, such as carbon nanotubes that readily penetrate mammalian cells. [19][20][21][22] Biosensor expression following transfection of plasmids may require some time (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) h or more), unless stable transfectants are generated, and the concentration of biosensor expressed from plasmid constructs is not always well determined. In contrast, the concentration of synthetic peptides can be predetermined and quantified following cellular internalization, offering a better control of biosensor in a given experiment. ...
... tides and biomaterials, and further constitute attractive scaffolds for biosensing applications. [8][9][10][11][12][13][14][15][16][17][18] Design of peptide biosensors should account for the biological context and the biomarker of interest, to define the specific needs and requirements, for instance whether they are meant for in vitro applications or for imaging purposes (Figure 1). [23,24] As for any biosensor, the first issue to address concerns the bioreceptor, i. e. the moiety that will enable recognition of the biomarker of interest and confer selectivity/ specificity. ...
Development of sensitive and selective strategies for detection of disease biomarkers constitutes a major challenge for diagnostics and companion assays. New strategies are required to advance personalized and point‐of‐care medicine. Protein kinases (PK) are hyperactivated in many human cancers, therefore constituting relevant biomarkers and attractive pharmacological targets.Yet quantification of PK expression levels alone does not convey an accurate readout of their activity, since PKs are subject to numerous interactions and post‐translational regulations. Hence technologies that report on PK function are essential for complete appreciation of their behaviour and dysregulation in physio‐pathological settings. This Review focuses on fluorescent peptide biosensors designed to report on PK activities, and in particular on a toolbox of biosensors tailored to monitor cyclin‐dependent kinases. These selective optical probes offer a straightforward means of quantifying differences in kinase activities between healthy and cancer cells, and can report on alterations in response to therapeutics in vitro, in living cells and in vivo.
... Over the past decade, a wide variety of fluorescent biosensors have been developed to monitor and image protein kinase activities, providing means of gaining insight into kinase behaviour, and in some cases offering new technologies for high-throughput screening or diagnostic approaches, as described in several comprehensive reviews [38][39][40][41][42][43][44]. The Fluorescent Biosensor Toolbox comprises a highly diverse panel of fluorescent and luminescent biosensors which differ in their nature, including genetically encoded, synthetic, peptide-based and nanomaterial-based biosensors ( Figure 3) [39][40][41][45][46][47][48][49][50][51][52][53][54][55]. ...
... In contrast, nongenetic synthetic fluorescent reporters based on polymeric or polypeptide scaffolds conjugated to small organic fluorophores are better suited for ex vivo diagnostics as they provide controlled platforms that can be readily implemented to probe targets of interest within complex samples. A vast array of synthetic biosensors have been engineered to report on PK activities, including peptides derived from PK substrates, conjugated to solvatochromic probes, which respond either directly to proximal phosphorylation or indirectly following interactions with phosphorecognition domains that alter biosensor fluorescence or through quenching/unquenching strategies, as described below (reviewed in [39][40][41][42][43][44][45][46][47][48][49][50][51]). Synthetic biosensors offer attractive opportunities for molecular diagnostics, monitoring of disease progression and response to therapeutics, and several synthetic fluorescent biosensors have been developed for predictive purposes, for instance, to detect dysregulated PK biomarkers in leukaemia [65] or for point-of-care monitoring of therapeutic drugs in plasma and urine [66]. ...
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.
... Indeed, high-throughput screening has been used successfully in a wide range of applications such as drug discovery (Prevel, Kurzawa et al. 2014), evolution of proteins (Packer and Liu 2015), enzyme engineering (Longwell, Labanieh et al. 2017), as well as screening and discovery of chemical probes (Inglese, Johnson et al. 2007), small molecules (Janzen 2014, Doyle, Pop et al. 2016, and lipopeptides (Biniarz, Lukaszewicz et al. 2017). ...
The incorporation of cell-free transcription and translation systems into high-throughput screening applications enables the in situ and on-demand expression of peptides and proteins. Coupled with modern microfluidic technology, the cell-free methods allow the screening, directed evolution, and selection of desired biomolecules in minimal volumes within a short timescale. Cell-free high-throughput screening applications are classified broadly into in vitro display and on-chip technologies. In this review, we outline the development of cell-free high-throughput screening methods. We further discuss operating principles and representative applications of each screening method. The cell-free high-throughput screening methods may be advanced by the future development of new cell-free systems, miniaturization approaches, and automation technologies.
... After the discovery that the green fluorescent protein was susceptible to mutations, the expansion of the fluorescent protein colour palette and enhancement of their optical properties provided a toolbox of great versatility (Shaner et al., 2005;Chudakov et al., 2010;Shcherbakova et al., 2014;Hochreiter et al., 2015). Fluorescent biosensor proteins constitute sensitive and selective tools which can readily be implemented to high throughput and high content screens in drug discovery programmes (Prevel et al., 2014). The value of such approaches is evident, as they allow a time-efficient high throughput screening of multitudinous chemicals for their biological properties. ...
Pharmaceuticals, such as beta-blockers, nonsteroidal anti-inflammatory drugs (NSAIDs) as well as their metabolites are introduced into the water cycle via municipal wastewater treatment plant (WWTP) effluents in all industrialized countries. As the amino acid sequences of the biological target molecules of these pharmaceuticals - the beta-1 adrenergic receptor for beta-blockers and the cyclooxygenase for NSAIDs - are phylogenetically conserved among vertebrates it is reasonable that wildlife vertebrates including fish physiologically respond in a similar way to them as documented in humans. Consequently, beta-blockers and NSAIDs both exhibit their effects according to their mode of action on one hand, but on the other hand that may lead to unwanted side effects in non-target species. To determine whether residuals of beta-1 adrenergic receptor antagonists and cyclooxygenase inhibitors may pose a risk to aquatic organisms, one has to know the extent to which such organisms respond to the total of active compounds, their metabolites and transformation products with the same modes of action. To cope with this demand, two cell-based assays were developed, by which the total beta-blocker and cyclooxygenase inhibitory activity can be assessed in a given wastewater or surface water extract in real time. The measured activity is quantified as metoprolol equivalents (MetEQ) of the lead substance metoprolol in the beta-blocker assay, and diclofenac equivalents (DicEQ) in the NSAID assay. Even though MetEQs and DicEQs were found to surpass the concentration of the respective lead substances (metoprolol, diclofenac), as determined by chemical analysis by a factor of two to three, this difference was shown to be reasonably explained by the presence and action of additional active compounds with the same mode of action in the test samples. Thus, both in vitro assays were proven to integrate effectively over beta-blocker and NSAID activities in WWTP effluents in a very sensitive and extremely rapid manner.
... The design of such compounds relies on identification of hydrophobic pockets distinct from the ATP-binding pocket, which are exposed only in inactive intermediates, or of allosteric sites. Alternatively, it requires the development of conformation-sensitive assays for high throughput screens [420,378,379] Compound 1 is a 2-aminopyrimidine analog identified as an ATP-non competitive inhibitor of CDK4 in a high throughput screen, which was further found to inhibit Rb phosphorylation in breast cancer cell lines, thereby offering promising perspectives for development of derivatives targeting this kinase [382]. ...
... Whilst targeting essential protein/protein interactions constitutes a potentially promising approach in terms of specificity, it remains difficult to identify small molecules which can effectively disrupt protein/protein interfaces [422][423][424][425][426]. However, it remains extremely challenging to design allosteric inhibitors through rational approaches, and their identification relies on the discovery of new allosteric sites within target kinases and calls for new strategies and tools for implementing throughput screens [378,379]. ...
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.
The cell cycle is a complex process that involves DNA replication, protein expression, and cell division. Dysregulation of the cell cycle is associated with various diseases. Cyclin-dependent kinases (CDKs) and their corresponding cyclins are major proteins that regulate the cell cycle. In contrast to inhibition, a new approach called proteolysis-targeting chimeras (PROTACs) and molecular glues can eliminate both enzymatic and scaffold functions of CDKs and cyclins, achieving targeted degradation. The field of PROTACs and molecular glues has developed rapidly in recent years. In this article, we aim to summarize the latest developments of CDKs and cyclin protein degraders. The selectivity, application, validation and the current state of each CDK degrader will be overviewed. Additionally, possible methods are discussed for the development of degraders for CDK members that still lack them. Overall, this article provides a comprehensive summary of the latest advancements in CDK and cyclin protein degraders, which will be helpful for researchers working on this topic.
Miniaturized, specific, rapid response and economical biosensors are finding applications in biotechnology, environmental studies, agriculture, food inspection and safety, disease diagnosis and medical utilities. Of the many categories of biosensors, optical biosensors have brought about an extra edge in sensing applications due to their selective, rapid and extremely sensitive measurements. Biosensors are analytical tools used to detect specific analytes such as cholesterol, urea, etc. having biomolecules such as nucleic acids, proteins, carbohydrates as key element for detecting these analytes along with a transducer and a data analysis and visualization tool. In case of optical biosensors the analyte is detected using light with either label based or label free techniques. In this paper some of the marked advances in the last decade in the field of optical biosensors have been reviewed with an emphasis on their fabrication approaches and growing application areas. Along with some of the carefully selected article on new developments in optical biosensors through the last decade, a brief historical review of optical biosensors since the breakthrough in optical biosensors in 1970s has also been presented. Another focus of the current review is the classification of biosensors, typical structures along with emerging developments in optical biosensing that are likely to impact the current decade. Major application areas and emerging applications through the last decade have been outlined to present a clear picture on the versatility of optical biosensors. Finally, the review also considers the challenges and future of emerging optical biosensing technologies in the current decade.
The cyclin-dependent kinases (CDKs) are promising therapeutic targets for cancer therapy. Herein, we describe our efforts toward the discovery of a series of 5-chloro-N4-phenyl-N2-(pyridin-2-yl)pyrimidine-2,4-diamine derivatives as dual CDK6 and 9 inhibitors. Intensive structural modifications lead to the identification of compound 66 as the most active dual CDK6/9 inhibitor with balancing potency against these two targets and good selectivity over CDK2. Further biological studies revealed that compound 66 directly bound to CDK6/9, resulting in suppression of their down-stream signaling pathway and inhibition of cell proliferation by blocking cell cycle progression and inducing cellular apoptosis. More importantly, compound 66 significantly inhibited tumor growth in an xenograft mouse model with no obvious toxicity, indicating the promising therapeutic potential of CDK6/9 dual inhibitors for cancer treatment. Therefore, the above results are of great importance in the development of dual CDK6/9 inhibitors for cancer therapy.
