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.

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High throughput screening assays aim to identify small molecules that interfere with protein function, activity, or conformation, which can serve as effective tools for chemical biology studies of targets involved in physiological processes or pathways of interest or disease models, as well as templates for development of therapeutics in medicinal chemistry. Fluorescent biosensors constitute attractive and powerful tools for drug discovery programs, from high throughput screening assays, to postscreen characterization of hits, optimization of lead compounds, and preclinical evaluation of candidate drugs. They provide a means of screening for inhibitors that selectively target enzymatic activity, conformation, and/or function in vitro. Moreover, fluorescent biosensors constitute useful tools for cell- and image-based, multiplex and multiparametric, high-content screening. Application of fluorescence-based sensors to screen large and complex libraries of compounds in vitro, in cell-based formats or whole organisms requires several levels of optimization to establish robust and reproducible assays. In this review, we describe the different fluorescent biosensor technologies which have been applied to high throughput screens, and discuss the prerequisite criteria underlying their successful application. Special emphasis is placed on protein kinase biosensors, since these enzymes constitute one of the most important classes of therapeutic targets in drug discovery.

Summary Background The cyclin‐dependent kinases (CDKs) CDK2 and CDK4 are involved in regulation of cell‐cycle progression, and psoriasis is characterized by hyperproliferation of basal epidermal cells. CDK inhibitory proteins (CKIs) such as p16INK4A (p16) bind CDK4/6 kinases and prevent their interaction with D‐type cyclins. CKIs such as p21Cip1 (p21) and p27Kip1 (p27) associate with CDK–cyclin complexes and prevent their activation. Objectives To gain insight into the molecular implication of CDK2 and CDK4 kinases in psoriasis, we sought to characterize expression of these kinases and associated cyclins, as well as of CKIs, and addressed the status of CDK2 and CDK4 activity in human psoriatic epidermis. Methods A cohort of 24 patients with psoriasis participated in the study. Biopsies were removed from a chronic plaque and from nonlesional skin. CDK2, CDK4, cyclin D1, cyclin E and CKI protein expression was assessed by immunoblotting, immunohistochemistry and immunofluorescence. CDK4 and CDK2 mRNA expression was determined by real‐time polymerase chain reaction. Specific kinase activities of CDK2 and CDK4 were evaluated using fluorescent peptide biosensors. Results CDK2–cyclin E expression and activity were significantly increased in psoriatic epidermis compared with uninvolved adjacent skin. In contrast, CDK4–cyclin D1 activity was inhibited, although its expression was increased in psoriatic epidermis and its transcription slightly inhibited. p27 expression was reduced, while p16 and p21 expression was induced in psoriatic epidermis. Conclusions Epidermal CDK2 activity is increased in psoriatic epidermis while CDK4 activity is completely inhibited. These alterations are not associated with changes in CDK transcription and instead involve post‐translational control mediated by decreased expression of p27 and p16 overexpression, respectively. What's already known about this topic? Cyclin‐dependent kinases (CDKs) are involved in cell‐cycle progression. The levels of cyclin partners and CDK inhibitors regulate their activity. Psoriasis is a chronic T‐cell‐driven inflammatory skin disease characterized by hyperproliferation of basal epidermal cells. What does this study add? Thanks to fluorescent peptide biosensors, this study demonstrates that epidermal CDK2 activity is increased in psoriatic epidermis while CDK4 activity is completely inhibited. These alterations involve post‐translational control mediated by decreased expression of p27, and p16 overexpression, respectively. What is the translational message? CDK2 and CDK4 are involved in regulation of cell‐cycle progression, and psoriasis is characterized by hyperproliferation of basal epidermal cells. Epidermal CDK2 activity is increased in psoriatic epidermis while CDK4 activity is completely inhibited. These alterations are not associated with changes in CDK transcription and instead involve post‐translational control mediated by decreased expression of p27 and p16 overexpression, respectively. Pharmacological modulation of CDK2 and CDK4 may c...

Cyclin-dependent kinases (CDKs) are involved in regulation of cell cycle and passage through the G1 restriction point relies on CDK4/6 and their association with cyclin D1 (CycD1) whereas entry into S phase requires CDK2 and its association with cyclin E (CycE). The levels of cyclin partners, their phosphorylation status, and the abundance of inhibitors of CDK (CDKI) regulate CDK activity. CKIs may be implicated in signaling pathways that are involved in psoriasis pathogenesis and regulation of G1 and S phases by CDKs is relevant for inflammatory skin diseases that are associated with proliferative cell disorders. To monitor CDK2 and CDK4 activity in human epidermis we used CDKACT fluorescent biosensors that undergo fluorescence enhancement upon phosphorylation by CDKs. By using confocal and Western blot analysis, we evaluated the expression of CDK4/2, Cyc D1/E and CDKI such as p16 INK4A (p16), p21 Cip1 (p21) and p27 Kip1 (p27). A cohort of 24 patients was asked to participate in the study. Punch biopsies were taken from a chronic plaque and from non-lesional skin of the same patient. CDK4 and its CycD1 partner were overexpressed in psoriatic epidermis. Epidermal QPCR analysis showed that the increased CDK4 expression was not due to enhanced transcription. Compared to normal skin there was no increase in CDK4/ CyclinD1 activity. CDK2 and CycE were overexpressed in psoriatic epidermis and this overexpression was correlated with an increase in CDK2-CycE activity. Increased CDK2 expression was not related to enhanced transcription. There was an increased expression of p16 and p21 whereas p27 was decreased in psoriatic epidermis. We have have shown that epidermal CDK2 activity was greatly increased in psoriatic epidermis while CDK4 activity was completely inhibited. These alterations are not associated with changes in CDK transcription and instead involve post-translational control that may be mediated by a dysregulation of CDKI expresion in psoriatic epidermis.

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Understanding the intricate steps of protein kinase regulation requires characterization of protein-protein interactions between the catalytic subunit, its regulatory partners and the substrate. Fluorescent probes are useful tools with which to study such interactions and to gain insight into their affinities and specificities. Solvatochromic probes, which display changes in their fluorescence emission in response to changes in the polarity of the medium, are particularly attractive. Here we describe conjugation of a switchable fluorescent dye, TP-2Rho, to peptide and protein derivatives of cyclin-dependent kinase 4 (CDK4) and its application to characterization of the interactions between the catalytic subunit of this kinase, its regulatory partner cyclin D1 and a peptide substrate. We demonstrate the sensitivity of TP-2Rho in relation to of those other dyes used for monitoring peptide-protein and protein-protein interactions. Moreover, we show that TP-Rho-labelled peptides can be introduced into living cells to probe endogenous CDK4/cyclin D.

We describe the identification and characterization of a series of covalent inhibitors of the C-terminal kinase domain (CTKD) of MSK1. The initial hit was identified via a high-throughput screening and represents a rare example of a covalent inhibitor which acts via an S N Ar reaction of a 2,5-dichloropyrimidine with a cysteine residue (Cys440). The covalent mechanism of action was supported by in vitro biochemical experiments and was confirmed by mass spectrometry. Ultimately, the displacement of the 2-chloro moiety was confirmed by crystallization of an inhibitor with the CTKD. We also disclose the crystal structures of three compounds from this series bound to the CTKD of MSK1, in addition to the crystal structures of two unrelated RSK2 covalent inhibitors bound to the CTKD of MSK1.