scholarly journals Development of Phosphodiesterase-Protein Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

2021 ◽  
Author(s):  
Nikhil Kumar Tulsian ◽  
Valerie Jia-En Sin ◽  
Ganesh Srinivasan Anand ◽  
Hwee-Ling Koh
Keyword(s):  
Protein Kinases ◽  
Protein Interactions ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Drug Targets ◽  
Cross Reactivity ◽  
Nucleotide Receptors ◽  
Macromolecular Assemblies ◽  
Fluorescence Polarization Assay ◽  
In Cells

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (Protein Kinases, PK) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PK generates an expanded active site which enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PK, and aid in signal termination. PDEs are important drug targets and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targets PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay has been adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode, and disrupt protein-protein interactions between PDEs and cyclic nucleotide activating protein kinases in a second mode. We tested this approach with three different systems: cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG and dual-specificity RegA-RD complexes and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

scholarly journals Development of Phosphodiesterase–Protein-Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

2021 ◽  
Vol 22 (10) ◽  
pp. 5242
Author(s):  
Nikhil K. Tulsian ◽  
Valerie Jia-En Sin ◽  
Hwee-Ling Koh ◽  
Ganesh S. Anand
Keyword(s):  
Protein Interactions ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Drug Targets ◽  
Cross Reactivity ◽  
Nucleotide Receptors ◽  
Protein Protein Interactions ◽  
Macromolecular Assemblies ◽  
Fluorescence Polarization Assay ◽  
In Cells

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE–PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE–PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems—cAMP-specific PDE8–PKAR, cGMP-specific PDE5–PKG, and dual-specificity RegA–RD complexes—and ranked inhibitors according to their inhibition potency. Targeting PDE–PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

A Fluorescence Polarization Assay for Cyclic Nucleotide Phosphodiesterases

2002 ◽  
Vol 7 (3) ◽  
pp. 215-222 ◽  
Author(s):  
Wei Huang ◽  
Yan Zhang ◽  
J. Richard Sportsman
Keyword(s):  
Cyclic Nucleotide ◽  
Fluorescence Polarization ◽  
Drug Targets ◽  
Second Messengers ◽  
Reaction Products ◽  
Cyclic Nucleotide Phosphodiesterases ◽  
Extracellular Signals ◽  
Fluorescence Polarization Assay ◽  
Hydrolysis Of ◽  
Nucleotide Phosphodiesterases

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the 3′-ester bond of cyclic AMP (cAMP) and cyclic GMP (cGMP), important second messengers in the transduction of a variety of extracellular signals. There is growing interest in the study of PDEs as drug targets for novel therapeutics. We describe the development of a homogeneous fluorescence polarization assay for PDEs based on the strong binding of PDE reaction products (i.e., AMP or GMP) onto modified nanoparticles through interactions with immobilized trivalent metal cations. This assay technology (IMAP) is applicable to both cAMP- and cGMP-specific PDEs. Results of the assay in 384- and 1536-well microplates are presented.

scholarly journals Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells.

1980 ◽  
Vol 87 (2) ◽  
pp. 336-345 ◽  
Author(s):  
C L Browne ◽  
A H Lockwood ◽  
J L Su ◽  
J A Beavo ◽  
A L Steiner
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Kinases ◽  
Cyclic Gmp ◽  
Cyclic Nucleotides ◽  
Mitotic Spindle ◽  
Cyclic Nucleotide ◽  
Dependent Protein Kinase ◽  
Mitotic Apparatus ◽  
Dependent Protein

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide-dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion.

scholarly journals A Cell-Based PDE4 Assay in 1536-Well Plate Format for High-Throughput Screening

2008 ◽  
Vol 13 (7) ◽  
pp. 609-618 ◽  
Author(s):  
Steven A. Titus ◽  
Xiao Li ◽  
Noel Southall ◽  
Jianming Lu ◽  
James Inglese ◽  
...  
Keyword(s):  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Compound Screening ◽  
A Cell

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,′5′-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5′nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein—coupled receptor as a driving force for cAMP production and a cyclic nucleotide—gated cation channel as a biosensor in 1536-well plates. ( Journal of Biomolecular Screening 2008:609-618)

scholarly journals Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

2018 ◽  
Author(s):  
Sindhuja Sridharan ◽  
Nils Kurzawa ◽  
Thilo Werner ◽  
Ina Guenthner ◽  
Dominic Helm ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Nucleic Acid Binding ◽  
Macromolecular Assemblies ◽  
Intrinsically Disordered ◽  
Cellular Environment ◽  
Nucleic Acid Binding Proteins ◽  
High Concentrations ◽  
In Cells

Nucleotide triphosphates (NTPs) regulate numerous biochemical processes in cells as (co-)substrates, allosteric modulators, biosynthetic precursors, and signaling molecules. Apart from its roles as energy source fueling cellular biochemistry, adenosine triphosphate (ATP), the most abundant NTP in cells, has been reported to affect macromolecular assemblies, such as protein complexes and membrane-less organelles. Moreover, both ATP and guanosine triphosphate (GTP) have recently been shown to dissolve protein aggregates. However, system-wide studies to characterize NTP- interactions under conditions approximating the native cellular environment are lacking, which limits our perspective of the diverse physiological roles of NTPs. Here, we have mapped and quantified proteome-wide NTP-interactions by assessing thermal stability and solubility of proteins using mechanically disrupted cells. Our results reveal diverse biological roles of ATP depending on its concentration. We found that ATP specifically interacts with proteins that utilize it as substrate or allosteric modulator at doses lower than 500 μM, while it affects protein-protein interactions of protein complexes at mildly higher concentrations (between 1-2 mM). At high concentrations (> 2 mM), ATP modulates the solubility state of a quarter of the insoluble proteome, consisting of positively charged, intrinsically disordered, nucleic acid binding proteins, which are part of membrane-less organelles. The extent of solubilization depends on the localization of proteins to different membrane-less organelles. Furthermore, we uncover that ATP regulates protein-DNA interactions of the Barrier to autointegration factor (BANF1). Our data provides the first quantitative proteome-wide map of ATP affecting protein structure and protein complex stability and solubility, providing unique clues on its role in protein phase transitions.

scholarly journals A human kinase yeast array for the identification of kinases modulating phosphorylation-dependent protein-protein interactions

2021 ◽  
Author(s):  
Stefanie Jehle ◽  
Natalia Kunowska ◽  
Nouhad Benlasfer ◽  
Jonathan Woodsmith ◽  
Gert Weber ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinases ◽  
Protein Interactions ◽  
Drug Targets ◽  
Kinase Activity ◽  
Protein Protein Interactions ◽  
Kinase Substrate ◽  
Human Kinase ◽  
A Minor ◽  
Activity Dependent

Protein kinases play an important role in cellular signaling pathways and their dysregulation leads to multiple diseases, making kinases prime drug targets. While more than 500 human protein kinases are known to collectively mediate phosphorylation of over 290,000 S/T/Y sites, the activities have been characterized only for a minor, intensively studied subset. To systematically address this discrepancy, we developed a human kinase array in Saccharomyces cerevisiae as a simple readout tool to systematically assess kinase activities. For this array, we expressed 266 human kinases in four different Saccharomyces cerevisiae strains and profiled ectopic growth as a proxy for kinase activity across 33 conditions. More than half of the kinases showed an activity-dependent phenotype across many conditions and in more than one strain. We then employed the kinase array to identify the kinase(s) that can modulate protein-protein-interactions (PPIs). Two characterized, phosphorylation-dependent PPIs with unknown kinase substrate relationships were analyzed in a phospho yeast two-hybrid assay. CK2α1 and SGK2 kinases can abrogate the interaction between the spliceosomal proteins AAR2 and PRPF8 and NEK6 kinase was found to mediate the estrogen receptor (ERα) interaction with 14 3 3 proteins. The human kinase yeast array can thus be used for a variety of kinase activity dependent readouts.

scholarly journals Pseudokinases: From Allosteric Regulation of Catalytic Domains and the Formation of Macromolecular Assemblies to Emerging Drug Targets

Catalysts ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 778 ◽  
Author(s):  
Andrada Tomoni ◽  
Jonathan Lees ◽  
Andrés G. Santana ◽  
Victor M. Bolanos-Garcia ◽  
Agatha Bastida
Keyword(s):  
Cell Proliferation ◽  
Protein Interactions ◽  
Drug Targets ◽  
Structural Features ◽  
Therapeutic Window ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Therapeutic Approaches ◽  
Macromolecular Assemblies ◽  
Catalytic Domains

Pseudokinases are a member of the kinase superfamily that lack one or more of the canonical residues required for catalysis. Protein pseudokinases are widely distributed across species and are present in proteins that perform a great diversity of roles in the cell. They represent approximately 10% to 40% of the kinome of a multicellular organism. In the human, the pseudokinase subfamily consists of approximately 60 unique proteins. Despite their lack of one or more of the amino acid residues typically required for the productive interaction with ATP and metal ions, which is essential for the phosphorylation of specific substrates, pseudokinases are important functional molecules that can act as dynamic scaffolds, competitors, or modulators of protein–protein interactions. Indeed, pseudokinase misfunctions occur in diverse diseases and represent a new therapeutic window for the development of innovative therapeutic approaches. In this contribution, we describe the structural features of pseudokinases that are used as the basis of their classification; analyse the interactome space of human pseudokinases and discuss their potential as suitable drug targets for the treatment of various diseases, including metabolic, neurological, autoimmune, and cell proliferation disorders.

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