scholarly journals Detection of Allosteric Kinase Inhibitors by Displacement of Active Site Probes

2012 ◽  
Vol 17 (6) ◽  
pp. 813-821 ◽  
Author(s):  
Connie S. Lebakken ◽  
Laurie J. Reichling ◽  
Jason M. Ellefson ◽  
Steven M. Riddle
Keyword(s):  
Active Site ◽  
Kinase Inhibitors ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Published Data ◽  
Binding Assays ◽  
Allosteric Inhibitors ◽  
Time Resolved ◽  
Inactive Form ◽  
Competitive Inhibitors

Non–adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)–based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes (“tracers”). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC50 values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.

scholarly journals Development and Applications of a Broad-Coverage, TR-FRET-Based Kinase Binding Assay Platform

2009 ◽  
Vol 14 (8) ◽  
pp. 924-935 ◽  
Author(s):  
Connie S. Lebakken ◽  
Steven M. Riddle ◽  
Upinder Singh ◽  
W. Jack Frazee ◽  
Hildegard C. Eliason ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Binding Assay ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinase Assay ◽  
Time Resolved ◽  
Traditional Activity

The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor® 647 conjugate of staurosporine (a “tracer”) from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (TR-FRET) format that simplifies instrumentation requirements and allows for the use of a substantially lower concentration of kinase than was required in the fluorescence-lifetime-based format. The authors use this new assay to demonstrate several aspects of the binding assay format that are advantageous relative to traditional activity-based assays. The TR-FRET binding format facilitates the assay of compounds against low-activity kinases, allows for the characterization of type II kinase inhibitors either using nonactivated kinases or by monitoring compound potency over time, and ensures that the signal being detected is specific to the kinase of interest and not a contaminating kinase.

scholarly journals Fluorescence Lifetime–Based Competitive Binding Assays for Measuring the Binding Potency of Protease Inhibitors In Vitro

2014 ◽  
Vol 19 (6) ◽  
pp. 870-877 ◽  
Author(s):  
Andreas Boettcher ◽  
Nathalie Gradoux ◽  
Edwige Lorthiois ◽  
Trixi Brandl ◽  
David Orain ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Serine Proteases ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Competitive Binding ◽  
Binding Assays ◽  
Time Resolved ◽  
Design And Synthesis ◽  
Competitive Binding Assays

Fluorescence lifetime (FLT)–based assays have developed to become highly attractive tools in drug discovery. All recently published examples of FLT-based assays essentially describe their use for monitoring enzyme-mediated peptide modifications, such as proteolytic cleavage or phosphorylation/dephosphorylation. Here we report the development of competitive binding assays as novel, inhibitor-centric assays, principally employing the FLT of the acridone dye Puretime 14 (PT14) as the readout parameter. Exemplified with two case studies on human serine proteases, the details of the rationale for both the design and synthesis of probes (i.e., active site–directed low-molecular-weight inhibitors conjugated to PT14) are provided. Data obtained from testing inhibitors with the novel assay format match those obtained with alternative formats such as FLT-based protease activity and time-resolved fluorescence resonance energy transfer–based competitive binding assays.

scholarly journals Readout Technologies for Highly Miniaturized Kinase Assays Applicable to High-Throughput Screening in a 1536-Well Format

2006 ◽  
Vol 11 (6) ◽  
pp. 617-633 ◽  
Author(s):  
Martin Klumpp ◽  
Andreas Boettcher ◽  
Damaris Becker ◽  
Gabriele Meder ◽  
Jutta Blank ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Assay Sensitivity ◽  
Time Resolved ◽  
And Performance ◽  
Maximal Relevance ◽  
Kinase Substrates

This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP™ technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns.

scholarly journals Identification of Small-Molecule Frequent Hitters from AlphaScreen High-Throughput Screens

2013 ◽  
Vol 19 (5) ◽  
pp. 715-726 ◽  
Author(s):  
Kenji Schorpp ◽  
Ina Rothenaigner ◽  
Elena Salmina ◽  
Jeanette Reinshagen ◽  
Terence Low ◽  
...  
Keyword(s):  
Small Molecule ◽  
Detection System ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Assays ◽  
Time Resolved ◽  
Protein Protein Interaction ◽  
Nickel Chelate ◽  
High Throughput Screens

Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)–compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni2+-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.

Multiplexing Terbium- and Europium-Based TR-FRET Readouts to Increase Kinase Assay Capacity

2010 ◽  
Vol 15 (8) ◽  
pp. 1008-1015 ◽  
Author(s):  
Robert A. Horton ◽  
Kurt W. Vogel
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinase Assay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Lipid Kinase ◽  
Identification And Characterization

Identification and characterization of kinase inhibitor potency and selectivity is often an iterative process in which a library of compounds is first screened against a single kinase, and hits from that screen are then profiled against other kinases to determine specificity. By developing kinase assays that employ either a terbium- or a europium-based time-resolved fluorescence resonance energy transfer (TR-FRET) readout, one can take advantage of the distinct emission properties of these labels to develop assays for 2 kinases that can be performed simultaneously in the same well. This not only increases the information content provided per assay well but can immediately provide information on compound specificity. The authors have applied this strategy to the development of multiplexed assays for 2 examples systems: EGFR and IKKβ, as well as lipid kinase family members mTOR and PIK3C3. They demonstrate the ability of these multiplexed assays to characterize selective kinase inhibitors in a dose-response mode, with no difference in results obtained from traditional single kinase assays performed separately.

Fluorescent ligands to investigate GPCR binding properties and oligomerization

2013 ◽  
Vol 41 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Martin Cottet ◽  
Orestis Faklaris ◽  
Amadine Falco ◽  
Eric Trinquet ◽  
Jean-Philippe Pin ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescent Imaging ◽  
Binding Assays ◽  
Time Resolved ◽  
Receptor Localization ◽  
Long Time ◽  
Fluorescent Ligands ◽  
Receptor Dimers ◽  
G Protein Coupled

Fluorescent ligands for GPCRs (G-protein-coupled receptors) have been synthesized for a long time but their use was usually restricted to receptor localization in the cell by fluorescent imaging microscopy. During the last two decades, the emergence of new fluorescence-based strategies and the concomitant development of fluorescent measurement apparatus have dramatically widened the use of fluorescent ligands. Among the various strategies, TR (time-resolved)-FRET (fluorescence resonance energy transfer) approaches exhibit an interesting potential to study GPCR interactions with various partners. We have derived various sets of ligands that target different GPCRs with fluorophores, which are compatible with TR-FRET strategies. Fluorescent ligands labelled either with a fluorescent donor (such as europium or terbium cryptate) or with a fluorescent acceptor (such as fluorescein, dy647 or Alexa Fluor® 647), for example, kept high affinities for their cognate receptors. These ligands turn out to be interesting tools to develop FRET-based binding assays. We also used these fluorescent ligands to analyse GPCR oligomerization by measuring FRET between ligands bound to receptor dimers. In contrast with FRET strategies, on the basis of receptor labelling, the ligand-based approach we developed is fully compatible with the study of wild-type receptors and therefore with receptors expressed in native tissues. Therefore, by using fluorescent analogues of oxytocin, we demonstrated the existence of oxytocin receptor dimers in the mammary gland of lactating rats.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Quantum Dot Bioconjugates as Energy Donors in Fluorescence Resonance Energy Transfer Assays

2003 ◽  
Vol 773 ◽  
Author(s):  
Aaron R. Clapp ◽  
Igor L. Medintz ◽  
J. Matthew Mauro ◽  
Hedi Mattoussi
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Genetically Engineered ◽  
Molar Ratio ◽  
Binding Assays ◽  
Specific Sequence ◽  
Donor Acceptor

AbstractLuminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.

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