The kinetics of binding to p38MAP kinase by analogues of BIRB 796

Regan, John; Pargellis, Christopher A.; Cirillo, Pier F.; Gilmore, Thomas D.; Hickey, Eugene R.; Peet, Gregory W.; Proto, Alfred; Swinamer, Alan; Moss, Neil

doi:10.1016/s0960-894x(03)00656-5

Cited by 106 publications

(116 citation statements)

References 20 publications

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“…Several p38 inhibitors are structurally and biophysically well-characterized (Table 1). [13][14][15] p38 inhibitors 1 and 4 bind to the DFG-in conformation [16] that is also observed for apo p38. [17] In contrast, p38 inhibitors 2 and 3 of the diarylurea class were found to bind to the DFG-out conformation.…”

mentioning

confidence: 72%

NMR Characterization of Kinase p38 Dynamics in Free and Ligand‐Bound Forms

et al. 2006

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In its apo state kinase p38 effects slow motions that can be detected in the NMR spectrum. One of the affected parts is the pharmacologically interesting DFG motif. Diarylurea inhibitors that bind to the DFG‐out conformation lock this motif in a defined state, whereas DFG‐in inhibitors that bind to the adjacent hinge region leave the flexibility of the DFG motif unaffected (see crystal structure of the complex of p38 with the inhibitor SB203580).

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mentioning

confidence: 72%

NMR Characterization of Kinase p38 Dynamics in Free and Ligand‐Bound Forms

et al. 2006

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“…bind catalytic sites at rates that approach diffusion limits (20,21,22), whereas DFG-out inhibitors of p38 MAP kinase have been shown to bind two to three orders of magnitude slower (23,24). Results of SPR kinetic analysis show that this is also the case for PYK2 (Fig.…”

Section: Surface Plasmon Resonance (Spr)-classical Kinase Inhibitorsmentioning

confidence: 84%

Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design

Han

Mistry

Chang

et al. 2009

Journal of Biological Chemistry

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Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, nonreceptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.Proline-rich tyrosine kinase 2 (PYK2) 2 and focal adhesion kinase (FAK) comprise the focal adhesion kinase subfamily of non-receptor tyrosine kinases. PYK2 and FAK are large multidomain proteins containing an N-terminal FERM domain, a central catalytic domain, and a C-terminal segment containing dual proline rich (PR) subdomains and a focal adhesion targeting (FAT) region (1, 2). While FAK is widely expressed, PYK2 expression is relatively restricted with highest levels in brain and the hematopoeitic system. Unlike FAK, optimal PYK2 activation is dependent on Ca 2ϩ mobilization. PYK2 (Ϫ/Ϫ) animals have been described previously, and develop normally (3, 4). Characterization of the immune system of PYK2(Ϫ/Ϫ) animals revealed the absence of marginal zone B-cells along with abnormal T-cell independent type II responses (4), and altered macrophage morphology, migration and signaling in response to cell attachment or chemokine treatment (3). These studies strengthen the link between PYK2 and signaling through chemokine and integrin receptors. In addition, PYK2(Ϫ/Ϫ) mice were shown to have increased susceptibility to diet-induced obesity and diabetes (5).Recently, the characterization of PYK2(Ϫ/Ϫ) mice showed a high bone mass phenotype resulting from increased osteogenesis and osteoblast activity. Using PYK2(Ϫ/Ϫ) mouse bone marrow cultures and hMSCs expressing a PYK2 shRNA, elimination or reduction of PYK2 protein levels resulted in significantly enhanced osteogeogenesis. Importantly, the daily administration of a pyrimidine-based PYK2 inhibitor, PF-431396, increased bone formation, and protected against bone loss in ovariectomized rats (6). PYK2(Ϫ/Ϫ) mice showed mild osteopetrosis which was attributed to the impairment in osteoclast function (7). Therefore, the high bone mass phenotype may result from both enhanced osteoblast and impaired osteocla...

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“…This property has been most extensively studied for BIRB 796, which binds to both active and nonactive forms of p38α, β, γ, and δ, 10,21 and this slow binding has been extensively demonstrated in both activity-based assays 10,21,22 as well as in experiments designed to directly measure compound binding. 10,23,24 We were able to reproduce these results in our TR-FRET-based format and observed a shift in the IC 50 of BIRB 796-dependent tracer displacement from approximately 1 µM to 20 nM over the course of 6 h (Fig. 3).…”

Section: Characterization Of Slow-binding Inhibitors To P38 and Brafmentioning

confidence: 52%

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

Lebakken¹,

Riddle²,

Singh³

et al. 2009

SLAS Discovery

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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 lowactivity 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. (Journal of Biomolecular Screening 2009:924-935)

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The kinetics of binding to p38MAP kinase by analogues of BIRB 796

Cited by 106 publications

References 20 publications

NMR Characterization of Kinase p38 Dynamics in Free and Ligand‐Bound Forms

NMR Characterization of Kinase p38 Dynamics in Free and Ligand‐Bound Forms

Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design

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

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