Structure, activity, regulation, and inhibitor discovery for a protein kinase associated with apoptosis and neuronal death

Velentza, Anastasia; Schumacher, Andrew; Watterson, D. Martin

doi:10.1016/s0163-7258(02)00190-0

Cited by 13 publications

(9 citation statements)

References 26 publications

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“…7 Although its potency was in the micromolar range, a single injection of this inhibitor attenuated brain tissue damage when administered before or 6 h after ischemic injury. 30 These therapeutic effects were validated in oxygen glucose deprivation and middle cerebral artery occlusion models.…”

Section: Resultsmentioning

confidence: 99%

Fluorescence Linked Enzyme Chemoproteomic Strategy for Discovery of a Potent and Selective DAPK1 and ZIPK Inhibitor

et al. 2013

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DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta and rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca2+-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.

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Section: Resultsmentioning

confidence: 99%

Fluorescence Linked Enzyme Chemoproteomic Strategy for Discovery of a Potent and Selective DAPK1 and ZIPK Inhibitor

et al. 2013

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“…Rho kinase and GSK3␤ assays were performed according to protocols provided by the manufacturer (Upstate Biotechnology, Lake Placid, NY). IC 50 values were calculated by a linear regression analysis and kinetics determined as described (10)(11)(12)(13). The MLCK inhibitor is competitive with the ATP substrate and has a K i of Ϸ5 M, similar in magnitude to the K m value for the substrate.…”

Section: Methodsmentioning

confidence: 99%

Protein kinase involved in lung injury susceptibility: Evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment

Wainwright

Rossi

Schavocky

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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133

147

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Acute lung injury (ALI) associated with sepsis and iatrogenic ventilator-induced lung injury resulting from mechanical ventilation are major medical problems with an unmet need for small molecule therapeutics. Prevailing hypotheses identify endothelial cell (EC) layer dysfunction as a cardinal event in the pathophysiology, with intracellular protein kinases as critical mediators of normal physiology and possible targets for drug discovery. The 210,000 molecular weight myosin light chain kinase (MLCK210, also called EC MLCK because of its abundance in EC) is hypothesized to be important for EC barrier function and might be a potential therapeutic target. To test these hypotheses directly, we made a selective MLCK210 knockout mouse that retains production of MLCK108 (also called smooth-muscle MLCK) from the same gene. The MLCK210 knockout mice are less susceptible to ALI induced by i.p. injection of the endotoxin lipopolysaccharide and show enhanced survival during subsequent mechanical ventilation. Using a complementary chemical biology approach, we developed a new class of small-molecule MLCK inhibitor based on the pharmacologically privileged aminopyridazine and found that a single i.p. injection of the inhibitor protected WT mice against ALI and death from mechanical ventilation complications. These convergent results from two independent approaches demonstrate a pivotal in vivo role for MLCK in susceptibility to lung injury and validate MLCK as a potential drug discovery target for lung injury.C urrent hypotheses (1, 2) identify dysfunction of the endothelial cell (EC) layer as a cardinal event in the pathophysiology of multiple medical conditions, including sepsis. The mortality associated with sepsis alone is similar (3) to that of acute myocardial infarction (MI). In contrast to MI, available therapies are limited for the treatment of sepsis and its associated tissue injuries (3, 4). Mechanical ventilation is often required for the support of patients with sepsis but is itself associated with additional, iatrogenic pulmonary injury that also appears to involve EC barrier dysfunction (5). Recent progress in the use of controlled ventilator strategies, such as positive end-expiratory pressure (PEEP), lessens the potential for ventilator-induced lung injury (VILI), but a need still exists for therapies that would prevent this clinical complication (5). Most recently, in vitro studies have linked a variety of EC signal transduction pathways to the physiological mechanisms of EC barrier function and identified several endothelial protein kinases as potential drug discovery targets (1). However, the integration of the knowledge regarding in vitro signal transduction pathways with in vivo pathophysiology related to compromised EC barrier function and the validation of potential EC therapeutic targets have not occurred.Homeostasis and resistance to tissue injury are maintained by a balance between intracellular EC cytoskeletal contractionrelaxation cycles and modulation of EC extracellular adhesion properties, whi...

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“…Several ongoing investigations are developing therapeutic approaches to neuronal death by modulating DAPK activity (3,5,29,(38)(39)(40). Our results suggest that DAPK is a good target for developing new therapies against memory disorders.…”

Section: Discussionmentioning

confidence: 65%

Deletion of the kinase domain from death-associated protein kinase enhances spatial memory in mice

Yukawa

Tanaka²,

Bai³

et al. 2006

Int J Mol Med

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Abstract. Death-associated protein kinase (DAPK) is a Ca 2+ /calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. DAPK is highly expressed in hippocampal neurons which are essential elements for memory formation. To examine if DAPK is implicated in spatial learning and memory, both wild-type and DAPK-mutant mice were subjected to Morris water maze tests. DAPKmutant mice were generated by deleting 74 amino acids from the catalytic kinase domain of DAPK, and were used to investigate roles of the DAPK kinase domain in regulating spatial memory. Both mutant and wild-type mice were able to learn the water maze tasks to locate a hidden escape platform. In the first probe test, mutant mice showed a more precise memory for platform position compared to wild-type mice. In the reversal training in which the platform was located opposite from the original position, DAPK-mutant mice exhibited superior spatial learning compared to wildtype mice. DAPK-mutant mice also showed a more precise memory than their wild-type littermates in the probe trial of reversal test. Thus, the present results revealed crucial implications of DAPK in regulating spatial memory in mice.

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Structure, activity, regulation, and inhibitor discovery for a protein kinase associated with apoptosis and neuronal death

Cited by 13 publications

References 26 publications

Fluorescence Linked Enzyme Chemoproteomic Strategy for Discovery of a Potent and Selective DAPK1 and ZIPK Inhibitor

Fluorescence Linked Enzyme Chemoproteomic Strategy for Discovery of a Potent and Selective DAPK1 and ZIPK Inhibitor

Protein kinase involved in lung injury susceptibility: Evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment

Deletion of the kinase domain from death-associated protein kinase enhances spatial memory in mice

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