Background
IPA-3 is an autoregulatory domain inhibitor of p21-activated kinase (Pak) with IC50 value of 2.5μM [1].
IPA-3 is a highly selective and non-ATP-competitive inhibitor that targets the autoregulatory mechanism of group I Paks. IPA-3 is screened out as an inhibitor of Pak1 by measuring ATP hydrolysis. In the in vitro assays, IPA-3 inhibits Pak1 autophosphorylation stimulated by Cdc42 or sphingosine. It shows an IC50 value of 2.5μM in the kinase assay. This inhibition of Pak1 is reported to be noncompetitive with ATP. Besides that, IPA-3 is found to remarkably inhibit the kinase activity of other group I Pak members, Pak2 and 3 at concentration of 10μM. Furthermore, 30μM IPA-3 can prevent both basal and PDGF-stimulated Pak activities in mouse embryonic fibroblasts [1].
参考文献:
[1] Deacon S W, Beeser A, Fukui J A, et al. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chemistry & biology, 2008, 15(4): 322-331.
Protocol
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Kinase experiment [1]:
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Kinase assays
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Pak1 (571 nM final) in Assay buffer was mixed with IPA-3 for 5 min at room temp, followed by addition of 4 μM Cdc42 and 8.3 μM MBP for an additional 5 min. Kinase reactions were started by addition of unlabeled ATP and 1–10 μCi [32P]-γ-ATP per reaction for 10 min at 30°C. Kinase reactions were stopped on dry ice. Under these conditions, MBP phosphorylation was linear with time.
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Cell experiment [1]:
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Cell lines
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Mouse embryonic fibroblasts
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Preparation method
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The solubility of this compound in DMSO is >16.1mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.
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Reacting condition
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30 μM for 10 min
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Applications
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IPA-3 selectively inhibits Pak1 activation in mammalian cells, and can inhibit activation of Group I Paks in cells.
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Animal experiment [2]:
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Animal models
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CD-1 mice
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Dosage form
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3.5 mg/kg, i.p.
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Application
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Inhibition of PAK1 by IPA-3 promoted recovery of neurological function, possibly by downregulating the expression of MMP-2, MMP-9, TNF-α, and IL-1β. IPA-3 may be a potential therapeutic drug for spinal cord injury.
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Other notes
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Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.
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参考文献:
[1] Deacon S W, Beeser A, Fukui J A, et al. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chemistry & biology, 2008, 15(4): 322 - 331.
[2] Ji X, Zhang Y, Zhang L, Chen H, Peng Y, Tang P. Inhibition of p21-Activated Kinase 1 by IPA-3 Promotes Locomotor Recovery After Spinal Cord Injury in Mice. Spine (Phila Pa 1976). 2016 Jun;41(11):919-25.
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