PAK1Dr. Peterson and Dr. Chernoff have devised a method to identify allosteric inhibitors of p21-activated kinases (Paks) that antagonize the conformational change required for Pak activation. The method may be extended to identify inhibitors of class I Paks, p21- and Rho-activated proteins, autoinhibited small GTPase effectors, and autoinhibited non-receptor serine/threonine kinases and phosphatases.
PAK2
Kinase domains are remarkably conserved across diverse kinase families and, consequently, many chemical kinase inhibitors block multiple kinases. Indeed, the development of specific inhibitors of kinases is a major challange for the development of an appropriate drug, and targeting the Pak inhibitor domain may provide greater specificity than targeting the catalytic site. In this method, compounds are screened for the ability to inhibit the wild-type PAK but not a constitutively active form. Candidate allosteric inhibitors are those that inhibit the wild-type but not the constitutively active form.
PAK3

Kinase assays need to be simple, inexpensive, and sensitive to the measured parameter but insensitive to solvent or biophysical properties of the compounds such as fluorescence. This invention provides a validated luminescence-based assay for kinase activity based on the measurement of residual ATP in the reaction following kinase-mediated ATP hydrolysis. This serves as a readout for the interaction of the PAK autoinhibitory domain with the kinase domain. This assay is remarkably homogeneous because the only manipulation is the addition of the detection mixture, and the assay is relatively insensitive to fluorescence or absorptive properties of individual compounds.

Compounds identified from this dual positive/negative selection screen may be further investigated for their inhibitory properties.

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Methods of Screening for Inhibitors of Autoinhibited Proteins

Topics in This Section

Developed by:

  • Jeffrey R. Peterson, PhD

  • Jonathan Chernoff, MD, PhD

  • Description:

    PAK1
    Dr. Peterson and Dr. Chernoff have devised a method to identify allosteric inhibitors of p21-activated kinases (Paks) that antagonize the conformational change required for Pak activation. The method may be extended to identify inhibitors of class I Paks, p21- and Rho-activated proteins, autoinhibited small GTPase effectors, and autoinhibited non-receptor serine/threonine kinases and phosphatases.
    PAK2
    Kinase domains are remarkably conserved across diverse kinase families and, consequently, many chemical kinase inhibitors block multiple kinases. Indeed, the development of specific inhibitors of kinases is a major challange for the development of an appropriate drug, and targeting the Pak inhibitor domain may provide greater specificity than targeting the catalytic site. In this method, compounds are screened for the ability to inhibit the wild-type PAK but not a constitutively active form. Candidate allosteric inhibitors are those that inhibit the wild-type but not the constitutively active form.
    PAK3

    Kinase assays need to be simple, inexpensive, and sensitive to the measured parameter but insensitive to solvent or biophysical properties of the compounds such as fluorescence. This invention provides a validated luminescence-based assay for kinase activity based on the measurement of residual ATP in the reaction following kinase-mediated ATP hydrolysis. This serves as a readout for the interaction of the PAK autoinhibitory domain with the kinase domain. This assay is remarkably homogeneous because the only manipulation is the addition of the detection mixture, and the assay is relatively insensitive to fluorescence or absorptive properties of individual compounds.

    Compounds identified from this dual positive/negative selection screen may be further investigated for their inhibitory properties.

    Applications:

    • Identification of allosteric kinase inhibitors

    Advantages:

    • This assay is remarkably homogeneous because the only manipulation is the addition of the detection mixture, and the assay relatively insensitive to fluorescence or absorptive properties of individual compounds.

    Relevant Articles: