ABSTRACT
A new class of quinoline-based kinase inhibitors has been discovered that both disrupt cyclin dependent 2 (CDK2) interaction with its cyclin A subunit and act as ATP competitive inhibitors. The key strategy for discovering this class of protein-protein disrupter compounds was to screen the monomer CDK2 in an affinity-selection/mass spectrometry-based technique and to perform secondary assays that identified compounds that bound only to the inactive CDK2 monomer and not the active CDK2/cyclin A heterodimer. Through a series of chemical modifications the affinity (Kd) of the original hit improved from 1 to 0.005µM.
Subject(s)
Cyclin A/antagonists & inhibitors , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Quinolines/pharmacology , Crystallography, X-Ray , Cyclin A/chemistry , Cyclin A/metabolism , Cyclin-Dependent Kinase 2/chemistry , Cyclin-Dependent Kinase 2/metabolism , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemistry , Quinolines/chemistry , Structure-Activity RelationshipABSTRACT
Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP)-based couple d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z' of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC trade mark and validated with the protein kinase C inhibitor staurosporine. The IC(50) value generated was comparable to the value obtained by the radioactive (33)P-gamma-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.
Subject(s)
Fluorescence Polarization Immunoassay/methods , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , 3-Phosphoinositide-Dependent Protein Kinases , Adenosine Triphosphate/metabolism , Humans , In Vitro Techniques , Lipid Metabolism , Phosphorylation , Protein Conformation , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.