title: Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors.
abstract: The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1-3 follows a sequential ordered Bi-Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in kcatfor PKN1 and PKN2, and a decrease in peptide KMfor PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (Ki<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.
authors.affiliation: Pfizer Oncology Research Unit, Pfizer Inc., San Diego, CA 92121, U.S.A.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Falk
authors.affiliation: Pfizer Worldwide Medicinal Chemistry, Pfizer Inc., San Diego, CA 92121, U.S.A.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: Pfizer Worldwide Medicinal Chemistry, Pfizer Inc., San Diego, CA 92121, U.S.A.
authors.forename: Ben
authors.initials: B
authors.lastname: Bolaños
authors.affiliation: Pfizer Oncology Research Unit, Pfizer Inc., Pearl River, NY 10965, U.S.A.
authors.forename: Keziban
authors.initials: K
authors.lastname: Unsal-Kacmaz
authors.affiliation: Pfizer Oncology Research Unit, Pfizer Inc., Pearl River, NY 10965, U.S.A.
authors.forename: Anke
authors.initials: A
authors.lastname: Klippel
authors.affiliation: Pfizer Oncology Research Unit, Pfizer Inc., San Diego, CA 92121, U.S.A.
authors.forename: Stephan
authors.initials: S
authors.lastname: Grant
authors.affiliation: Pfizer Worldwide Medicinal Chemistry, Pfizer Inc., San Diego, CA 92121, U.S.A.
authors.forename: Alexei
authors.initials: A
authors.lastname: Brooun
authors.affiliation: Pfizer Oncology Research Unit, Pfizer Inc., San Diego, CA 92121, U.S.A. sergei.timofeevski@pfizer.com.
authors.forename: Sergei
authors.initials: S
authors.lastname: Timofeevski
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D016718:Arachidonic Acid / Q000737:chemistry*; D002384:Catalysis; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry; D007700:Kinetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D047428:Protein Kinase Inhibitors / Q000737:chemistry*
chemical_list: D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; D016718:Arachidonic Acid; D000255:Adenosine Triphosphate; C061570:protein kinase N; D011493:Protein Kinase C
keywords: AGC kinase; cancer; kinase inhibitor; kinetic mechanism; lipid; protein kinase N (PKN)