title: Design, synthesis, and in vitro evaluation of a fluorescently labeled irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKACα).
abstract: The design and development of irreversible kinase inhibitors is an expanding frontier of kinase drug discovery. The current approach to develop these inhibitors utilizes ATP-competitive inhibitor scaffolds to target non-catalytic cysteines in the kinase ATP-binding site. However, this approach is limited as not all kinases have a cysteine in the ATP-binding site that can be targeted. In this work, we report a complementary approach to developing irreversible kinase inhibitors that utilizes the substrate-binding site. Using the catalytic subunit of cAMP-dependent protein kinase (PKACα) as a model system, we have designed and synthesized an irreversible inhibitor based on the substrate-competitive inhibitor scaffold PKI(14-22) that covalently modifies non-catalytic Cys199 in the PKACα substrate-binding site. The new compound inhibits PKACα (IC50 = 11.8 ± 1.1 nM), is ∼100-fold selective for PKACα in a kinase panel, and covalently labels the kinase as demonstrated by fluorescence, mass spectrometry, and kinetics experiments. This study demonstrates the feasibility of utilizing this new approach to develop irreversible inhibitors for any of the eighty-nine kinases that possess a similar non-catalytic cysteine in their substrate-binding sites.
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Coover
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Luzi
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: Sudha
authors.initials: S
authors.lastname: Korwar
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: Maria E
authors.initials: ME
authors.lastname: Casile
authors.affiliation: Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Lyons
authors.affiliation: Institute for Structural Biology, Drug Discovery, and Development, Virginia Commonwealth University, Richmond, VA, USA. kcellis@vcu.edu.
authors.forename: Darrell L
authors.initials: DL
authors.lastname: Peterson
authors.affiliation: Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, USA and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA, USA and Institute for Structural Biology, Drug Discovery, and Development, Virginia Commonwealth University, Richmond, VA, USA. kcellis@vcu.edu.
authors.forename: Keith C
authors.initials: KC
authors.lastname: Ellis
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D060326:Chemistry Techniques, Synthetic; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D015195:Drug Design; D005456:Fluorescent Dyes / Q000737:chemistry*; D007659:Ketones / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular
chemical_list: D005456:Fluorescent Dyes; D007659:Ketones; D017868:Cyclic AMP-Dependent Protein Kinases