title: Phenylalanine-Based Inactivator of AKT Kinase: Design, Synthesis, and Biological Evaluation.
abstract: Strategies to inhibit kinases by targeting the substrate binding site offer many advantages, including naturally evolved selectivity filters, but normally suffer from poor potency. In this work we propose a strategy to design and prepare covalent substrate-competitive kinase inhibitors as a method to improve potency. We have chosen AKT as the model kinase for this work. Using the AKT-GSK3β cocrystal structure and a reactive cysteine near the substrate binding site, we have identified phenylalanine (Phe) as an appropriate scaffold for the covalent inactivator portion of these inhibitors. By synthesizing compounds that incorporate cysteine-reactive electrophiles into phenylalanine and testing these compounds as AKT inhibitors, we have identified Boc-Phe-vinyl ketone as a submicromolar inactivator of AKT. We also show that Boc-Phe-vinyl ketone (1) potently inhibits AKT1 and inhibits cell growth in HCT116 and H460 cells nearly as well as AKT inhibitors GSK690693 and MK-2206, (2) is selective for kinases that possess an activation loop cysteine such as AKT, (3) requires the vinyl ketone for inactivation, (4) has inactivation that is time-dependent, and (5) alkylates Cys310 of AKT as shown by mass spectrometry. Identification of Boc-Phe-vinyl ketone as a covalent inactivator of AKT will allow the development of peptide and small-molecule substrate-competitive covalent kinase inhibitors that incorporate additional substrate binding elements to increase selectivity and potency. This proof-of-principle study also provides a basis to apply this strategy to other kinases of the AGC and CAMK families.
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, and the Massey Cancer Center, Virginia Commonwealth University , Richmond, Virginia 23298-0540, United States.
authors.forename: Thuy
authors.initials: T
authors.lastname: Nguyen
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, and the Massey Cancer Center, Virginia Commonwealth University , Richmond, Virginia 23298-0540, United States.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Coover
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, and the Massey Cancer Center, Virginia Commonwealth University , Richmond, Virginia 23298-0540, United States.
authors.forename: Jenson
authors.initials: J
authors.lastname: Verghese
authors.affiliation: Department of Pharmacology & Toxicology, School of Medicine, and the Massey Cancer Center, Virginia Commonwealth University , Richmond, Virginia 23298-0540, United States.
authors.forename: Richard G
authors.initials: RG
authors.lastname: Moran
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, and the Massey Cancer Center, Virginia Commonwealth University , Richmond, Virginia 23298-0540, United States.
authors.forename: Keith C
authors.initials: KC
authors.lastname: Ellis
keywords: AKT kinase; covalent inhibitor; substrate competitive