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.

Characterization of PKACα enzyme kinetics and inhibition in an HPLC assay with a chromophoric substrate

“…As the chromophore, we have chosen to use rhodamine B, based on its strong absorbance at ~560 nm, low cost (~$0.50/g), and our previous experience with making rhodamine B-labeled peptide inhibitors. [12] To join the rhodamine B chromophore to the peptide, we utilized a methylaminobutyric acid (MAB) linker. Combining these elements yields the Rh-MAB-Kemptide substrate ( 1 , Fig.…”

“…S1 of the Supplementary Information) following a previously published protocol. [12] Following purification by prep-scale RP-HPLC, Rh-MAB-Kemptide ( 1 ) was characterized by MALDI-MS.…”

“…Subsequent amino acids Leu, Ser, Ala, Arg, Arg, and Leu (0.2 M in DMF) were incorporated using a standard coupling cycle (deprotection: 20% piperidine in DMF; coupling: 0.2 M DIC and 0.2 M HOBt; washes: DMF) at 50 °C, with double couplings used for Arg. Following the deprotection of the Fmoc from the Leu, the resin was removed from the peptide synthesizer and the N-terminus coupled to the previously reported rhodamine-MAB-NHS ester fragment [12] (250 mg, 0.5 mmol, see Fig. S1 in the Supplementary Information) with NEt 3 (0.14 mL, 1 mmol) in DMF (5 mL).…”

“…In our work to develop novel substrate competitive kinase inhibitors,[12–14] we have used an NADH-coupled enzyme assay [15] for kinetics analyses and the commercially available Z′-LYTE FRET-based assay [16] for inhibition studies. In our hands, we have found that each of these assays has significant drawbacks.…”

Kinetics and inhibition studies of the L205R mutant of cAMP‐dependent protein kinase involved in Cushing's syndrome

“…1A), an important site for substrate, regulatory subunit, and inhibitor recognition. The hydrophobic side chain of Ile22 of PKI (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) binds in the P+1-binding site, creating a positive binding interaction and contributing to overall inhibitor binding. In the cocrystal structure of the L205R-PKACa mutant with PKI(5-24) and ATP ( Fig.…”