title: Design, synthesis, and evaluation of a selective chemosensor for leucine-rich repeat kinase 2.
abstract: We describe the design, synthesis, and evaluation of a selective activity probe for leucine-rich repeat kinase 2 (LRRK2), a possible molecular target for the treatment of Parkinson's disease. Our optimal chemosensor design, termed Nictide-S2, incorporates a phosphorylation-sensitive sulfonamido-oxine fluorophore at an engineered cysteine within the substrate sequence. This design allows for the direct, real-time analysis of LRRK2 kinase activity with a detection limit of 2.5 nM. Under optimized conditions, we measured a Z' factor of 0.7 demonstrating the potential utility of this assay for inhibitor screening. Off-target kinases capable of phosphorylating Nictide-S2 are identified and an optimized inhibitor cocktail for suppressing background signal is provided. The resulting chemosensor could be utilized to identify LRRK2 inhibitors as well as selectively report on LRRK2 activity in the presence of off-target kinases.
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, United States.
authors.forename: David A
authors.initials: DA
authors.lastname: Szalewski
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, United States.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Beck
authors.affiliation: Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, United States. Electronic address: cstains2@unl.edu.
authors.forename: Cliff I
authors.initials: CI
authors.lastname: Stains
mesh_terms: D015374:Biosensing Techniques; D015195:Drug Design; D005456:Fluorescent Dyes; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D015125:Oxyquinoline / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis* / Q000494:pharmacology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D013449:Sulfonamides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; C000599938:Nictide-S2; D010446:Peptide Fragments; D010455:Peptides; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D015125:Oxyquinoline; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases
keywords: Fluorescence-based biosensor; Inhibitor; Kinase activity assay; LRRK2; Parkinson’s disease