title: Design and synthesis of AX7574: a microcystin-derived, fluorescent probe for serine/threonine phosphatases.
abstract: The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resulting conjugate specifically labeled the active site of protein phosphatases 1 (PP-1) with a 1:1 stoichiometry and IC50 of 4.0 nM. AX7574 was used to isolate and identify PP-1, PP-2A, PP-4, and PP-6 in Jurkat cells. Finally, AX7574 was able to record changes in the phosphatase activity levels of calyculin A treated Jurkat cells versus untreated control cells.
authors.affiliation: ActivX Biosciences, Inc., 11025 N. Torrey Pines Road; La Jolla, California 92037, USA. kevins@activx.com
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Shreder
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Liu
authors.forename: Tyzoon
authors.initials: T
authors.lastname: Nomanhboy
authors.forename: Stacy R
authors.initials: SR
authors.lastname: Fuller
authors.forename: Melissa S
authors.initials: MS
authors.lastname: Wong
authors.forename: Wen Zhi
authors.initials: WZ
authors.lastname: Gai
authors.forename: Jiangyue
authors.initials: J
authors.lastname: Wu
authors.forename: Phillip S
authors.initials: PS
authors.lastname: Leventhal
authors.forename: Jennie R
authors.initials: JR
authors.lastname: Lill
authors.forename: Sergio
authors.initials: S
authors.lastname: Corral
mesh_terms: D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D020128:Inhibitory Concentration 50; D019169:Jurkat Cells; D007700:Kinetics; D008387:Marine Toxins; D013058:Mass Spectrometry; D052998:Microcystins; D015335:Molecular Probes / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000138:chemical synthesis* / Q000737:chemistry; D015394:Molecular Structure; D010080:Oxazoles / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000138:chemical synthesis* / Q000737:chemistry*; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000378:metabolism*; D020543:Proteome / Q000187:drug effects / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: C495576:AX 7574; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008387:Marine Toxins; D052998:Microcystins; D015335:Molecular Probes; D010080:Oxazoles; D010456:Peptides, Cyclic; D020543:Proteome; C078588:microcystin; C059041:calyculin A; D010749:Phosphoprotein Phosphatases