Design and synthesis of AX7574: a microcystin-derived, fluorescent probe for serine/threonine phosphatases.
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.
Shreder
Kevin R
KR
ActivX Biosciences, Inc., 11025 N. Torrey Pines Road; La Jolla, California 92037, USA. kevins@activx.com
Liu
Yongsheng
Y
Nomanhboy
Tyzoon
T
Fuller
Stacy R
SR
Wong
Melissa S
MS
Gai
Wen Zhi
WZ
Wu
Jiangyue
J
Leventhal
Phillip S
PS
Lill
Jennie R
JR
Corral
Sergio
S
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
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