Substrate optimization for monitoring cathepsin C activity in live cells.
A series of peptidic fluorogenic substrates were synthesized to develop a flow cytometry assay (FACS) to monitor the proteolytic activity of cathepsin C in live cells. Of the 16 substrates tested, (NH(2)-aminobutyric-homophenylalanine)(2)-rhodamine demonstrated the best reactivity and selectivity profile in the FACS assay using the B721 human B-lymphoblastoid cell line. The resulting FACS assay was validated through correlation of the IC(50) values with a competitive radiolabeling assay against a series of small molecule inhibitors of cathepsin C.
Li
Jun
J
Genomics Institute of the Novartis Research Foundation (GNF), 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.
Petrassi
H Michael
HM
Tumanut
Christine
C
Masick
Brian T
BT
Trussell
Christopher
C
Harris
Jennifer L
JL
D020789:Cathepsin C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D020128:Inhibitory Concentration 50; D007553:Isotope Labeling; D011868:Radioisotopes / Q000737:chemistry; D012235:Rhodamines / Q000138:chemical synthesis / Q000737:chemistry*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D011868:Radioisotopes; D012235:Rhodamines; D020789:Cathepsin C