Evaluation of alpha,beta-unsaturated ketone-based probes for papain-family cysteine proteases.
The field of activity-based proteomics makes use of small molecule active site probes to monitor distinct subsets of enzymatic proteins. While a number of reactive functional groups have been applied to activity-based probes (ABPs) that target diverse families of proteases, there remains a continual need for further evaluation of new probe scaffolds and reactive functional groups for use in ABPs. In this study we evaluate the utility of the, alpha,beta-unsaturated ketone reactive group for use in ABPs targeting the papain-family of cysteine proteases. We find that this reactive group shows highly selective labeling of cysteine cathepsins in both intact cells and total cell extracts. We observed a variable degree of background labeling that depended on the type of tag and linker used in the probe synthesis. The relative ease of synthesis of this class of compounds provides the potential for further derivatization to generate new families of cysteine protease ABPs with unique specificity and labeling properties.
Yang
Zhimou
Z
Department of Pathology, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.
Fonović
Marko
M
Verhelst
Steven H L
SH
Blum
Galia
G
Bogyo
Matthew
M
D000818:Animals; D020134:Catalytic Domain; D002401:Cathepsin B / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007202:Indicators and Reagents; D007659:Ketones / Q000138:chemical synthesis / Q000737:chemistry*; D051379:Mice; D015335:Molecular Probes / Q000737:chemistry*; D010206:Papain / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007659:Ketones; D015335:Molecular Probes; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D010206:Papain