Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay

Vosyka, O., Vinothkumar, K.R., Wolf, E.V., Brouwer, A.J., Liskamp, R.M.J. and Verhelst, S.H.L. (2013) Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay. Proceedings of the National Academy of Sciences of the United States of America, 110(7), pp. 2472-2477. (doi:10.1073/pnas.1215076110)

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Abstract

Rhomboid proteases are evolutionary conserved intramembrane serine proteases. Because of their emerging role in many important biological pathways, rhomboids are potential drug targets. Unfortunately, few chemical tools are available for their study. Here, we describe a mass spectrometry-based assay to measure rhomboid substrate cleavage and inhibition. We have identified isocoumarin inhibitors and developed activity-based probes for rhomboid proteases. The probes can distinguish between active and inactive rhomboids due to covalent, reversible binding of the active-site serine and stable modification of a histidine residue. Finally, the structure of an isocoumarin-based inhibitor with Escherichia coli rhomboid GlpG uncovers an unusual mode of binding at the active site and suggests that the interactions between the 3-substituent on the isocoumarin inhibitor and hydrophobic residues on the protease reflect S′ subsite binding. Overall, these probes represent valuable tools for rhomboid study, and the structural insights may facilitate future inhibitor design.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Liskamp, Professor Robert
Authors: Vosyka, O., Vinothkumar, K.R., Wolf, E.V., Brouwer, A.J., Liskamp, R.M.J., and Verhelst, S.H.L.
College/School:College of Science and Engineering > School of Chemistry
Journal Name:Proceedings of the National Academy of Sciences of the United States of America
Publisher:National SAcademy of Sciences
ISSN:0027-8424
ISSN (Online):1091-6490

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