10.1002/anie.201906334
Angewandte Chemie International Edition
COMMUNICATION
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Figure 4. (A) Dose-response (top) and limit of detection (bottom) studies of
labelling of recombinant PAD1 by 21. F, fluorograph; C, coomassie stain. (B)
Selectivity of labelling of PADs1-4 by 21 and 22. (C) Schematic representation
of labelling of PAD1 by 21 in HEK293TPAD1 cells. (D) Dose-response of
labelling of PAD1 in HEK293TPAD1 cells by 21. An immunoblot using anti-
FLAG antibody is used as a loading control. (E) Target engagement assay in
HEK293TPAD1 cells using 21 (5 µM) and increasing concentrations of 1. (F)
Selective enrichment of PAD1 in HEK293TPAD1 cells by 21. Enrichment ratio
is determined from the proportion of heavy and light formaldehyde labelling of
probe-treated and control samples, respectively.
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Experimental Section
Synthesis and characterization of inhibitors and probes (Figures S14-
S57), in vitro PAD inactivation, cell-based assays, and the labelling of
recombinant and cellular PAD1 are discussed in supporting information.
Conflict of Interest
P.R.T. founded Padlock Therapeutics and is entitled to
payments from Bristol Myers Squibb if certain milestones are
met. P.R.T. is
a
consultant for Celgene and Disarm
Therapeutics. We are filing a patent based on these results.
Keywords: Activity-based protein profiling (ABPP) • Covalent
protein modification • Halogen bonding • Histone citrullination •
Protein arginine deiminase (PAD)
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