A fluopol-ABPP HTS assay to identify PAD inhibitors

Abstract

Protein Arginine Deiminase (PAD) activity is dysregulated in numerous diseases, e.g., Rheumatoid Arthritis. Herein we describe the development of a fluorescence polarization-Activity Based Protein Profiling (fluopol-ABPP) based high throughput screening assay that can be used to identify PAD-selective inhibitors. Using this assay, streptonigrin was identified as a potent, selective, and irreversible PAD4 inactivator.

Fig 1

(a) Structure of Rhodamine-conjugated Fluoro-amidine (RFA). (b) The fluopol-ABPP assay. The compound is either an inhibitor of the enzyme (top) or is inactive (bottom).

Fig. 2

RFA reacts with PAD4 in a time dependent manner. A strong, time-dependent increase in fluopol was observed at 300 min.

Fig. 3

Structures of ‘hits’ and gel-based secondary screen. (a) The numbers below the structure are fluoPol-ABPP primary hit number (top) and NIH validation collection compound number (bottom). n/a = not available. (b) Inhibition of RFA labeling of PAD4 by the primary ‘hits’. Lane 1 is a DMSO control (C), lane 2 is a positive control, i.e. F-amidine (FA), and lanes 3-9 are compounds NIH3-NIH9 (10 μM final).

Fig. 4

Streptonigrin is an irreversible PAD4 inactivator. (a) The preformed PAD4•streptonigrin complex was dialyzed for 20 h and the % activity remaining determined. (b) Plot of the pseudo-first order rate constants of inactivation, i.e. k_obs, versus streptonigrin concentration.

Fig. 5

Bioavailability of streptonigrin in (a) HL-60 granulocytes and (b) MCF-7 cells. Cell lines were treated with either Cl-amidine (10 μM) or streptonigrin (1, 10, or 100 nM) and then probed with anti-citrulline H3 antibody (top). The blot was stripped and re-probed with anti-H3 antibody to show equal loading of protein (bottom).