A fluoroacetamidine-based inactivator of protein arginine deiminase 4: design, synthesis, and in vitro and in vivo evaluation

Abstract

Protein arginine deiminase 4 (PAD4) is a calcium-dependent transcriptional corepressor that has been implicated in the onset and progression of rheumatoid arthritis. Herein we describe the synthesis and in vitro evaluation of a fluoroacetamidine-containing compound, N-alpha-benzoyl-N5-(2-fluoro-1-iminoethyl)-l-ornithine amide, 1, hereafter referred to as F-amidine, that is the most potent PAD4 inhibitor ever described. Additional studies described herein indicate that F-amidine can also inhibit PAD4 activity in vivo. The bioavailability of this compound suggests that F-amidine will be a powerful chemical probe of PAD4 function that can be used to dissect the roles of this enzyme in both rheumatoid arthritis and transcriptional control. The fact that inhibition is of an irreversible nature suggests that, with appropriate functionalization, F-amidine analogues will be robust activity-based protein-profiling and proteomic capture reagents.

Figure 1

Reaction catalyzed by PAD4. R = peptide backbone.

Figure 2

Structure of F-amidine and benzoyl arginine amide (BAA), a nonphysiological PAD4 substrate.

Figure 3

Potential mechanism of PAD4 inactivation by F-amidine.

Figure 4

F-Amidine is an irreversible inactivator of PAD4. (A) Plots of product formation versus time in the absence and presence of increasing concentrations of F-amidine. (B) Rapid dilution of preformed PAD4·F-amidine complexes into assay buffer containing excess substrate. (C) Concentration dependence of k_obs.

Figure 5

F-Amidine inhibits the p300GBD–GRIP1 interaction in CV-1 cells in a concentration-dependent manner. A mammalian two-hybrid assay was used to monitor interactions between GRIP1 and the p300GBD and the enhancement afforded by either wild-type or mutant PAD4. F-Amidine was added to the cell culture medium at the concentrations (in μM) indicated in the figure.