Inhibitors and inactivators of protein arginine deiminase 4: functional and structural characterization

Abstract

Protein arginine deiminase 4 (PAD4) is a transcriptional coregulator that catalyzes the calcium-dependent conversion of specific arginine residues in proteins to citrulline. Recently, we reported the synthesis and characterization of F-amidine, a potent and bioavailable irreversible inactivator of PAD4. Herein, we report our efforts to identify the steric and leaving group requirements for F-amidine-induced PAD4 inactivation, the structure of the PAD4-F-amidine x calcium complex, and in vivo studies with N-alpha-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine), a PAD4 inactivator with enhanced potency. The PAD4 inactivators described herein will be useful pharmacological probes in characterizing the incompletely defined physiological role(s) of this enzyme. In addition, they represent potential lead compounds for the treatment of rheumatoid arthritis because a growing body of evidence supports a role for PAD4 in the onset and progression of this chronic autoimmune disorder.

Figure 1

PAD4 hydrolyzes the guanidinium group of an Arg residue to form citrulline (Cit) and ammonia, which is subsequently protonated to form the ammonium ion. R = peptide backbone.

Figure 2

Structures of (halo)acetamidine-based PAD4 inhibitors and/or inactivators and potential mechanisms of inactivation. (A) Structure of BAA and the (halo)acetamidine-based inhibitors and inactivators of PAD4. (B) Two potential mechanisms of PAD4 inactivation. Mechanism 1 involves direct substitution of the halide, whereas mechanism 2 involves the formation of a tetrahedral intermediate, which first evolves into a three-membered sulfonium ring and subsequently rearranges to the thioether with the collapse of the tetrahedral intermediate. The latter mechanism is invoked to account for the poor leaving group potential of fluoride.

Figure 3

Calcium and substrate dependence of Cl-amidine-induced inactivation. (A) PAD4 was preincubated with increasing concentrations of Cl-amidine in the absence and presence of calcium and then BAEE added to initiate the IC₅₀ assay. Calcium was also added at this time to the sample preincubated in the absence of this metal ion to upregulate the activity of PAD4. (B) Substrate protection was assayed by observing the time-dependent inactivation properties of Cl-amidine (100 μM). For these studies, product formation in the presence and absence of Cl-amidine was quantified as a function of time using two different concentrations of BAEE (2 and 10 mM) as the substrate.

Figure 4

Cl-amidine is an irreversible time- and concentration-dependent inactivator of PAD4. (A) Rapid dilution of the preformed PAD4·Cl-amidine complex, and controls containing no inhibitor, into assay buffer containing excess substrate did not show any recovery of activity. (B) Dialysis of preformed PAD4–Cl-amidine complexes for 0, 3.5, and 20 h did not show any recovery of activity. (C) Plots of product formation vs time in the absence and presence of increasing concentrations of Cl-amidine. (D) Plot of k_obs vs the concentration of Cl-amidine.

Figure 5

Cl-amidine inhibits the PAD4-mediated enhancement of the p300GBD–GRIP1 interaction in CV-1 cells. (A) Schematic depiction of the mammalian two-hybrid assay used to evaluate the efficiency of the p300GBD–GRIP1 interaction. (B) CV-1 cells were transfected with a luciferase reporter construct as well as plasmids encoding the proteins depicted in this panel. The indicated concentrations of Cl-amidine were added to the cell culture medium and the mixtures incubated for 40 h. Cell extracts were then prepared, and the luciferase activity present in these extracts was quantified. The catalytically defective PAD4 C645S mutant was also transfected into this system to demonstrate that the decrease in the strength of the p300GBD–GRIP1 interaction is not a nonspecific effect.

Figure 6

Structure of the PAD4–F-amidine·calcium complex. (A) The 2F_o − F_c (blue) and F_o − F_c (red) electron density maps of the active site, with contour levels of 1.0σ and 3.0σ, respectively. PAD4 and F-amidine are shown in stick format, colored yellow and white, respectively. The green dotted line indicates the electron density for the covalent bond formed between Cys645 and F-amidine. (B) Active site structure of the PAD4–F-amidine·calcium complex. F-Amidine and PAD4 are shown in stick format (colored orange and white, respectively). Red dashed lines indicate potential hydrogen bonds.

Figure 7

Structural comparison of the PAD4·Ca²⁺·F-amidine and PAD4·Ca²⁺·BAA complexes.