The development of N-α-(2-carboxyl)benzoyl-N(5)-(2-fluoro-1-iminoethyl)-l-ornithine amide (o-F-amidine) and N-α-(2-carboxyl)benzoyl-N(5)-(2-chloro-1-iminoethyl)-l-ornithine amide (o-Cl-amidine) as second generation protein arginine deiminase (PAD) inhibitors

Abstract

Protein arginine deiminase (PAD) activity is upregulated in a number of human diseases, including rheumatoid arthritis, ulcerative colitis, and cancer. These enzymes, there are five in humans (PADs 1-4 and 6), regulate gene transcription, cellular differentiation, and the innate immune response. Building on our successful generation of F- and Cl-amidine, which irreversibly inhibit all of the PADs, a structure-activity relationship was performed to develop second generation compounds with improved potency and selectivity. Incorporation of a carboxylate ortho to the backbone amide resulted in the identification of N-α-(2-carboxyl)benzoyl-N(5)-(2-fluoro-1-iminoethyl)-l-ornithine amide (o-F-amidine) and N-α-(2-carboxyl)benzoyl-N(5)-(2-chloro-1-iminoethyl)-l-ornithine amide (o-Cl-amidine), as PAD inactivators with improved potency (up to 65-fold) and selectivity (up to 25-fold). Relative to F- and Cl-amidine, the compounds also show enhanced potency in cellulo. As such, these compounds will be versatile chemical probes of PAD function.

Figure 1

Protein arginine deiminase reaction and inhibitors. (A) The guanidinium group of an arginine residue is converted into a ureido group to form citrulline. (B) Structures of Cl- and F-amidine.

Figure 2

Structures of side chain analogs (A) and backbone analogs (B).

Figure 3

Crystal structure of PAD4•F-amidine complex. This figure was prepared with UCSF Chimera using the coordinates for the PAD4•F-amidine complex (PDBID: 2DW5).

Figure 4

Inactivation of PAD4 with o-F- and o-Cl-amidine. The residual activity of PAD4 was measured versus time with increasing concentrations of (A) o-F-amidine and (C) o-Cl-amidine. The rates (k_obs) were plotted versus concentration of (B) o-F-amidine and (D) o-Cl-amidine to obtain the inactivation parameters.

Figure 5

Overlays of the crystal structure of PAD4 with (A) F-amidine and o-F-amidine bound and (B) Cl-amidine and o-Cl-amidine bound. (C) Stereoview of the crystal structure of PAD4 bound to o-F-amidine.

Figure 6

(A) Levels of citrullinated histone H3 in HL-60 granulocytes after treatment with o-F- and o-Cl-amidine. (B) o-F- and o-Cl-amidine induce the differentiation of HL-60 cells. HL-60 cells were exposed to either o-F-, or o-Cl-amidine over the course of 48 hours with time points taken after 6, 12, 24, and 48 h.

Figure 7

Effects of o-F-amidine (A) and o-Cl-amidine (B) in combination with doxorubicin on cell viability of HL-60 cells. Cell viability was measured, using a standard MTT assay, after a 24 h incubation with the inhibitors. Each data point represents an average of three trials (p < 0.05). The combination of o-F-amidine (C) and o-Cl-amidine (D) with doxorubicin resulted in synergistic killing of HL-60 cells. Synergistic, additive, and subadditive effects of the combination therapy were determined by a comparison of the O/E ratios, where O/E < 0.8 is considered synergistic, O/E = 0.8-1.2 is additive, and an O/E > 1.2 is subadditive.

Figure 8

o-F-amidine is more potent at driving apoptosis in inflammatory cells than Cl-amidine. TK6 cells, a human lymphoblastoid cell line, were exposed to either Cl-amidine (50 μg/ml) or o-F-amidine (50 μg/ml) for indicated time periods. (A) Representative scatterplots from one of three separate experiments. (B) Average percentage apoptosis (from percentages in the 4^th quadrant) for Cl-amidine and o-F-amidine as indicated from 3 experiments. (C) PARP cleavage by Cl-amidine and o-F-amidine.