Background/Purpose: Peptidyl arginine deaminase 4 (PAD4) catalyzes the conversion of arginine residues to citrulline, a post-translational modification that has been implicated in the auto-immune response in ACPA⁺ rheumatoid arthritis (RA). Genetic association studies identify PADI4 as a susceptibility gene for RA and studies with Padi4^-/- mice highlight a pathological role of PAD4 citrullination in inflammation and arthritis. Moreover, histone H3 has been identified as a key substrate for PAD4 in human myeloid cells, particularly granulocytes, and citrullinated histone H3 (citHH3) has recently been implicated in the process of neutrophil extracellular trap (NET) formation and pathological inflammation (1,2). Despite the strong therapeutic rationale for PAD4 inhibitors for RA, development of a potent PAD4 inhibitor (PAD4i) with desirable pharmacological properties remains challenging. Here, we describe the development of a potent and selective allosteric inhibitor PAD4i that demonstrates robust in vitro and in vivo inhibition of histone citrullination.

Methods: PAD4 inhibitors were evaluated for selectivity in a biochemical assay using a small molecule substrate (BAEE) as previously described (3). The inhibition of PAD4 activity in RA synovial fluid was tested using the ABAP assay (4). Inhibition of citrullinated histone was assessed in a) HL60 cells or primary neutrophils, stimulated with calcium ionophore, and measured by Cellomics imaging, b) monocytes stimulated with TLR ligand, c) neutrophils following NK-mediated ADCC, d) peritoneal or bronchial lavage cells following LPS challenge in mice, as measured by flow cytometry.

Results: Our PAD4i has >500-fold selectivity over PAD2 in a biochemical assay. This compound shows potent inhibition of RA synovial-fluid derived PAD4 activity. PAD4i fully suppresses citrullination of histone H3 in human primary neutrophils and HL-60 cell (EC50s < 10 nM). CitHH3 has recently been described to be increased in monocytes from RA patients and At-Risk-Individuals (ARI)(5); our PAD4i is able to potently suppress citHH3 in RA PBMC-derived monocytes. Moreover, hypercitrullination in synovial neutrophils is hypothesized to be driven by immune-mediated membranolytic mechanisms, including pore forming granules such as perforin and granzyme (6). We developed an NK-cell mediated ADCC assay which induces citrullination in target neutrophils and showed this to be robustly inhibited by PAD4i. Potent inhibition of citHH3 in cellular assays translates to dose-dependent inhibition of citHH3 in vivo in peritoneal cells during LPS-induced peritonitis and in bronchial lavage cells in an inhaled LPS model.

Conclusion: In summary, our data show that Gilead’s PAD4 inhibitor is a potent and selective molecule to inhibit citrullination in inflammatory diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-potent-inhibitor-of-pad4-suppresses-histone-citrullination-in-vitro-and-in-vivo/