Human proteins are subjected to more than 200 known post-translational modifications (PTMs) (e.g., phosphorylation, glycosylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, and citrullination) and these PTMs can alter protein structure and function with consequent effects on the multitude of pathways necessary for maintaining the physiological homeostasis. When dysregulated, however, the enzymes that catalyze these PTMs can impact the genesis of countless diseases. In this review, we will focus on protein citrullination, a PTM catalyzed by the Protein Arginine Deiminase (PAD) family of enzymes. Specifically, we will describe the roles of the PADs in both normal human physiology and disease. The development of PAD inhibitors and their efficacy in a variety of autoimmune disorders and cancer will also be discussed.

The protein arginine deiminases (PADs) are known to play a crucial role in the onset and progression of multiple inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and cancer. However, it is not known how each of the five PAD isozymes contributes to disease pathogenesis. As such, potent, selective, and bioavailable PAD inhibitors will be useful chemical probes to elucidate the specific roles of each isozyme. Since D-amino amino acids often possess enhanced in cellulo stability, and perhaps unique selectivities, we synthesized a series of D-amino acid analogs of our pan-PAD inhibitor Cl-amidine, hypothesizing that this change would provide inhibitors with enhanced pharmacokinetic properties. Herein, we demonstrate that d-Cl-amidine and d-o-F-amidine are potent and highly selective inhibitors of PAD1. The pharmacokinetic properties of d-Cl-amidine were moderately improved over those of l-Cl-amidine, and this compound exhibited similar cell killing in a PAD1 expressing, triple-negative MDA-MB-231 breast cancer cell line. These inhibitors represent an important step in our efforts to develop stable, bioavailable, and highly selective inhibitors for all of the PAD isozymes.

Protein Arginine Deiminases (PADs) catalyze the post-translational conversion of peptidyl-Arginine to peptidyl-Citrulline in a calcium-dependent, irreversible reaction. Evidence is emerging that PADs play a role in carcinogenesis. To determine the cancer-associated functional implications of PADs, we designed a small molecule PAD inhibitor (called Chor-amidine or Cl-amidine), and tested the impact of this drug on the cell cycle. Data derived from experiments in colon cancer cells indicate that Cl-amidine causes a G1 arrest, and that this was p53-dependent. In a separate set of experiments, we found that Cl-amidine caused a significant increase in microRNA-16 (miRNA-16), and that this increase was also p53-dependent. Because miRNA-16 is a putative tumor suppressor miRNA, and others have found that miRNA-16 suppresses proliferation, we hypothesized that the p53-dependent G1 arrest associated with PAD inhibition was, in turn, dependent on miRNA-16 expression. Results are consistent with this hypothesis. As well, we found the G1 arrest is at least in part due to the ability of Cl-amidine-mediated expression of miRNA-16 to suppress its' G1-associated targets: cyclins D1, D2, D3, E1, and cdk6. Our study sheds light into the mechanisms by which PAD inhibition can protect against or treat colon cancer.

Ulcerative colitis (UC) is a chronic disease, in which the lining of the colon becomes inflamed and develops ulcers leading to abdominal pain, diarrhea, and rectal bleeding. The extent of these symptoms depends on disease severity. The protein arginine deiminase (PAD) family of enzymes converts peptidyl-Arginine to peptidylCitrulline through citrullination. PADs are dysregulated, with abnormal citrullination in many diseases, including UC and colorectal cancer (CRC). We have developed the small molecule, pan-PAD inhibitor, Chlor-amidine (Cl-amidine), with multiple goals, including treating UC and preventing CRC. Building off our recent results showing that: 1) Cl-amidine suppresses colitis in vivo in a dextran sulfate sodium (DSS) mouse model; and 2) Cl-amidine induces microRNA (miR)-16 in vitro causing cell cycle arrest, we tested the hypothesis that Cl-amidine can prevent tumorigenesis and that miR-16 induction, by Cl-amidine, may be involved in vivo. Consistent with our hypothesis, we present evidence that Cl-amidine, delivered in the drinking water, prevents colon tumorigenesis in our mouse model of colitis-associated CRC where mice are given carcinogenic azoxymethane (AOM), followed by multiple cycles of 2% DSS to induce colitis. To begin identifying mechanisms, we examined the effects of Cl-amidine on miR-16. Results show miR-16 suppression during the colitis-to-cancer sequence in colon epithelial cells, which was rescued by drinking Cl-amidine. Likewise, Ki67 and cellular proliferation targets of miR-16 (Cyclins D1 and E1) were suppressed by Clamidine. The decrease in cell proliferation markers and increase in tumor suppressor miRNA expression potentially define a mechanism of how Cl-amidine is suppressing tumorigenesis in vivo.

BackgroundUlcerative colitis (UC) is a chronic inflammatory bowel disease that is associated with an increased risk of colorectal cancer in 8-10 years after disease onset. Current colitis treatment strategies do not offer a cure for the disease, but only treat the symptoms with limited success and dangerous side-effects. Also, there is no preventive treatment for either UC or colorectal cancer. Quinacrine is an anti-malarial drug with versatile use in the treatment of diseases involving inflammatory response such as rheumatoid arthritis and lupus erythematosus. It also has putative anti-cancer effect. Quinacrine's anti-inflammatory, anti-oxidant properties, and anti-tumorigenic properties make it a potential small molecule preventive agent for both UC and associated colorectal cancer.ResultsThere were obvious changes in the CDI, histology, and inflammatory load in quinacrine-treated groups in a dose and time dependent manner in both models of UC, induced by chemical or haptenating agent.MethodsWe tested quinacrine at two different doses as a colitis treatment agent in two mouse models of UC - the dextran sulfate sodium and oxazolone. The clinical disease index (CDI), histological changes of the colon, levels of inflammatory markers (Cox-2, iNOS, p53) and overall health vitals were evaluated.ConclusionsWe demonstrate that quinacrine successfully suppresses colitis without any indication of toxicity or side-effects in two mouse models of UC.