Background/Purpose: Citrullination of proteins is catalyzed by a family of enzymes called the peptidylarginine deiminases (PADs). While the citrullinated proteins may have physiological roles in differentiation, development, cell death, and gene regulation, the pathological protein citrullination has been associated with a range of diseases including rheumatoid arthritis (RA), multiple sclerosis, alzheimer’s disease, psoriasis and cancer. Notably, the citrulinated proteins are of interest in the pathology in rheumatid arthritis and the citrullinated proteins were detected in the synovium of RA patients. One of the PAD enzymes, PAD4, was reported to be associated with RA and higher levels of PAD4 expression is reported in the synovium. PAD4 co-localizes with citrullinated proteins in the synovium. We hypothesized that inhibition of PAD4 would be beneficial in the treatment of RA and other autoimmune disorders. Our medicinal chemistry efforts have led to the identification and characterization of a series of novel and selective PAD4 inhibitors. The optimization and development of these inhibitors for a variety of autoimmune disorders is underway

Methods: A variety of approaches including the Structure Based Drug Design (SBDD) was employed to identify the hit series and to optimize the leads. The enzymatic activity of hPAD4, PAD1 and PAD2 was assessed using fluorescence based ammonia release assay. The level of citrullinated histones, which is a direct reflection of PAD4 activity, was measured in human neutrophils by ELISA. The mouse model of collagen-induced arthritis, oxazolone induced colitis and Imiquimod induced psoriasis were used to profile the lead compounds

Results: The lead compound PADi showed an IC₅₀ of 190 nM against PAD4 in the biochemical assay. It was selective against PAD1 and PAD2 enzymes. In the human neutrophil-based assay, the compound showed significant inhibition of H3-Histone citrullination with an IC₅₀ of 320 nM. PADi showed oral bioavailability of greater than 50% across species. Treatment with PADi showed significant improvement in disease index across the various animal models, with efficacies comparable to the standard of care. In Collagen Induced Arthritis and Oxazolone induced colitis models, 50 mpk (BID, P.O.) dose showed >70% reversal of clinical score, with no effect on body weight. The observed pharmacological benefit was accompanied by reduction of histone citrullination in tissue samples, indicating target engagement. In Imiquimod induced psoriasis model, application of 500 ug of compound topically showed a reduction of psoriatic index with a significant decrease in ear thickness and ear weight.

Conclusion: We have identified and optimized a few novel chemical series of PAD4 inhibitors. One of our lead compound, PADi, a selective PAD4 inhibitor, is efficacious in the disease model of arthritis, colitis by oral administration and in psoriasis by topical application. These results suggest that PAD4 inhibition could be a novel therapeutic strategy against unmet medical needs in autoimmune disorders. Further characterization and advanced pre-clinical studies of PADi is in progress.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-selective-orally-active-pad4-inhibitors-for-the-treatment-of-autoimmune-disorders/