Background/Purpose: Protein arginine deiminases (PAD) 4 is an enzyme that catalyzes the conversion of protein-embedded arginine to citrulline. It is essential for neutrophil extracellular traps (NETs) formation which is implicated in multiple immune-mediated pathological conditions. Importantly, PAD4 deficiency does not lead to increased infection of immune suppression, suggesting that PAD4 is an attractive therapeutic target for autoimmune and inflammatory diseases. However, the development of a drug-like PAD4 inhibitor has been challenging. Here, we report the discovery and characterization of a potent, selective and orally bioavailable small molecule PAD4 inhibitor.

Methods: By leveraging Computer Accelerated Rational Design (rCARD^TM) and Structure-Based Drug Discovery (SBDD) platforms, we rationally designed and synthesized a small molecule RGT-691. The biochemical activities against PAD4 and PAD2 were measured by ammonia release assays using BAEE as the substrate. The cellular activity was determined by inhibition of citrullination of histone 3 (Cit H3) in differentiated HL-60 cells stimulated with calcium ionophore (A23187). The absorption (MDCK based permeability assay and kinetic solubility), distribution (free fraction in plasma), and metabolism (liver microsomes, hepatocytes, plasma stability) were characterized in in vitro assays. The pharmacokinetic study was performed in preclinical species to evaluate the exposure and bioavailability. Proof-of-mechanism study was performed using the LPS challenged airway inflammation mouse model in which the bronchial alveolar lavage fluid (BALF) was collected to measure the concentration of Cit H3 and pro-inflammatory cytokines as efficacy readouts.

Results: The biochemical and cellular activities of RGT-691 were determined. Its IC₅₀ in PAD4 ammonia release assay is 46 + 12 nM; while its IC₅₀ against PAD2 is > 10 mM. It demonstrated a cellular IC₅₀ of 22 + 11 nM in A23187 stimulated differentiated HL-60 cells. The binding mode of RGT-691 with PAD4 was determined at 2.8 Å by crystallography, providing structural basis for the high potency and selectivity of RGT-691 for PAD4. RGT-691 demonstrated moderate permeability and good solubility. PK studies in mice showed good drug exposures via oral dosing and achieved desired efficacious exposure. In LPS induced airway inflammation model, RGT-691 potently inhibited neutrophil trafficking to lung, reduced the level of dsDNA and Cit H3. Furthermore, it suppressed the pro-inflammatory cytokines including TNF-a and IL-6 in a dose-dependent manner.

Conclusion: In summary, RGT-691 is a potent, selective, orally bioavailable small molecule PAD4 inhibitor which provides therapeutic opportunities in multiple NETs-driven autoimmune and inflammatory diseases.

« Back to ACR Convergence 2023

ACR Meeting Abstracts - https://acrabstracts.org/abstract/discovery-and-characterization-of-a-selective-orally-bioavailable-pad4-inhibitor-to-target-nets-driven-autoimmune-and-inflammatory-diseases/