Abstract 2646: Novel, isoform selective PAD4 inhibitors for the treatment of cancer

Abstract

Abstract Background: Peptidyl Arginine Deiminase 4 (PAD4/PADI4) is an enzyme that converts protein arginine or mono-methylarginine to citrulline. The PAD4-mediated hyper citrullination reaction in neutrophils causes the release of nuclear chromatin to form a chromatin network termed Neutrophil Extracellular Traps (NETs). NETs have been shown to be associated with several pathological processes including, fibrosis, ischemic stroke, preeclampsia, thrombosis, cancers, and other autoimmune diseases. In the context of cancer, tumors secrete chemokines such as CXCL1 and GCSF to recruit and prime neutrophils to form NETs, which in turn, facilitate cancer metastasis by forming Circulating Tumor Cell (CTC) clusters and potentially increasing tumor cell extravasation. Therefore, cancer cell endogenous PAD4 seems to play a significant role in metastasis, and inhibition of PAD4 could be a novel strategy to inhibit cancer progression and metastasis. Here, we describe a series of novel small molecule PAD4 inhibitors that are selective and orally bioavailable for the treatment of cancer. Methods: Potency and selectivity were evaluated in ammonia release assay using recombinant PAD enzymes. Cellular potency was evaluated by assessing the modulation of citrullination in a human neutrophil-based assay. ADME surrogates and PK studies were conducted using appropriate preclinical models. In vivo efficacy studies were conducted in syngeneic mice tumor models. Results: Multiple leads with strong in vitro potency on PAD4 (< 0.200 µM) were identified. One of the leads (JTxPAD4i) showed an IC50 of 0.102 µM against recombinant human PAD4 in the biochemical assay and a similar potency on mouse PAD4. It was selective against PAD1, PAD2, and PAD3 enzymes (IC50 > 10 µM). In the human neutrophil-based assay, JTxPAD4i inhibited H3-Histone citrullination with an IC50 of 0.125 µM. It showed an oral bioavailability of greater than 50% across species, including mouse, rat, and dog. Oral dosing with JTxPAD4i at 50 mg/kg, BID, in 4T1 mice syngeneic breast cancer model resulted in ~50% tumor growth inhibition (TGI) as compared to ~35% TGI with anti-PD1 antibody and was well tolerated. TGI was associated with strong inhibition of histone citrullination and myeloperoxidase (MPO) that are markers for NET formation. There was also a strong and significant inhibition in inflammatory cytokines, including IL-6, IL-17, and IL-1β in the tumor. Similar TGI was observed in additional breast and lung cancer syngeneic models with more than one PAD4 inhibitor from our lead series. Further studies with our lead molecules are underway to elucidate the mechanism of action of these PAD4 inhibitors. Conclusion: These studies clearly emphasize the potential of PAD4 inhibition in the treatment of cancer and our lead PAD4 inhibitors are being profiled in advanced toxicology studies to be developed as a clinical candidate. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Chandru G, Mohd Zainuddin, Naveen Sadhu, Ramachandraiah Gosu, Luca Rastelli, Amir Siddiqui, Sharad Singh, Rudresh G, Sadanand Mullurwar, Saravanan Kandan, Santosh Vishwakarma, Gurulingappa Hallur, Ravi Trivedi, Jeyaraj D A. Novel, isoform selective PAD4 inhibitors for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2646.