PAD4-IN-4 (compound 28) is a potent PAD4 inhibitor with an IC50 of 0.79±0.09 μM. It reshapes the neutrophil phenotype, increases the proportion of dendritic cells and M1 macrophages, reduces the number of myeloid-derived suppressor cells, and enhances the tumor immune microenvironment. PAD4-IN-4 is applicable for research in triple-negative breast cancer.

PAD2:2.97 μM, PAD4:0.79 μM

PAD4-IN-4 demonstrates potent inhibitory activity against PAD2 and PAD4, with IC50 values of 2.97±0.29 μM and 0.79±0.09 μM, respectively, exhibiting the highest selectivity towards PAD4 (3.8 μM). It significantly suppresses in vitro proliferation of TNBC cells (4T1 IC50: 2.39±0.54 μM; MDA-MB468 IC50: 2.34±0.23 μM), while showing relatively low toxicity towards normal breast cells (MCF-10A IC50: 8.39±0.60 μM). PAD4-IN-4 (0.5, 1, 2 μM; 48 h) enhances anti-metastatic activity in TNBC cells. Additionally, PAD4-IN-4 (0.5, 1, 2 μM; 48 h) is an effective inhibitor of PAD4, capable of blocking histone citrullination and the formation of neutrophil extracellular traps (NETs).

PAD4-IN-4, administered intravenously at doses of 1, 5, and 10 mg/kg every other day for a total of nine treatments, effectively inhibits lung metastasis of TNBC in a dose-dependent manner, demonstrating high anti-TNBC activity without significant toxicity in BALB/c mice bearing the orthotopic 4T1-luc xenograft model. It modulates the tumor microenvironment by altering immune cell ratios and reshaping neutrophil phenotype and function to favor an anti-tumor state.