Anoctamin1 (ANO1), a calcium-activated chloride channel, is frequently overexpressed in several cancers, including human prostate cancer and oral squamous cell carcinomas. ANO1 plays a critical role in tumor growth and maintenance of these cancers. In this study, we have isolated two new compounds (1 and 2) and four known compounds (3–6) from Mallotus apelta. These compounds were evaluated for their inhibitory effects on ANO1 channel activity and their cytotoxic effects on PC-3 prostate cancer cells. Interestingly, compounds 1 and 2 significantly reduced both ANO1 channel activity and cell viability. Electrophysiological study revealed that compound 2 (Ani-D2) is a potent and selective ANO1 inhibitor, with an IC50 value of 2.64 μM. Ani-D2 had minimal effect on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity and intracellular calcium signaling. Notably, Ani-D2 significantly reduced ANO1 protein expression levels and cell viability in an ANO1-dependent manner in PC-3 and oral squamous cell carcinoma CAL-27 cells. In addition, Ani-D2 strongly reduced cell migration and induced activation of caspase-3 and cleavage of PARP in PC-3 and CAL-27 cells. This study revealed that a novel ANO1 inhibitor, Ani-D2, has therapeutic potential for the treatment of several cancers that overexpress ANO1, such as prostate cancer and oral squamous cell carcinoma.

Lupus nephritis (LN) is the most frequent phenotype in patients with systemic lupus erythematosus (SLE) and has a high rate of progression to end-stage renal disease, in spite of intensive treatment and maintenance therapies. Recent evidence suggests that protease-activated receptor-2 (PAR2) is a therapeutic target for glomerulonephritis. In this study, we performed a cell-based high-throughput screening and identified a novel potent PAR2 antagonist, punicalagin (PCG, a major polyphenol enriched in pomegranate), and evaluated the effects of PCG on LN. The effect of PCG on PAR2 inhibition was observed in the human podocyte cell line and its effect on LN was evaluated in NZB/W F1 mice. In the human podocyte cell line, PCG potently inhibited PAR2 (IC50 = 1.5 ± 0.03 µM) and significantly reduced the PAR2-mediated activation of ERK1/2 and NF-κB signaling pathway. In addition, PCG significantly decreased PAR2-induced increases in ICAM-1 and VCAM-1 as well as in IL-8, IFN-γ, and TNF-α expression. Notably, the intraperitoneal administration of PCG significantly alleviated kidney injury and splenomegaly and reduced proteinuria and renal ICAM-1 and VCAM-1 expression in NZB/W F1 mice. Our results suggest that PCG has beneficial effects on LN via inhibition of PAR2, and PCG is a potential therapeutic agent for LN.

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Background and Purpose Protease‐activated receptor 1 (PAR1) is a GPCR expressed in several skin cell types, including keratinocyte and dermal fibroblast. PAR1 activation plays a crucial role in the process of skin wound healing such as thrombosis, inflammation, proliferation and tissue repair. In the present study, we identified a novel positive allosteric modulator of PAR1, GB83, and investigated its effect on skin wound healing. Experimental Approach The enhancement of PAR1 activity by GB83 was measured using Fluo‐4 calcium assay. In silico docking analysis of GB83 in PAR1 was performed using dock ligands method (CDOCKER) with CHARMm force field. Effects of GB83 on cell viability and gene expression were observed using MTS assay and quantitative real‐time PCRs, respectively. SKH‐1 hairless mice were used to investigate the wound healing effect of GB83. Key Results We demonstrated that GB83 did not activate PAR1 by itself but strongly enhanced PAR1 activation by thrombin and PAR1‐activating peptide (AP). In silico docking analysis revealed that GB83 can bind to the PAR1 binding site of vorapaxar. GB83 significantly promoted PAR1‐mediated cell viability and migration. In addition, the enhancement of PAR1 activity by GB83 strongly increased gene expression of TGF‐β, fibronectin and type I collagen in vitro and promoted skin wound healing in vivo. Conclusion and Implications Our results revealed that GB83 is the first positive allosteric modulator of PAR1 and it can be a useful pharmacological tool for studying PAR1 and a potential therapeutic agent for skin wound healing.