Thrombin stimulates platelets via a dual receptor system of protease-activated receptors (PARs): PAR1 and PAR4. PAR1 activation induces a rapid and transient signal associated with the initiation of platelet aggregation, whereas PAR4 activation results in a prolonged signal, required for later phases, that regulates the stable formation of thrombus. In this study, we observed differential signaling pathways for thrombin-induced PAR1 and PAR4 activation in a human megakaryoblastic leukemia cell line, MEG-01. Interestingly, thrombin induced both calcium signaling and morphological changes in MEG-01 cells via the activation of PAR1 and PAR4, and these intracellular events were very similar to those observed in platelets shown in previous studies. We developed a novel image-based assay to quantitatively measure the morphological changes in living cells, and observed the underlying mechanism for PAR1- and PAR4-mediated morphological changes in MEG-01 cells. Selective inhibition of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, showed that thrombin-induced morphological changes were primarily mediated by PAR4 activation. Treatment of a set of kinase inhibitors and 2-aminoethoxydiphenyl borate (2-APB) revealed that thrombin-mediated morphological changes were primarily regulated by calcium-independent pathways and PAR4 activation-induced PI3K/Akt and RhoA/ROCK signaling pathways in MEG-01 cells. These results indicate the importance of PAR4-mediated signaling pathways in thrombin-induced morphological changes in MEG-01 cells and provide a useful in vitro cellular model for platelet research.

Chloroquine (CQ) is an antimalaria drug that has been widely used for decades. However, CQ-induced pruritus remains one of the major obstacles in CQ treatment for uncomplicated malaria. Recent studies have revealed that MrgprX1 plays an essential role in CQ-induced itch. To date, a few MrgprX1 antagonists have been discovered, but they are clinically unavailable or lack selectivity. Here, a cell-based high-throughput screening was performed to identify novel antagonists of MrgprX1, and the screening of 2543 compounds revealed two novel MrgprX1 inhibitors, berbamine and closantel. Notably, berbamine potently inhibited CQ-mediated MrgprX1 activation (IC₅₀ = 1.6 μM) but did not alter the activity of other pruritogenic GPCRs. In addition, berbamine suppressed the CQ-mediated phosphorylation of ERK1/2. Interestingly, CQ-induced pruritus was significantly reduced by berbamine in a dose-dependent manner, but berbamine had no effect on histamine-induced, protease-activated receptors 2-activating peptide-induced, and deoxycholic acid-induced itch in mice. These results suggest that berbamine is a novel, potent, and selective antagonist of MrgprX1 and may be a potential drug candidate for the development of therapeutic agents to treat CQ-induced pruritus.

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor (GPCR) activated by proteolytic cleavage of its N-terminal domain. Once activated, PAR2 is rapidly desensitized and internalized by phosphorylation and β-arrestin recruitment. Due to its irreversible activation mechanism, some agonists that rapidly desensitized PAR2 have been misconceived as antagonists, and this has impeded a better understanding of the pathophysiological role of PAR2. In the present study, we found that GB83, initially identified as a PAR2 antagonist, is a bona fide agonist of PAR2 that induces unique cellular signaling, distinct from trypsin and PAR2-activating peptide (AP). Activation of PAR2 by GB83 markedly elicited an increase in intracellular calcium levels and phosphorylation of MAPKs, but in a delayed and sustained manner compared to the rapid and transient signals induced by trypsin and PAR2-AP. Interestingly, unlike PAR2-AP, GB83 and trypsin induced sustained receptor endocytosis and PAR2 colocalization with β-arrestin. Moreover, the recovery of the localization and function of PAR2 was significantly delayed after stimulation by GB83, which may be the reason why GB83 is recognized as an antagonist of PAR2. Our results revealed that GB83 is a bona fide agonist of PAR2 that uniquely modulates PAR2-mediated cellular signaling and is a useful pharmacological tool for studying the pathophysiological role of PAR2.

Anoctamin1 (ANO1), a calcium-activated chloride channel, is overexpressed in a variety of cancer cells, including prostate cancer, and is involved in cancer cell proliferation, migration, and invasion. Inhibition of ANO1 in these cancer cells exhibits anticancer effects. In this study, we conducted a screening to identify novel ANO1 inhibitors with anticancer effects using PC-3 human prostate carcinoma cells. Screening of 2978 approved and investigational drugs revealed that hemin is a novel ANO1 inhibitor with an IC₅₀ value of 0.45 μM. Notably, hemin had no significant effect on intracellular calcium signaling and cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP (cAMP)-regulated chloride channel, and it showed a weak inhibitory effect on ANO2 at 3 μM, a concentration that completely inhibits ANO1. Interestingly, hemin also significantly decreased ANO1 protein levels and strongly inhibited the cell proliferation and migration of PC-3 cells in an ANO1-dependent manner. Furthermore, it strongly induced caspase-3 activation, PARP degradation, and apoptosis in PC-3 cells. These findings suggest that hemin possesses anticancer properties via ANO1 inhibition and could be considered for development as a novel treatment for prostate cancer.

Atopic dermatitis is a chronic inflammatory skin disorder characterized by persistent inflammation and severe pruritus. Current anti-inflammatory agents carry risks of long-term adverse effects, while antihistamines provide limited relief of pruritus. Protease-activated receptor 2 (PAR2) has emerged as a critical mediator of both inflammation and pruritus, representing a promising therapeutic target. In this study, we investigated the therapeutic potential of punicalagin (PCG), a potent PAR2 antagonist, in atopic dermatitis. PCG fully and potently inhibited trypsin-induced PAR2 activation in HaCaT cells with an IC₅₀ of 1.30 µM, exhibiting over 40-fold greater selectivity over PAR1. PCG significantly inhibited PAR2-induced phosphorylation of ERK1/2 and NF-κB in both HaCaT and human dermal fibroblast cells and reduced IL-8 secretion in HaCaT cells. In addition, PCG did not significantly affect other pruritus-related GPCRs including H1R, H4R, TGR5, 5HT2A, 5HT2B, and MRGPRX2 at 30 µM. Notably, PCG strongly blocked PAR2-AP-induced scratching in mice. In addition, PCG improved skin lesions, reduced dermatitis severity scores, and alleviated scratching behavior in a DNFB-induced atopic dermatitis model. These effects were associated with reduced epidermal thickness, decreased serum TSLP levels, and inhibition of PAR2-dependent calcium signaling in dorsal root ganglion neurons. These findings demonstrate that PCG is a selective PAR2 antagonist that effectively alleviates both inflammatory and pruritic symptoms of atopic dermatitis, suggesting its potential as a novel therapeutic agent.

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor, plays a central role in coordinating multiple phases of cutaneous wound healing, including hemostasis, cell proliferation, migration, and extracellular matrix remodeling. Despite its therapeutic potential, PAR1-selective positive allosteric modulators (PAMs) remain limited. Here, we characterized the wound healing efficacy of gestodene, a third-generation progestin previously identified as a selective PAM of PAR1. Gestodene exhibited no intrinsic agonist activity but selectively potentiated PAR1-activating peptide (PAR1-AP)-induced calcium signaling without affecting PAR2 or PAR4 responses. Consistently, gestodene induced a concentration-dependent leftward shift in the PAR1-AP dose–response curve. Notably, gestodene enhanced PAR1-dependent cell proliferation, migration, and ERK1/2 activation, effects abolished by PAR1 knockout or pharmacological inhibition with vorapaxar in human keratinocytes (HaCaT) and dermal fibroblasts (HDF). Gestodene also potentiated the expression of wound healing-associated genes, including matrix metalloproteinases (MMP-1, -2, -3, -10), fibronectin, and type I collagen (COL1A1). In a murine wound model, topical administration of gestodene accelerated wound closure, achieving complete re-epithelialization by Day 8 and significantly enhancing collagen deposition, effects reversed by vorapaxar. Collectively, these findings demonstrate that gestodene accelerates cutaneous wound healing through PAR1-selective positive allosteric modulation and supports its potential as a drug repositioning candidate for wound repair.