From a Designer Drug to the Discovery of Selective Cannabinoid Type 2 Receptor Agonists with Favorable Pharmacokinetic Profiles for the Treatment of Systemic Sclerosis.

Synthetic cannabinoids, as exemplified by SDB-001 (1), bind to both CB1 and CB2 receptors and exert cannabimimetic effects similar to (-)-trans-Δ9-tetrahydrocannabinol, the main psychoactive component present in the cannabis plant. As CB1 receptor ligands were found to have severe adverse psychiatric effects, increased attention was turned to exploiting the potential therapeutic value of the CB2 receptor. In our efforts to discover novel and selective CB2 receptor agonists, 1 was selected as a starting point for hit molecule identification and a class of 1H-pyrazole-3-carboxamide derivatives were thus designed, synthesized, and biologically evaluated. Systematic structure-activity relationship investigations resulted in the identification of the most promising compound 66 as a selective CB2 receptor agonist with favorable pharmacokinetic profiles. Especially, 66 treatment significantly attenuated dermal inflammation and fibrosis in a bleomycin-induced mouse model of systemic sclerosis, supporting that CB2 receptor agonists might serve as potential therapeutics for treating systemic sclerosis.

Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis.

BACKGROUND: Increasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis. PURPOSE: The purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis. METHODS: A gaussia princeps luciferase-based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and ß-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining. RESULTS: Celastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions. CONCLUSION: Taken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.

Arenobufagin, isolated from toad venom, inhibited epithelial-to-mesenchymal transition and suppressed migration and invasion of lung cancer cells via targeting IKKß/NFκB signal cascade.

ETHNOPHARMACOLOGICAL RELEVANCE: Lung cancer is the leading cause of cancer incidence and mortality worldwide. Arenobufagin (Arg), a representative natural bufadienolide compound, is one of the major bioactive components isolated from toad venom ("Chan Su"named in Chinese to treat multifarious clinical neoplasms in China). However, the underlying molecular mechanisms that Arg inhibited the metastasis of lung cancer cells remain poorly understood. MATERIALS AND METHODS: The mobility capacities of lung cancer cells treated with Arg were evaluated using wound healing assay. The anti-migratory and anti-invasive effects of Arg on lung cancer cells were investigated by transwell invasion assay and matrigel invasion assay. iTRAQ-labeled LC-MS proteomics was used to analyze the potential proteins related to metastasis in lung cancer cells treated with Arg and differentially-expressed proteins related to EMT and NFκB signaling cascade were further confirmed by Western blotting assay. The changed subcellular localization of p65 in lung cancer A549 and H1299 cells treated with Arg was detected by immunofluorescence staining. Molecular docking and molecular dynamic (MD) simulation assay were performed to verify the binding between Arg and IKKα/IKKß. siRNA knockdown was used to check whether Arg inhibited EMT of lung cancer cells via targeting NFκB signaling cascade, which was further verified by in vivo study of lung cancer cell xenograft mice model and pulmonary metastasis mice model accompanying with immunohistochemical and hematoxylin-eosin (HE) staining. RESULTS: Arg suppressed the wound closure of lung cancer cells using wound healing assay. Moreover, Arg significantly inhibited the migration and invasion of lung cancer cells by transwell invasion assay and matrigel invasion assay. 24 unique differentially-expressed proteins related to metastasis in lung cancer cells treated with Arg were identified using iTRAQ-labeled LC-MS proteomics and 14 differentially-expressed proteins related to EMT were further confirmed by Western blotting assay. Arg significantly decreased the phosphorylation of IKKß, IκBα and p65 in the cytoplasm of lung cancer cells by Western blotting assay, and remarkably reduced the release of p65 from the cytoplasm to the nucleus. Arg could be bound in the ATP binding pocket of IKKα and IKKß by molecular docking assay, and MD simulation assay further demonstrated that Arg binding to the ATP-binding pocket of IKKß was very stable in 300 ns MD simulation, compared with the binding of Arg and IKKα. IKKß/NFκB signaling cascade was also involved in the inhibitory effect of Arg on EMT of lung cancer cells by siRNA knockdown assay. The study of lung cancer cell xenograft mice model and pulmonary metastasis mice model in vivo indicated that Arg inhibited EMT and suppressed migration and invasion of lung cancer cells via downregulating IKKß/NFκB signaling cascade. CONCLUSION: In the present study, we explored the molecular mechanism of Arg prohibiting the metastasis of lung cancer cells in vitro and in vivo, which displayed Arg could target IKKß to inactive NFκB signaling cascade and further change the expression of proteins related to EMT. These results highlight the potential of toad venom as a potential chemotherapeutic agent and warrant its development as the clinical therapy for lung cancer.

Dynamic PIP2 interactions with voltage sensor elements contribute to KCNQ2 channel gating.

The S4 segment and the S4-S5 linker of voltage-gated potassium (Kv) channels are crucial for voltage sensing. Previous studies on the Shaker and Kv1.2 channels have shown that phosphatidylinositol-4,5-bisphosphate (PIP2) exerts opposing effects on Kv channels, up-regulating the current amplitude, while decreasing the voltage sensitivity. Interactions between PIP2 and the S4 segment or the S4-S5 linker in the closed state have been highlighted to explain the effects of PIP2 on voltage sensitivity. Here, we show that PIP2 preferentially interacts with the S4-S5 linker in the open-state KCNQ2 (Kv7.2) channel, whereas it contacts the S2-S3 loop in the closed state. These interactions are different from the PIP2-Shaker and PIP2-Kv1.2 interactions. Consistently, PIP2 exerts different effects on KCNQ2 relative to the Shaker and Kv1.2 channels; PIP2 up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. Disruption of the interaction of PIP2 with the S4-S5 linker by a single mutation decreases the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2-S3 loop does not alter voltage sensitivity. These results provide insight into the mechanism of PIP2 action on KCNQ channels. In the closed state, PIP2 is anchored at the S2-S3 loop; upon channel activation, PIP2 interacts with the S4-S5 linker and is involved in channel gating.