Anoctamin1 (ANO1)/transmembrane protein 16A (TMEM16A), a calcium-activated chloride channel (CaCC), is involved in many physiological functions such as fluid secretion, smooth muscle contraction, nociception and cancer progression. To date, only a few ANO1 inhibitors have been described, and these have low potency and selectivity for ANO1. Here, we performed a high-throughput screening to identify highly potent and selective small molecule inhibitors of ANO1. Three novel ANO1 inhibitors were discovered from screening of 54,400 synthetic small molecules, and they were found to fully block ANO1 channel activity with an IC50 < 3 μM. Electrophysiological analysis revealed that the most potent inhibitor, 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9), completely inhibited ANO1 chloride current with submicromolar potency. Notably, unlike previous small-molecule ANO1 inhibitors identified to date, Ani9 displayed high selectivity for ANO1 as compared to ANO2, which shares a high amino acid homology to ANO1. In addition, Ani9 did not affect the intracellular calcium signaling and CFTR chloride channel activity. Our results suggest that Ani9 may be a useful pharmacological tool for studying ANO1 and a potential development candidate for drug therapy of cancer, hypertension, pain, diarrhea and asthma.
Anoctamin 1 (ANO1), a calcium-activated chloride channel, is highly amplified in prostate cancer, the most common form of cancer and leading causes of cancer death in men, and downregulation of ANO1 expression or its functional activity is known to inhibit cell proliferation, migration and invasion in prostate cancer cells. Here, we performed a cell-based screening for the identification of ANO1 inhibitors as potential anticancer therapeutic agents for prostate cancer. Screening of ~300 selected bioactive natural products revealed that luteolin is a novel potent inhibitor of ANO1. Electrophysiological studies indicated that luteolin potently inhibited ANO1 chloride channel activity in a dose-dependent manner with an IC50 value of 9.8 μM and luteolin did not alter intracellular calcium signaling in PC-3 prostate cancer cells. Luteolin inhibited cell proliferation and migration of PC-3 cells expressing high levels of ANO1 more potently than that of ANO1-deficient PC-3 cells. Notably, luteolin not only inhibited ANO1 channel activity, but also strongly decreased protein expression levels of ANO1. Our results suggest that downregulation of ANO1 by luteolin is a potential mechanism for the anticancer effect of luteolin.
In this work, we developed novel stimuli-responsive injectable hydrogels composed of a highly biocompatible cartilage acellularized matrix (CAM) and a water-soluble cross-linker containing a diselenide bridge by using ultrafast norbornene (Nb)-tetrazine (Tz) click chemistry. The cross-linking reaction between the Nb groups of the CAM and Tz groups of the cross-linker evolved nitrogen gas and resulted in injectable hydrogels with highly porous structures. The synthesized hydrogels demonstrated high drug loading efficiencies (up to 93%), good swelling ratios, and useful mechanical properties. The doxorubicin (DOX)-loaded hydrogels released minimal amounts of DOX in the simulated physiological medium; however, sustained release of DOX was detected under reducing conditions, revealing more than 90% DOX release after 96 h. Interestingly, the indocyanine green (ICG)-incorporated hydrogels produced reactive oxygen species upon exposure to NIR light and exhibited burst release (>50% DOX release) of DOX during the first 4 h, followed by a sustained release phase. In vitro cytocompatibility tests showed that the synthesized CAM-Nb and hydrogels are essentially nontoxic to HFF-1 fibroblast cells and human colorectal adenocarcinoma cells (HT-29), indicating their excellent bioorthogonality and biocompatibility. Furthermore, DOX-loaded and DOX + ICG-loaded hydrogels inhibited the metabolic activities of HT-29 cells after GSH or NIR exposure and induced antitumor effects similar to those of free DOX. Therefore, these biocompatible and reduction-responsive injectable hydrogels, which exhibited on-demand drug release after NIR exposure, could be promising candidates for minimally invasive local delivery of cancer therapeutics.
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