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Abstract: TMEM16A mediates the calcium-activated transmembrane flow of chloride ions and a variety of physiological functions. The binding of cytoplasmic calcium ions of TMEM16A and the consequent conformational changes of it are the key issues to explore the structure-function relationship. In recent years, researchers have explored this issue through electrophysiological experiments, structure resolving, molecular dynamic simulation, and other methods. The structures of TMEM16 family members determined by cryo-Electron microscopy (cryo-EM) and X-ray crystallization provide the primary basis for the investigation of the molecular mechanism of TMEM16A. However, the binding and activation mechanism of calcium ions in TMEM16A are still unclear and controversial. This review discusses four Ca2+ sensing sites of TMEM16A and analyzes activation properties of TMEM16A by them, which will help understand the structure-function relationship of TMEM16A and throw light on the molecular design targeting the TMEM16A channel.

Cancer chemotherapy drugs are widely criticized for their serious side effects and low cure rate. Therefore, adjuvant therapy as a combination with chemotherapy administration is being accepted by many patients. However, unclear drug targets and mechanisms limit the application of adjuvant treatment. In this study, we confirmed TMEM16A is a key drug target for lung adenocarcinoma, and narirutin is an effective anti-lung adenocarcinoma natural product. Virtual screening and fluorescence experiments confirmed that narirutin inhibits the molecular target TMEM16A, which is specific high-expression in lung adenocarcinoma. Molecular dynamics simulations and electrophysiological experiments revealed the precise molecular mechanism of narirutin regulating TMEM16A. The anticancer effect of narirutin and its synergistic effect on cisplatin were explored by cell proliferation, migration, and apoptosis assays. The signaling pathways regulated by narirutin were analyzed by western blot. Tumor xenograft mice experiments demonstrated the synergistic anticancer effect of narirutin and cisplatin, and the side effects of high concentrations of cisplatin were almost eliminated. Pharmacokinetic experiments showed the biological safety of narirutin is satisfactory in vivo. Based on the significant anticancer effect and high biosafety, naringin has great potential as a functional food in the adjuvant treatment of lung cancer.

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch-clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing i) a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I, and ii) a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide (NEM), as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His-191 in Cav3.2 reduced the channel’s sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to both MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2, such that it become sensitive to ambient zinc.

The calcium-activated chloride channel, also known as TMEM16A, shows both calcium and membrane potential dependent activation. The channel is expressed broadly and contributes to a variety of physiological processes, and it is expected to be a target for the treatment of diseases such as hypertension, pain, cystic fibrosis and lung cancer. A thorough understanding of the structural characteristics of TMEM16A is important to reveal its physiological and pathological roles. Recent studies have released several Cryo-EM structures of the channel, revealed the structural basis and mechanism of the gating of the channel. This review focused on the understandings of the structural basis and molecular mechanism of the gating and permeation of TMEM16A channel, which will provide important basis for the development of drugs targeting TMEM16A.

Methamphetamine (METH), an illicit psycho-stimulant, is widely known as an addictive drug that may cause neurotoxic effects. Previous researches on METH abuse have mainly focused on neurotransmitters, such as dopamine and glutamate. However, there is growing evidence that neuroinflammation also plays an important role in the etiology and pathophysiology of brain dysfunction induced by METH abuse. This has cast a spotlight on the research of microglia and astrocyte, which are critical mediators of neuroimmune pathology in recent years. In the central nervous system (CNS) immunity, abnormalities of the microglia and astrocytes have been observed in METH abusers from both postmortem and preclinical studies. The bidirectional communication between neurons and glia is essential for the homeostasis and biological function of the CNS while activation of glia induces the release of cytokines and chemokines during pathological conditions, which will affect the neuron-glia interactions and lead to adverse behavioral consequences. However, the underlying mechanisms of interaction between neurons and glia in METH-induced neuroinflammation remain elusive. Notably, discovering and further understanding glial activity and functions, as well as the crosstalk between neurons and glia may help to explain the pathogenesis of METH abuse and behavioral changes in abusers. In this review, we will discuss the current understanding of the crosstalk between neurons and glia in METH-induced neuroinflammation. We also review the existing microglia-astrocyte interaction under METH exposure. We hope the present review will lead the way for more studies on the development of new therapeutic strategies for METH abuse in the near future.

TMEM16A, the calcium-activated chloride channel, is broadly expressed and plays pivotal roles in diverse physiological processes. To understand the structural and functional relationships of TMEM16A, it is necessary to fully clarify the structural basis of the gating of the TMEM16A channel. Herein, we performed the protein electrostatic analysis and molecular dynamics simulation on the TMEM16A in the presence and absence of Ca2+. Data showed that the separation of TM4 and TM6 causes pore expansion, and Q646 may be a key residue for the formation of π-helix in the middle segment of TM6. Moreover, E705 was found to form a group of H-bond interactions with D554/K588/K645 below the hydrophobic gate to stabilize the closed conformation of the pore in the Ca2+-free state. Interestingly, in the Ca2+ bound state, the E705 side chain swings 100o to serve as Ca2+-binding coordination and released K645. K645 is closer to the hydrophobic gate in the calcium-bound state, which facilitates the provision of electrostatic forces for chloride ions as the ions pass through the hydrophobic gate. Our findings provide the structural-based insights to understanding the mechanisms of gating of TMEM16A.

Lung cancer has the highest mortality among cancers worldwide due to its high incidence and lack of the effective cures. We have previously demonstrated that the membrane ion channel TMEM16A is a potential drug target for the treatment of lung adenocarcinoma and have identified a pocket of inhibitor binding that provides the basis for screening promising new inhibitors. However, conventional drug discovery strategies are lengthy and costly, and the unpredictable side effects lead to a high failure rate in drug development. Therefore, finding new therapeutic directions for already marketed drugs may be a feasible strategy to obtain safe and effective therapeutic drugs. Here, we screened a library of over 1400 Food and Drug Administration-approved drugs through virtual screening and activity testing. We identified a drug candidate, Zafirlukast (ZAF), clinically approved for the treatment of asthma, that could inhibit the TMEM16A channel in a concentration-dependent manner. Molecular dynamics simulations and site-directed mutagenesis experiments showed that ZAF can bind to S387/N533/R535 in the nonselective inhibitor binding pocket, thereby blocking the channel pore. Furthermore, we demonstrate ZAF can target TMEM16A channel to inhibit the proliferation and migration of lung adenocarcinoma LA795 cells. In vivo experiments showed that ZAF can significantly inhibit lung adenocarcinoma tumor growth in mice. Taken together, we identified ZAF as a novel TMEM16A channel inhibitor with excellent anticancer activity, and as such, it represents a promising candidate for future preclinical and clinical studies.

Transmembrane 16A (TMEM16A), also known as anoctamin 1, is the molecular basis of the calcium-activated chloride channels. TMEM16A is present in interstitial cells of Cajal, which are the pacemaker cells that control smooth muscle contraction. TMEM16A is implicated in gastrointestinal disorders. Activation of TMEM16A is believed to promote the gastrointestinal muscle contraction. Here, we report a highly efficient, nontoxic, and selective activator of TMEM16A, canthaxanthin (CX). The study using molecular docking and site-directed mutation revealed that CX-specific binging site in TMEM16A is K769. CX was also found to promote the contraction of smooth muscle cells in gastrointestinal tract through activation of TMEM16A channels, which provides an excellent basis for development of CX as a chemical tool and potential therapeutic for gastrointestinal dysfunction.

Recently, we and other groups revealed that aberrant expression of Kv10.1 channel, a voltage-gated potassium ion channel, contributes to a variety of tumorigenesis process. Potent and selective inhibitor of Kv10.1 is urgently needed, both as pharmacological tools for studying the physiological functions of this enigmatic channel and as potential leads for development of anti-tumor drugs. In this study, Procyanidin B1, a natural compound extracted from the grape seed, was identified as a potent, specific inhibitor, which can inhibit the Kv10.1 channel in a concentration-dependent manner (IC50 = 10.38 ± 0.87 μM), but has negligible effects on other potassium channels, including Kir2.1, HERG or KCNQ1. It was demonstrated that Procyanidin B1 directly binds to Kv10.1 channel and inhibits its currents, without increasing intracellular Ca2+. Further, three amino acids, I550, T552, and Q557 in the C-linker domain of Kv10.1 were found critical for forming the binding pocket of Procyanidin B1 with Kv10.1 channel. In addition, Procyanidin B1 inhibits migration and proliferation of liver cancer cells (HuH-7 cells, HepG2 cells) through inhibiting Kv10.1, but not in Kv10.1 negatively expressed cell lines. Next, we assayed the tumor suppressing effect of Procyanidin B1 on cell line-derived xenograft mouse model. Our data showed that 15 mg/kg Procyanidin B1 can significantly suppress the growth of the tumor (HepG2) with an inhibition rate of about 60.25%. Compared with cisplatin, Procyanidin B1 has no side effect on the normal metabolism of the mice. The present work indicated that Procyanidin B1 is a proming liver cancer anti-tumor drug, and also confirmed that Kv10.1 can serve as a potential, tumor-specific drug target.

TMEM16A is a calcium-activated chloride channel that is associate with several diseases, including pulmonary diseases, hypertension, diarrhea and cancer. The CaCCinh-A01 (A01) is widely recognized as an efficient blocker of TMEM16A and has been used as a tool drug to inhibit TMEM16A currents in the laboratory. A01 also has excellent pharmacokinetic properties and can be developed as a drug to target TMEM16A. However, the molecular mechanism how A01 inhibits TMEM16A is still elusive, which slows down its drug development process. Here, calculations identified that the binding pocket of A01 was located above the pore, and it was also discovered that the binding of A01 to TMEM16A not only blocked the pore but also led to its collapse. The interaction model analysis predicted that R515/K603/E623 were crucial residues for the binding between TMEM16A and A01, and the site-directed mutagenesis studies confirmed the above results. The binding mode and quantum chemical calculations showed that the carboxyl and the amide oxygen atom of A01 were the key interaction sites between TMEM16A and A01. Therefore, our study proposed the inhibitory mechanism of TMEM16A current by A01 and revealed how A01 inhibits TMEM16A at the molecular level. These findings will shed light on both the development of A01 as a potential drug for TMEM16A dysfunction-related disorders and drug screening targeting the pocket.

Myristyl serinate was firstly enzyme-synthesized from myristyl alcohol and l -serine in selected solvents (single and two-phase solvents). The effects of reaction system, two-phase system composition, temperature, pH, and reaction time on myristyl serinate synthesis were investigated. The results showed that two-phase solvent reaction system could significantly increase the enzymatic conversion ratio and the optimal ratio of two-phase solvent was 80:20 (dichloromethane:phosphate buffer). The optimal conditions for myristyl serinate enzymatic synthesis were substrate molar ratio of 1:1, Novozym 435 load of 5% (w/w), a solvent dosage of 20 ml, 30 °C and reaction time of 36 h. Under these conditions, 72.25% of myristyl alcohol could be converted to myristyl serinate, which was purified through column chromatography and identified by HPLC/IR/LC-MS/NMR.

Azoreductase from Pseudomonas putida WLY, which is a kind of induced and extracellular enzyme, was purified by chromatographic methods. The purified azoreductase, with a molecular weight of 28,000Da, gave a single band on SDS-PAGE. The maximal azoreductase activity was observed at pH 7.0 and 35°C. The specific activity of the purified enzyme was 8.22 x 104U mg-1. The Km value for X-3B was 39|xM and the maximal velocity (Fmax) was 12|imol of X-3B min 1 mg" ' The purified azoreductase catalyses the reductive cleavage of the azo bond of X-3B in the presence of NADH as electron donor and yields aniline. The purified azoreductase is inhibited by several metal ions, including Fe2 + and Ca2 + , and is activated by Mg2 + . The result of eliminating the plasmid of P. putida WLY showed that the azoreductase coding gene locates in the plasmid DNA.