Identification of a druggable pocket of the calcium-activated chloride channel TMEM16A in its open state.

The calcium-activated chloride channel TMEM16A is a potential drug target to treat hypertension, secretory diarrhea, and several cancers. However, all reported TMEM16A structures are either closed or desensitized, and direct inhibition of the open state by drug molecules lacks a reliable structural basis. Therefore, revealing the druggable pocket of TMEM16A exposed in the open state is important for understanding protein-ligand interactions and facilitating rational drug design. Here, we reconstructed the calcium-activated open conformation of TMEM16A using an enhanced sampling algorithm and segmental modeling. Furthermore, we identified an open-state druggable pocket and screened a potent TMEM16A inhibitor, etoposide, which is a derivative of a traditional herbal monomer. Molecular simulations and site-directed mutagenesis showed that etoposide binds to the open state of TMEM16A, thereby blocking the ion conductance pore of the channel. Finally, we demonstrated that etoposide can target TMEM16A to inhibit the proliferation of prostate cancer PC-3 cells. Together, these findings provide a deep understanding of the TMEM16A open state at an atomic level and identify pockets for the design of novel inhibitors with broad applications in chloride channel biology, biophysics, and medicinal chemistry.

Expression, purification, characterization and crystallization of Panax quinquefolius ginsenoside glycosyltransferase Pq3-O-UGT2.

Pq3-O-UGT2, derived from Panax quinquefolius, functions as a ginsenoside glucosyltransferase, utilizing UDP-glucose (UDPG) as the sugar donor to catalyze the glycosylation of Rh2 and F2. An essential step in comprehending its catalytic mechanism involves structural analysis. In preparation for structural analysis, we expressed Pq3-O-UGT2 in the Escherichia coli (E. coli) strain Rosetta (DE3). The recombinant Pq3-O-UGT2 was purified through Ni-NTA affinity purification, a two-step ion exchange chromatography, and subsequently size-exclusion chromatography (SEC). Notably, the purified Pq3-O-UGT2 showed substantial activity toward Rh2 and F2, catalyzing the formation of Rg3 and Rd, respectively. This activity was discernible within a pH range of 4.0-9.0 and temperature range of 30-55 °C, with optimal conditions observed at pH 7.0-8.0 and 37 °C. The catalytic efficiency of Pq3-O-UGT2 toward Rh2 and F2 was 31.43 s-1 mΜ-1 and 169.31 s-1 mΜ-1, respectively. We further crystalized Pq3-O-UGT2 in both its apo form and co-crystalized forms with UDPG, Rh2 and F2, respectively. High-quality crystals were obtained and X-ray diffraction data was collected for all co-crystalized samples. Analysis of the diffraction data revealed that the crystal of Pq3-O-UGT2 co-crystalized with UDP-Glc belonged to space group P1, while the other two crystals belonged to space group P212121. Together, this study has laid a robust foundation for subsequent structural analysis of Pq3-O-UGT2.

Highly efficient overexpression and purification of multisubunit tethering complexes in Saccharomyces cerevisiae.

Vesicle trafficking is a fundamental cellular process that ensures proper material exchange between organelles in eukaryotic cells, and multisubunit tethering complexes (MTCs) are essential in this process. The heterohexameric homotypic fusion and protein sorting (HOPS) complex, which functions in the endolysosomal pathway, is a member of MTCs. Despite its critical role, the complex composition and low-expression level of HOPS have made its expression and purification extremely challenging. In this study, we present a highly efficient strategy for overexpressing and purifying HOPS from Saccharomyces cerevisiae. We achieved HOPS overexpression by integrating a strong promoter TEF1 before each subunit using the gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) system. The HOPS complex was subsequently purified using Staphylococcus aureus protein A (ProtA) affinity purification and size-exclusion chromatography, resulting in high purity and homogeneity. We obtained two-fold more HOPS using this method than that obtained using the commonly used GAL1 promoter-controlled HOPS overexpression. Negative staining electron microscopy analysis confirmed the correct assembly of HOPS. Notably, we also successfully purified two other MTCs, class C core vacuole/endosome tethering (CORVET) and Golgi-associated retrograde protein (GARP) using this approach. Our findings facilitate further in vitro biochemical characterization and functional studies of MTCs and provide a useful guide for the preparation of other heterogenic multisubunit complexes.

Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene PAL promotes ginsenoside Rg3 accumulation in ginseng plant chassis.

The ginsenoside Rg3 found in Panax species has extensive pharmacological properties, in particular anti-cancer effects. However, its natural yield in Panax plants is limited. Here, we report a multi-modular strategy to improve yields of Rg3 in a Panax ginseng chassis, combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene, phenylalanine ammonia lyase (PAL). We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2, a glycosyltransferase that directly catalyzes the biosynthesis of Rg3 from Rh2 . Next, we used clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg3 . Overexpression of PAL accelerated the formation of the xylem structure, significantly improving ginsenoside Rg3 accumulation (to 6.19-fold higher than in the control). We combined overexpression of the ginsenoside aglycon synthetic genes squalene epoxidase, Pq3-O-UGT2, and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rg3 accumulation. Finally, we produced ginsenoside Rg3 at a yield of 83.6 mg/L in a shake flask (7.0 mg/g dry weight, 21.12-fold higher than with wild-type cultures). The high-production system established in this study could be a potential platform to produce the ginsenoside Rg3 commercially for pharmaceutical use.

Activation of TMEM16A by natural product canthaxanthin promotes gastrointestinal contraction.

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.

Recent advances in TMEM16A: Structure, function, and disease.

TMEM16A (also known as anoctamin 1, ANO1) is the molecular basis of the calcium-activated chloride channels, with ten transmembrane segments. Recently, atomic structures of the transmembrane domains of mouse TMEM16A (mTMEM16A) were determined by single-particle electron cryomicroscopy. This gives us a solid ground to discuss the electrophysiological properties and functions of TMEM16A. TMEM16A is reported to be dually regulated by Ca2+ and voltage. In addition, the dysfunction of TMEM16A has been found to be involved in many diseases including cystic fibrosis, various cancers, hypertension, and gastrointestinal motility disorders. TMEM16A is overexpressed in many cancers, including gastrointestinal stromal tumors, gastric cancer, head and neck squamous cell carcinoma (HNSCC), colon cancer, pancreatic ductal adenocarcinoma, and esophageal cancer. Furthermore, overexpression of TMEM16A is related to the occurrence, proliferation, and migration of tumor cells. To date, several studies have shown that many natural compounds and synthetic compounds have regulatory effects on TMEM16A. These small molecule compounds might be novel drugs for the treatment of diseases caused by TMEM16A dysfunction in the future. In addition, recent studies have shown that TMEM16A plays different roles in different diseases through different signal transduction pathways. This review discusses the topology, electrophysiological properties, modulators and functions of TMEM16A in mediates nociception, gastrointestinal dysfunction, hypertension, and cancer and focuses on multiple regulatory mechanisms regarding TMEM16A.

Drug repurposing and molecular mechanisms of the antihypertensive drug candesartan as a TMEM16A channel inhibitor.

TMEM16A, a Ca2+-activated chloride channel (CaCC), and its pharmacological inhibitors can inhibit the growth of lung adenocarcinoma cells. However,the poor efficacy, safety, and stability of TMEM16A inhibitors limit the development of these agents. Therefore, finding new therapeutic directions from already marketed drugs is a feasible strategy to obtain safe and effective therapeutic drugs. Here, we screened a library contain more than 2400 FDA, EMA, and NMPA-approved drugs through virtual screening. We identified a drug candidate, candesartan (CDST), which showed strong inhibitory effect on the TMEM16A in a concentration-dependent manner with an IC50 of 24.40 ± 3.21 µM. In addition, CDST inhibited proliferation, migration and induced apoptosis of LA795 cells targeting TMEM16A, and significantly inhibited lung adenocarcinoma tumor growth in vivo. The molecular mechanism of CDST inhibiting TMEM16A channel indicated it bound to R515/R535/E623/E624 in the drug pocket, thereby blocked the pore. In conclusion, we identified a novel TMEM16A channel inhibitor, CDST, which exhibited excellent inhibitory activity against lung adenocarcinoma. Considering that CDST has been used in clinical treatment of hypertension, it may play an important role in the combined treatment of hypertension and lung adenocarcinoma as a multi-target drug in the future.

Structure-function and engineering of plant UDP-glycosyltransferase.

Natural products synthesized by plants have substantial industrial and medicinal values and are therefore attracting increasing interest in various related industries. Among the key enzyme families involved in the biosynthesis of natural products, uridine diphosphate-dependent glycosyltransferases (UGTs) play a crucial role in plants. In recent years, significant efforts have been made to elucidate the catalytic mechanisms and substrate recognition of plant UGTs and to improve them for desired functions. In this review, we presented a comprehensive overview of all currently published structures of plant UGTs, along with in-depth analyses of the corresponding catalytic and substrate recognition mechanisms. In addition, we summarized and evaluated the protein engineering strategies applied to improve the catalytic activities of plant UGTs, with a particular focus on high-throughput screening methods. The primary objective of this review is to provide readers with a comprehensive understanding of plant UGTs and to serve as a valuable reference for the latest techniques used to improve their activities.

Molecular mechanism of ion channel protein TMEM16A regulated by natural product of narirutin for lung cancer adjuvant treatment.

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.

Recent progress in structural studies on TMEM16A channel.

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.