CXCL5 activates CXCR2 in nociceptive sensory neurons to drive joint pain and inflammation in experimental gouty arthritis.

Gouty arthritis evokes joint pain and inflammation. Mechanisms driving gout pain and inflammation remain incompletely understood. Here we show that CXCL5 activates CXCR2 expressed on nociceptive sensory neurons to drive gout pain and inflammation. CXCL5 expression was increased in ankle joints of gout arthritis model mice, whereas CXCR2 showed expression in joint-innervating sensory neurons. CXCL5 activates CXCR2 expressed on nociceptive sensory neurons to trigger TRPA1 activation, resulting in hyperexcitability and pain. Neuronal CXCR2 coordinates with neutrophilic CXCR2 to contribute to CXCL5-induced neutrophil chemotaxis via triggering CGRP- and substance P-mediated vasodilation and plasma extravasation. Neuronal Cxcr2 deletion ameliorates joint pain, neutrophil infiltration and gait impairment in model mice. We confirmed CXCR2 expression in human dorsal root ganglion neurons and CXCL5 level upregulation in serum from male patients with gouty arthritis. Our study demonstrates CXCL5-neuronal CXCR2-TRPA1 axis contributes to gouty arthritis pain, neutrophil influx and inflammation that expands our knowledge of immunomodulation capability of nociceptive sensory neurons.

Computational insights into the conformational transition of STING: Mechanistic, energetic considerations, and the influence of crucial mutations.

STING (stimulator of interferon genes) is a crucial protein in the innate immune system's response to viral and bacterial infections. In this study, we investigated the mechanistic and energetic mechanism of the conformational transition process of STING activated by cGAMP binding. We found that the STING connector region undergoes an energetically unfavorable rotation during this process, which is compensated by the favorable interaction between cGAMP and the STING ligand binding domain. We further studied several disease-causing mutations and found that the V155 M mutation facilitates a smoother transition in the STING connector region. However, the V147L mutation exhibits unfavorable conformational transition energy, suggesting it may hinder STING activation pathway that relies on connector region rotation. Despite being labeled as hyperactive, the widespread prevalence of V147L/V147I mutations across species implies a neutral character, indicating complexity in its role. Overall, our analysis deepens the understanding of STING activation within the connector region, and targeting this region with compounds may provide an alternative approach to interfering with STING's function.

Liensinine, a Novel and Food-Derived Compound, Exerts Potent Antihepatoma Efficacy via Inhibiting the Kv10.1 Channel.

Plant metabolites from natural product extracts offer unique advantages against carcinogenesis in the development of drugs. The target-based virtual screening from food-derived compounds represents a promising approach for tumor therapy. In this study, we performed virtual screening to target the presumed inhibitor-binding pocket and identified a highly potent Kv10.1 inhibitor, liensinine (Lien), which can inhibit the channel in a dose-dependent way with an IC50 of 0.24 ± 0.07 µM. Combining molecular dynamics simulations with mutagenesis experiments, our data show that Lien interacts with Kv10.1 by binding with Y539, T543, D551, E553, and H601 in the C-linker domain of Kv10.1. In addition, the interaction of sequence alignment and 3D structural modeling revealed differences between the C-linker domain of the Kv10.1 channel and the Kv11.1 channel. Furthermore, antitumor experiments revealed that Lien suppresses the proliferation and migration of HCC both in vitro and in vivo. In summary, the food-derived compound, Lien, may serve as a lead compound for antihepatoma therapeutic drugs targeting Kv10.1.

Corydaline binds to a druggable pocket of hEAG1 channel and inhibits hepatic carcinoma cell viability.

Ether-à-go-go (EAG) potassium channels play a crucial role in the regulation of neuronal excitability and cancer progression, rendering them potential drug targets for cancer therapy. However, the scarcity of information regarding the selection sites on hEAG1 has posed a challenge in the discovery of new hEAG1 inhibitors. In this study, we introduced a novel natural product, corydaline, which selectively inhibits the hEAG1 channel without sensitivity to other KCNH channels. The IC50 of corydaline for the hEAG1 channel was 11.3 ± 0.6 µM, whereas the IC50 for hEAG2 and hERG1 were 73.6 ± 9.9 µM and 111.4 ± 8.5 µM, respectively. Molecular dynamics simulations together with site-directed mutagenesis, have unveiled that the site corydaline forms interactions with Lys217, Phe273, Pro276, Trp295 and Arg366, situated within the intracellular transmembrane segments S1-S4 of the voltage-sensor domain, be considered a novel drug pocket for hEAG1. Additionally, the intergaration of sequence alignment and 3D structural modeling revealed differences between the voltage sensor domain of hEAG1 channel and other EAG channels, suggesting the feasibility of a VSD modulation approach that could potentially lead to the selective inhibition of hEAG1 channels. Furthermore, antitumor experiments demonstrated that corydaline can inhibit the proliferation and migration of hepatic carcinoma cells by targeting hEAG1. The identification of this novel druggable pocket offers the possibility for drug screening against diseases linked to abnormal hEAG1 channels.

Preparation and epitope mapping of monoclonal antibodies against African swine fever virus p22 protein.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is now widespread in many countries and severely affects the commercial rearing of swine. Rapid and early diagnosis is crucial for the prevention of ASF. ASFV mature virions comprise the inner envelope protein, p22, making it an excellent candidate for the serological diagnosis and surveillance of ASF. In this study, the prokaryotic-expressed p22 recombinant protein was prepared and purified for immunization in mice. Four monoclonal antibodies (mAbs) were identified using hybridoma cell fusion, clone purification, and immunological assays. The epitopes of mAbs 14G1 and 22D8 were further defined by alanine-scanning mutagenesis. Our results showed that amino acids C39, K40, V41, D42, C45, G48, E49, and C51 directly bound to 14G1, while the key amino acid epitope for 22D8 included K161, Y162, G163, D165, H166, I167, and I168. Homologous and structural analysis revealed that these sites were highly conserved across Asian and European ASFV strains, and the amino acids identified were located on the surface of p22. Thus, our study contributes to a better understanding of the antigenicity of the ASFV p22 protein, and the results could facilitate the prevention and control of ASF.

Host serine protease ACOT2 assists DENV proliferation by hydrolyzing viral polyproteins.

Dengue fever is a mosquito-borne tropical disease caused by the dengue virus (DENV). The replication of DENV relies on the processing of its genome-encoded polyprotein by both viral protease NS3 (NS3pro) and host proteases. However, the impact of host proteases on DENV proliferation is not well understood. In this study, we utilized fluorophosphonate-based probes (FPs) to investigate the up-regulation of host serine proteases during DENV infection in detail. Among the identified proteases, acyl-CoA thioesterase 2 (ACOT2), an enzyme that hydrolyzes acyl-CoA molecules to generate fatty acids and free CoA, exhibited cleavage activity against DENV polypeptide substrates. Enzymatic assays and virological experiments confirmed that ACOT2 contributes to DENV propagation during the replication stage by cleaving the viral polyprotein. Docking models provided insights into the binding pocket of viral polypeptides and the catalytic mechanism of ACOT2. Notably, this study is the first to demonstrate that ACOT2 functions as a serine protease to hydrolyze protein substrates. These findings offer novel insights into DENV infection, host response, as well as the potential development of innovative antiviral strategies.IMPORTANCEDENV, one of the major pathogens of Dengue fever, remains a significant public health concern in tropical and subtropical regions worldwide. How DENV efficiently hijacks the host and accesses its life cycle with delicate interaction remains to be elucidated. Here, we deconvoluted that the host protease ACOT2 assists the DENV replication and characterized the ACOT2 as a serine protease involved in the hydrolysis of the DENV polypeptide substrate. Our results not only further the understanding of the DENV life cycle but also provide a possibility for the usage of activity-based proteomics to reveal host-virus interactions.

Dauricine interferes with SARS-CoV-2 variants infection by blocking the interface between RBD and ACE2.

The continued viral evolution results in the emergence of various SARS-CoV-2 variants, such as delta or omicron, that are partially resistant to current vaccines and antiviral medicines, posing an increased risk to global public health and raising the importance of continuous development of antiviral medicines. Inhibitor screening targeting the interactions between the viral spike proteins and their human receptor ACE2 represents a promising approach for drug discovery. Here, we demonstrate that the evolutionary trend of the SARS-CoV-2 variants is associated with increased electrostatic interactions between S proteins and ACE2. Virtual screening based on the ACE2-RBD binding interface identified nine monomers of Traditional Chinese medicine (TCM). Furthermore, live-virus neutralization assays revealed that Dauricine, one of the identified monomers, exhibited an antiviral activity with an IC50 range of 18.2 to 33.3 µM for original strain, Delta, and Omicron strains, respectively. The computational study showed that the polycyclic and methoxy groups of Dauricine adhere to the RBD surface through π-π and electrostatic interactions. The discovery of Dauricine is a successful attempt to target viral entry, which will not only help society to respond quickly to viral variants, but also accelerate variant drug development thereby reducing the pressure on health authorities to respond to outbreaks.

Deubiquitinating PABPC1 by USP10 upregulates CLK2 translation to promote tumor progression in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant with limited treatment options. Deubiquitinases (DUBs), which cleave ubiquitin on substrates, can regulate tumor progression and are appealing therapeutic targets, but there are few related studies in PDAC. In our study, we screened the expression levels and prognostic value of USP family members based on published databases and selected USP10 as the potential interventional target in PDAC. IHC staining of the PDAC microarray revealed that USP10 expression was an adverse clinical feature of PDAC. USP10 promoted tumor growth both in vivo and in vitro in PDAC. Co-IP experiments revealed that USP10 directly interacts with PABPC1. Deubiquitination assays revealed that USP10 decreased the K27/29-linked ubiquitination level of the RRM2 domain of PABPC1. Deubiquitinated PABPC1 was able to couple more CLK2 mRNA and eIF4G1, which increased the translation efficiency. Replacing PABPC1 with a mutant that could not be ubiquitinated impaired USP10 knock-down-mediated tumor suppression in PDAC. Targeting USP10 significantly delayed the growth of cell-derived xenograft and patient-derived xenograft tumors. Collectively, our study first identified USP10 as the DUB of PABPC1 and provided a rationale for potential therapeutic options for PDAC with high USP10 expression.

Inhibition of epithelial-to-mesenchymal transition augments antitumor efficacy of nanotherapeutics in pancreatic ductal adenocarcinoma.

Intrinsic drug resistance mechanisms of tumor cells often reduce intracellular drug concentration to suboptimal levels. Epithelial-to-mesenchymal transition (EMT) is a pivotal process in tumor progression and metastasis that confers an aggressive phenotype as well as resistance to chemotherapeutics. Therefore, it is imperative to develop novel strategies and identify new targets to improve the overall efficacy of cancer treatment. We developed SN38 (active metabolite of irinotecan)-assembled glycol chitosan nanoparticles (cSN38) for the treatment of pancreatic ductal adenocarcinoma (PDAC). Furthermore, cSN38 and the TGF-ß1 inhibitor LY364947 formed composite nanoparticles upon self-assembly (cSN38 + LY), which obviated the poor aqueous solubility of LY364947 and enhanced drug sensitivity. The therapeutic efficacy of cSN38 + LY nanotherapeutics was studied in vitro and in vivo using suitable models. The cSN38 nanoparticles exhibited an antitumor effect that was significantly attenuated by TGF-ß-induced EMT. The cellular uptake of SN38 was impeded during EMT, which affected the therapeutic efficacy. The combination of LY364947 and cSN38 markedly enhanced the cellular uptake of SN38, increased cytotoxic effects, and inhibited EMT in PDAC cells in vitro. Furthermore, cSN38 + LY significantly inhibited PDAC xenograft growth in vivo. The cSN38 + LY nanoparticles increased the therapeutic efficacy of cSN38 via repressing the EMT of PDAC cells. Our findings provide a rationale for designing nanoscale therapeutics to combat PDAC.

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.

Computational and experimental studies of salvianolic acid A targets 3C protease to inhibit enterovirus 71 infection.

Hand, foot, and mouth disease (HFMD) is a common childhood infectious disease caused by enterovirus (EV) infection. EV71 is one of the major pathogens causing hand, foot, and mouth disease and is more likely to cause exacerbation and death than other enteroviruses. Although a monovalent vaccine for EV71 has been developed, there are no clinically available anti-EV71 specific drugs. Here, we performed virtual screening and biological experiments based on the traditional Chinese medicine monomer library. We identified a traditional Chinese medicine monomer, Salvianolic acid A (SA), a polyphenolic compound isolated from Salvia miltiorrhiza. Salvianolic acid A inhibits EV71 virus infection in a concentration-dependent manner, and its antiviral activity is higher than that of other reported natural polyphenols and has a high biosafety. Furthermore, molecular dynamics simulations showed that salvianolic acid A can anchor to E71, a member of the enzyme catalytic triad, and cause H40 to move away from the catalytic center. Meanwhile, molecular mechanics generalized born surface area (MMGBSA) and steered molecular dynamics (SMD) results showed that the P1 group of SA was most easily unbound to the S1 pocket of 3Cpro, which provided theoretical support to further improve the affinity of salvianolic acid A with 3Cpro. These findings suggest that salvianolic acid A is a novel EV71 3Cpro inhibitor with excellent antiviral activity and is a promising candidate for clinical studies.

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.

Intracellular zinc protects Kv7 K+ channels from Ca2+/calmodulin-mediated inhibition.

Zinc (Zn) is an essential trace element; it serves as a cofactor for a great number of enzymes, transcription factors, receptors, and other proteins. Zinc is also an important signaling molecule, which can be released from intracellular stores into the cytosol or extracellular space, for example, during synaptic transmission. Amongst cellular effects of zinc is activation of Kv7 (KCNQ, M-type) voltage-gated potassium channels. Here, we investigated relationships between Kv7 channel inhibition by Ca2+/calmodulin (CaM) and zinc-mediated potentiation. We show that Zn2+ ionophore, zinc pyrithione (ZnPy), can prevent or reverse Ca2+/CaM-mediated inhibition of Kv7.2. In the presence of both Ca2+ and Zn2+, the Kv7.2 channels lose most of their voltage dependence and lock in an open state. In addition, we demonstrate that mutations that interfere with CaM binding to Kv7.2 and Kv7.3 reduced channel membrane abundance and activity, but these mutants retained zinc sensitivity. Moreover, the relative efficacy of ZnPy to activate these mutants was generally greater, compared with the WT channels. Finally, we show that zinc sensitivity was retained in Kv7.2 channels assembled with mutant CaM with all four EF hands disabled, suggesting that it is unlikely to be mediated by CaM. Taken together, our findings indicate that zinc is a potent Kv7 stabilizer, which may protect these channels from physiological inhibitory effects of neurotransmitters and neuromodulators, protecting neurons from overactivity.

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.

Near-Infrared Light-Responsive Nanoinhibitors for Tumor Suppression through Targeting and Regulating Anion Channels.

The gated state of anion channels is involved in the regulation of proliferation and migration of tumors. Specific regulators are urgently needed for efficacious cancer ablation. For this purpose, it is essential to understand the molecular mechanisms of interaction between the regulators and anion channels and apply this knowledge to regulate anion channels. Transmembrane 16A (TMEM16A) is the molecular basis of the calcium-activated chloride channels. It is an anion channel activated by Ca2+, and the inhibition of TMEM16A is associated with a decrease in tumorigenesis. Herein, we characterized a natural compound procyanidin (PC) as an efficacious and selective inhibitor of TMEM16A with an IC50 of 10.6 ± 0.6 µM. Our research revealed the precise sites (D383, R535, and E624) of electrostatic interactions between PC and TMEM16A. Near-infrared (NIR)-light-responsive photothermal conjugated polymer nanoparticles encapsulating PC (CPNs-PC) were established to remotely target and regulate the TMEM16A anion channel. Upon NIR irradiation, CPNs-PC downregulated the signaling pathway downstream of TMEM16A and arrested the cell cycle progression of cancer cells and improved the bioavailability of PC. The tumor inhibition ratio of CPNs-PC was superior to PC by 13.4%. Our findings enabled the development of a strategy to accurately and remotely regulate anion channels to promote tumor regression using NIR-light-responsive conjugated polymer nanoparticles containing specific inhibitors of TMEM16A.

Multi-target tracheloside and doxorubicin combined treatment of lung adenocarcinoma.

Chemotherapy is one of the main methods for malignant lung cancer treatment. However, the side effects of chemotherapy drugs are serious and it is prone to drug resistance. Therefore, multi-drug combination chemotherapy is popular in lung cancer treatment. This study found that tracheloside (TCS) was a novel inhibitor of TMEM16A which was specific high expressed in lung cancer tissues. TCS concentration dependently inhibited TMEM16A with an IC50 of 3.09 ± 0.21 µM. It inhibited lung cancer cells proliferation, migration, and induced cells apoptosis targeting TMEM16A. In addition, molecular docking combined with site-directed mutagenesis confirmed that the binding sites of TCS to TMEM16A were S387, E623, E624. Subsequently, multi-target combined drug administration was conducted based on the different drug targets of TCS and doxorubicin (DOX). Both in vitro and in vivo experiments indicated that the combined administration of low concentration of TCS and DOX achieved satisfactory anticancer effect, and it offset the side effects caused by high concentration of DOX. Therefore, TCS is a safe and efficient anticancer lead compound which can enhance the effect of DOX.

Molecular mechanism of CD44 homodimerization modulated by palmitoylation and membrane environments.

The homodimerization of CD44 plays a key role in an intercellular-to-extracellular signal transduction and tumor progression. Acylated modification and specific membrane environments have been reported to mediate translocation and oligomerization of CD44; however, the underlying molecular mechanism remains elusive. In this study, extensive molecular dynamics simulations are performed to characterize the dimerization of palmitoylated CD44 variants in different bilayer environments. CD44 forms homodimer depending on the cysteines on the juxta-membrane domains, and the dimerization efficiency and packing configurations are defected by their palmitoylated modifications. In the phase-segregated (raft included) membrane, homodimerization of the palmitoylated CD44 is hardly observed, whereas PIP2 addition compensates to realize dimerization. However, the structure of CD44 homodimer formed in the phase-segregated bilayer turns susceptive and PIP2 addition allows for an extensive conformation of the cytoplasmic domain, a proposal prerequisite to access the cytoskeleton linker proteins. The results unravel a delicate competitive relationship between PIP2, lipid raft, and palmitoylation in mediating protein homodimerization, which helps to clarify the dynamic dimer conformations and involved cellular signaling of the CD44 likewise proteins.

Anoctamin 1 controls bone resorption by coupling Cl- channel activation with RANKL-RANK signaling transduction.

Osteoclast over-activation leads to bone loss and chloride homeostasis is fundamental importance for osteoclast function. The calcium-activated chloride channel Anoctamin 1 (also known as TMEM16A) is an important chloride channel involved in many physiological processes. However, its role in osteoclast remains unresolved. Here, we identified the existence of Anoctamin 1 in osteoclast and show that its expression positively correlates with osteoclast activity. Osteoclast-specific Anoctamin 1 knockout mice exhibit increased bone mass and decreased bone resorption. Mechanistically, Anoctamin 1 deletion increases intracellular Cl- concentration, decreases H+ secretion and reduces bone resorption. Notably, Anoctamin 1 physically interacts with RANK and this interaction is dependent upon Anoctamin 1 channel activity, jointly promoting RANKL-induced downstream signaling pathways. Anoctamin 1 protein levels are substantially increased in osteoporosis patients and this closely correlates with osteoclast activity. Finally, Anoctamin 1 deletion significantly alleviates ovariectomy induced osteoporosis. These results collectively establish Anoctamin 1 as an essential regulator in osteoclast function and suggest a potential therapeutic target for osteoporosis.

Protein disulfide isomerase modulation of TRPV1 controls heat hyperalgesia in chronic pain.

Protein disulfide isomerase (PDI) plays a key role in maintaining cellular homeostasis by mediating protein folding via catalyzing disulfide bond formation, breakage, and rearrangement in the endoplasmic reticulum. Increasing evidence suggests that PDI can be a potential treatment target for several diseases. However, the function of PDI in the peripheral sensory nervous system is unclear. Here we report the expression and secretion of PDI from primary sensory neurons is upregulated in inflammatory and neuropathic pain models. Deletion of PDI in nociceptive DRG neurons results in a reduction in inflammatory and neuropathic heat hyperalgesia. We demonstrate that secreted PDI activates TRPV1 channels through oxidative modification of extracellular cysteines of the channel, indicating that PDI acts as an unconventional positive modulator of TRPV1. These findings suggest that PDI in primary sensory neurons plays an important role in development of heat hyperalgesia and can be a potential therapeutic target for chronic pain.

Zafirlukast inhibits the growth of lung adenocarcinoma via inhibiting TMEM16A channel activity.

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.