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.

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.

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.

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.

Theaflavin binds to a druggable pocket of TMEM16A channel and inhibits lung adenocarcinoma cell viability.

As a calcium-activated chloride channel regulated by the intracellular Ca2+ concentration and membrane potential, TMEM16A has attracted considerable attention and has been proposed as a novel anticancer drug target. We have previously reported that the pocket above the ion conductance pore could be a nonselective inhibitor-binding pocket. However, whether this pocket is druggable remains unexplored. In this study, we performed virtual screening to target the presumed inhibitor-binding pocket and identified a highly effective TMEM16A inhibitor, theaflavin (TF: a tea polyphenol in black tea). Molecular dynamics simulations revealed that theaflavin adopts a "wedge insertion mode" to block the ion conduction pore and induces pore closure. Moreover, the binding mode showed that the TF pedestal plays an important role in pore blockade, and R515, R535, T539, K603, E623, and E633 were determined to be most likely to interact directly with the pedestal. Mutagenesis experiment results corroborated the mechanism through which TF binds to this pocket. Combined with the quantitative calculation results, our data indicated that the three hydroxyl groups on the pedestal may be the most crucial pharmacophores for TMEM16A inhibition by TF. Finally, antitumor experiments revealed that TF could target TMEM16A to inhibit the proliferation and migration of LA795 cells, indicating the potential therapeutic effect of TF on the growth of lung adenocarcinoma with high TMEM16A expression. The successful application of drug screening strategies based on this binding pocket highlights new directions for discovering superior modulators and contributes to the development of novel therapeutics for lung adenocarcinoma.

The Crosstalk Between Neurons and Glia in Methamphetamine-Induced Neuroinflammation.

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.

Delineating an extracellular redox-sensitive module in T-type Ca2+ channels.

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 a high-affinity metal-binding site formed by the extracellular IS1-IS2 and IS3-IS4 loops of domain I and 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, 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 His191 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 MTSES and SP. We hypothesize that oxidative modification of IS1-IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

Emerging Modulators of TMEM16A and Their Therapeutic Potential.

Calcium-activated chloride channels (CaCCs) are widespread chloride channels which rely on calcium activation to perform their functions. In 2008, TMEM16A (also known as anoctamin1, ANO1) was identified as the molecular basis of the CaCCs, which provided the possibility to study the physiological function of CaCCs. TMEM16A is widely expressed in various cells and controls basic physiological functions, including neuronal and cardiac excitability, nerve transduction, smooth muscle contraction, epithelial Cl- secretion and fertilization. However, the abnormal function of TMEM16A may cause a variety of diseases, including asthma, gastrointestinal motility disorder and various cancers. Therefore, TMEM16A is a putative drug target for many diseases, and it is important to determine specific and efficient modulators of TMEM16A channel. In recent years, we and others have screened several natural modulators of TMEM16A against cancers and gastrointestinal motility dysfunction. This article reviews the screening methods, efficacy of TMEM16A modulators and pharmacological effects of TMEM16A modulators on different diseases. GRAPHIC ABSTACT.

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.

TMEM16A, a Homoharringtonine Receptor, as a Potential Endogenic Target for Lung Cancer Treatment.

Lung cancer has the highest rate of incidence and mortality among all cancers. Most chemotherapeutic drugs used to treat lung cancer cause serious side effects and are susceptible to drug resistance. Therefore, exploring novel therapeutic targets for lung cancer is important. In this study, we evaluated the potential of TMEM16A as a drug target for lung cancer. Homoharringtonine (HHT) was identified as a novel natural product inhibitor of TMEM16A. Patch-clamp experiments showed that HHT inhibited TMEM16A activity in a concentration-dependent manner. HHT significantly inhibited the proliferation and migration of lung cancer cells with high TMEM16A expression but did not affect the growth of normal lung cells in the absence of TMEM16A expression. In vivo experiments showed that HHT inhibited the growth of lung tumors in mice and did not reduce their body weight. Finally, the molecular mechanism through which HHT inhibits lung cancer was explored by western blotting. The findings showed that HHT has the potential to regulate TMEM16A activity both in vitro and in vivo and could be a new lead compound for the development of anti-lung-cancer drugs.

The Molecular Mechanism of Ginsenoside Analogs Activating TMEM16A.

The calcium-activated chloride channel TMEM16A is involved in many physiological processes, and insufficient function of TMEM16A may lead to the occurrence of various diseases. Therefore, TMEM16A activators are supposed to be potentially useful for treatment of TMEM16A downregulation-inducing diseases. However, the TMEM16A activators are relatively rare, and the underlying activation mechanism of them is unclear. In the previous work, we have proved that ginsenoside Rb1 is a TMEM16A activator. In this work, we explored the activation mechanism of ginsenoside analogs on TMEM16A through analyzing the interactions between six ginsenoside analogs and TMEM16A. We identified GRg2 and GRf can directly activate TMEM16A by screening five novel ginsenosids analogs (GRb2, GRf, GRg2, GRh2, and NGR1). Isolated guinea pig ileum assay showed both GRg2 and GRf increased the amplitude and frequency of ileum contractions. We explored the molecular mechanisms of ginsenosides activating TMEM16A by combining molecular simulation with electrophysiological experiments. We proposed a TMEM16A activation process model based on the results, in which A697 on TM7 and L746 on TM8 bind to the isobutenyl of ginsenosides through hydrophobic interaction to fix the spatial location of ginsenosides. N650 on TM6 and E705 on TM7 bind to ginsenosides through electrostatic interaction, which causes the inner half of α-helix 6 to form physical contact with ginsenosides and leads to the pore opening. It should be emphasized that TMEM16A can be activated by ginsenosides only when both the above two conditions are satisfied. This is the first, to our knowledge, report of TMEM16A opening process activated by small-molecule activators. The mechanism of ginsenosides activating TMEM16A will provide important clues for TMEM16A gating mechanism and for new TMEM16A activators screening.

Molecular Mechanisms and Structural Basis of Retigabine Analogues in Regulating KCNQ2 Channel.

KCNQ2 channel is one of the important members of potassium voltage-gated channel. KCNQ2 is closely related to neuronal excitatory diseases including epilepsy and neuropathic pain, and also acts as a drug target of the anti-epileptic drug, retigabine (RTG). In the past few decades, RTG has shown strong efficacy in the treatment of refractory epilepsy but has been withdrawn from clinical use due to its multiple adverse effects in clinical phase III trials. To overcome the drawbacks of RTG, several RTG analogues have been developed with different activation potency to KCNQ2. However, the detailed molecular mechanism by which these RTG analogues regulate KCNQ2 channel remains obscure. In this study, we used molecular simulations to analyse the interaction mode between the RTG analogues and KCNQ2, and to determine their molecular mechanism of action. Our data show that the van der Waals interactions, hydrophobic interactions, hydrogen bond, halogen bond, and π-π stacking work together to maintain the binding stability of the drugs in the binding pocket. On an atomic scale, the amide group in the carbamate and the amino group in the 2-aminophenyl moiety of RTG and RL648_81 are identified as key interaction sites. Our finding provides insight into the molecular mechanism by which KCNQ2 channels are regulated by RTG analogues. It also provides direct theoretical support for optimizing design of the KCNQ2 channel openers in the future, which will help treat refractory epilepsy caused by nerve excitability.

Molecular mechanism of CaCCinh-A01 inhibiting TMEM16A channel.

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.

Arctigenin, a novel TMEM16A inhibitor for lung adenocarcinoma therapy.

TMEM16A plays critical roles in physiological process and may serve as drug targets for diverse diseases. Recently, TMEM16A has started to be regarded as potential primary lung adenocarcinoma targets. Here, we identified that arctigenin, a natural compound, is a novel TMEM16A inhibitor, and it can suppress lung adenocarcinoma growth through inhibiting TMEM16A both in vitro and in vivo. Our data also showed that the IC50 of actigenin to TMEM16A whole-cell current was 19.29 ± 4.69 µM, and the putative binding sites of arctigenin in TMEM16A were R515 and R535. Arctigenin concentration-dependently inhibited the proliferation and migration of LA795, however, the inhibition effect can be abolished by knockdown of the endogenous TMEM16A with shRNA. Further, we injected arctigenin on xenograft mouse model which exhibited significant antitumor activity with no adverse effect. At last, western blotting results showed the mechanism of arctigenin inhibiting lung adenocarcinoma was through inhibiting MAPK pathway. In summary, TMEM16A is a novel drug target for lung adenocarcinoma treatment. Arctigenin can be used as a lead compound for the development of lung adenocarcinoma therapy drugs.

Matrine is a novel inhibitor of the TMEM16A chloride channel with antilung adenocarcinoma effects.

Calcium-activated chloride channels (CaCCs) are ion channels with key roles in physiological processes. They are abnormally expressed in various cancers, including esophageal squamous cell cancer, head and neck squamous cell carcinoma, colorectal cancer, and gastrointestinal stromal tumors. The CaCC component TMEM16A/ANO1 was recently shown to be overexpressed in lung adenocarcinoma cells and may serve as a tumorigenic protein. In this study, we determined that matrine is a potent TMEM16A inhibitor that exerts anti-lung adenocarcinoma effects. Patch clamp experiments showed that matrine inhibited TMEM16A current in a concentration-dependent manner with an IC 50 of 27.94 ± 4.78 µM. Molecular simulation and site-directed mutation experiments demonstrated that the matrine-sensitive sites of the TMEM16A channel involve the amino acids Y355, F411, and F415. Results of cell viability and wound healing assays showed that matrine significantly inhibited the proliferation and migration of LA795 cells, which exhibit high TMEM16A expression. In contrast, matrine has only weak inhibitory effect on CCD-19Lu and HeLa cells lacking TMEM16A expression. Matrine-induced effects on the proliferation and migration of LA795 cells were abrogated upon shRNA-mediated TMEM16A knockdown in LA795 cells. Finally, in vivo experiments demonstrated that matrine dramatically inhibited the growth of lung adenocarcinoma xenograft tumors in mice but did not affect mouse body weight. Collectively, these data indicate that matrine is an effective and safe TMEM16A inhibitor and that TMEM16A is the target of matrine anti-lung adenocarcinoma activity. These findings provide new insight for the development of novel treatments for lung adenocarcinoma.

Entering the spotlight: Chitosan oligosaccharides as novel activators of CaCCs/TMEM16A.

Calcium-activated chloride channels (CaCCs)/TMEM16A control diverse fundamental physiological functions, and abnormal function of TMEM16A will lead to various diseases including asthma, hypertension, gastrointestinal hypomotility and cancers. Therefore, TMEM16A as drug targets for related diseases has been increasingly concerned by researchers. In this work, COS were reported as novel natural activators of TMEM16A. It was demonstrated that COS can activate TMEM16A in a concentration dependent manner, with an EC50 of 74.5 µg/mL. Then, fluorescence experiments and inside-out patch clamp experiments were combined to confirm that COS can directly activate TMEM16A. Further, we compared the activation effects of COS monomers DP2 to DP6, with DP3 the best activator. Molecular simulation was performed to find that the binding sites between DP3 and TMEM16A are E143 and E146 in TMEM16A, and it was speculated that COS and TMEM16A may be combined by electrostatic interaction. Finally, we verified that guinea pig ileum contraction was promoted by COS and the monomers through activating TMEM16A. Collectively, COS are novel efficient natural activators of TMEM16A, with potential to be developed to treatment diseases caused by down-regulation of TMEM16A including gastrointestinal hypomotility.

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.

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.

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.

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.