Discovery of Two Novel Scorpion Venom Peptides Activating TRPML2 to Impair ZIKV Internalization

TL;DR

Researchers discovered two scorpion venom peptides that activate a cellular channel to block Zika virus entry, offering new tools for studying cell biology and developing antiviral treatments.

Contribution

First report of animal venom-derived peptides acting as specific TRPML2 agonists with antiviral activity against ZIKV.

Findings

BmP05 and BmKK12 peptides strongly activate TRPML2 and increase Ca2+ influx.

BmP05 and BmKK12 inhibit ZIKV replication at non-cytotoxic concentrations.

Peptides MMTX and BmTX1 showed weaker or no antiviral effects.

Abstract

The endo-lysosomal channel TRPML2 regulates key processes like membrane trafficking and autophagy, which are hijacked by many RNA viruses during endocytic entry. However, the development of TRPML2-targeted therapeutics has been hindered by a notable lack of high-affinity and selective peptide-based activators. Scorpion venom peptides, honed by evolution for exceptional specificity toward diverse membrane ion channels, represent a promising, underexplored natural library for discovering novel pharmacological probes and drug leads. Here, we screened and identified seven candidate peptides interacting with TRPML2 using co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the Mesobuthus martensii venom. Based on molecular docking analysis, the top four candidates—MMTX, BmP05, BmTX1, and BmKK12—were selected for chemical synthesis, oxidatively cyclized to form their native disulfide-bridged conformations, and subsequently purified and characterized by analytical HPLC and MS. Calcium imaging confirmed that two of the four oxidized peptides, BmP05 and BmKK12, exhibited superior potency in inducing a sharp increase in Ca2+ influx. Crucially, BmP05 and BmKK12 demonstrated potent, concentration-dependent inhibition of Zika virus (ZIKV) replication at the RNA level at non-cytotoxic concentrations, whereas the weaker activators MMTX and BmTX1 did not. The current study first reports animal venom-derived peptides that function as specific TRPML2 agonists with concomitant antiviral activity. Together, our findings provide not only new molecular probes for dissecting TRPML2 biology but also a pioneering strategy for developing host-directed, broad-spectrum therapeutics against viruses dependent on endo-lysosomal entry.

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1. Introduction

The transient receptor potential mucolipin (TRPML) channels, including TRPML1, TRPML2, and TRPML3, are non-selective cation channels primarily localized to endosomal and lysosomal membranes. They are pivotal regulators of vesicular trafficking, membrane fusion, pH homeostasis, autophagy and calcium signaling within the endo-lysosomal system [1,2,3,4]. Among them, TRPML2 has garnered increased attention due to its unique physiological roles. It is recognized as an interferon-inducible factor and is upregulated by Toll-like receptor (TLR) activators, positioning it as a key player in innate immune responses [5,6,7]. Functionally, TRPML2 critically regulates vesicular dynamics, including through its interaction with Rab4-positive recycling endosomes to coordinate intracellular trafficking [8]. Notably, this channel has a dual role in viral infections: it enhances the infectivity of enveloped viruses like dengue virus (DENV), influenza A virus, Zika virus (ZIKV), and yellow fever virus by promoting their trafficking from early to late endosomes, yet its pharmacological activation can paradoxically exert broad-spectrum antiviral effects [9,10,11,12]. Therefore, targeting the TRPML2-mediated endocytic pathway presents a promising strategy for broad-spectrum antiviral drug discovery, as it disrupts a critical cellular process shared by many viruses.

Structural biology breakthroughs have provided atomic-level insights into ’the gating mechanism of TRPML2. Several studies have elucidated that the opening of the TRPML channel pore is governed by conformational changes within its core region formed by the S5 and S6 transmembrane helices [13,14,15]. Key aromatic residues, such as Y423 and F497, directly mediate agonist-induced dilation of the pore’s lower gate, a mechanism allosterically linked to the conserved S4–S5 linker. Recently, Cryo-Electron Microscopy (Cryo-EM) studies reveal that channel activation involves a unique π-helix-to-α-helix transition in the S6 transmembrane segment, which is induced by selective agonists like ML2-SA1 [16]. Furthermore, the lysosomal lipid PI(3,5)P2 can allosterically modulate this process by binding to residues such as Arg310, indicating a complex lipid-dependent regulation of channel opening. This detailed structural understanding underscores the potential for developing highly specific channel modulators. While small-molecule agonists (e.g., ML2-SA1, ML-SA1) have demonstrated antiviral efficacy, the quest for next-generation activators, particularly peptide-based ones, is driven by the need for superior subtype selectivity and reduced off-target effects. Given their potential for high-specificity binding, peptide activators of TRPML2 could enable precise channel modulation, offering a novel therapeutic approach for viral and immune-related diseases.

As one class of ancient arachnids with over 400 million years of evolution, scorpions have developed a complex arsenal of venoms for defense and predation [17,18,19]. Their venoms are rich in peptides (typically 20–90 amino acids) characterized by high potency and exquisite specificity for various ion channels and receptors on cell membranes, through which they exert potent neurotoxic effects [20,21,22]. Advancements in transcriptomics and other omics technologies have significantly accelerated the identification of these peptides, overcoming historical challenges of low abundance and purification difficulties [23,24,25]. Consequently, scorpion venom is now recognized as a vast library of pharmacologically active compounds with demonstrated therapeutic potential, particularly in antimicrobial applications [26,27,28,29]. Beyond this, massive scorpion venom peptides have also emerged as an invaluable resource for discovering novel ion channel modulators, targeting voltage-gated sodium (Na_v_), potassium (K_v_), chloride (Cl^−^) and calcium (Ca^2+^) channels, which underscore the capacity of them to serve as indispensable tools for probing channel structure–function relationships and as leads for drug discovery [30,31,32,33].

Notably, recent research underscores their capability to interact with transient receptor potential (TRP) channels. For instance, the peptide BmP01, synergizing with the venom’s weakly acidic environment, potently activates the TRPV1 channel to induce pain, revealing a sophisticated “molecular combination punch” mechanism [34]. WaTx, a selective cell-penetrating scorpion toxin, targets the irritant receptor, TRPA1, via a biochemical mechanism divergent from that of classical irritants [35]. Furthermore, we have systematically summarized that a growing group of scorpion toxins (TRPTx) targeting TRP channels highlights their importance as molecular probes and therapeutic leads for related diseases [36]. Therefore, systematic screening of the vast untapped library of scorpion venom peptides might address the lack of specific TRPML2 tools, advancing our understanding of its role in endo-lysosomal biology and viral pathogenesis.

In this study, using co-immunoprecipitation coupled with liquid chromatography-mass spectrometry (LC-MS/MS), we first identified seven scorpion venom peptides from Mesobuthus martensii that interact with TRPML2. Following molecular docking, four top candidates—MMTX, BmP05, BmTX1, and BmKK12—were synthesized, cyclized, and verified by HPLC/MS. Calcium imaging confirmed that two of the four oxidized peptides, BmP05 and BmKK12 exhibited superior potency in inducing a sharp increase in Ca^2+^ influx. Meanwhile, both BmP05 and BmKK12 potently inhibited ZIKV replication in a concentration-dependent manner, surpassing MMTX and BmTX1. This work reports the first animal venom-derived peptides that act as specific TRPML2 agonists and exhibit anti-ZIKV activity, providing valuable probes for studying endo-lysosomal biology.

2. Results

2.1. Screening of TRPML2-Interacting Peptides from the Venom of Scorpion Mesobuthus martensii

To identify potential peptide modulators of the TRPML2 channel—a key regulator in viral endocytosis and a potential target for broad-spectrum antiviral therapy—we performed affinity-based screening [37]. Lysates from HEK293T cells stably expressing FLAG-tagged human TRPML2 were incubated with the venom of the scorpion Mesobuthus martensii, followed by co-immunoprecipitation (co-IP) using an anti-FLAG resin (Figure 1A). Analysis of the eluates by SDS-PAGE and subsequent LC-MS/MS identified nineteen venom-derived peptides that potentially interacted with TRPML2 (Figure 1B,C). Based on sequence homology and structural characteristics, these peptides include known types such as sodium channel toxins and potassium channel toxins. The peptides exhibited a broad length distribution: twelve contained over 40 residues, with the longest being 66 amino acids (e.g., BmKAS); the remaining seven were shorter chains, the shortest comprising only 29 residues (e.g., MMTX). Hence, this collection of structurally diverse peptides provides a valuable molecular resource for further investigation into their specific interactions with TRPML2.

2.2. Seven Candidate Scorpion Venom Peptides Targeting TRPML2 Was Verified Through Molecular Docking

To prioritize candidates with the highest potential for channel modulation, we referenced established structural trends in TRP-targeting scorpion toxins. Active peptides, such as BmP01 (TRPV1) and WaTX (TRPA1), typically form compact, disulfide-stabilized cores of 25–35 amino acids—a size compatible with precise interaction at channel pores or regulatory domains [34,35]. Based on this structural feature, we selected 7 peptides with lengths between 29 and 37 amino acids as the core candidate molecules for subsequent interaction verification and functional studies (Table 1). Bioinformatic analysis revealed that these candidate peptides contain three intramolecular disulfide bonds, a structural hallmark of scorpion toxic peptides that confers high stability and is often essential for functional interactions with ion channels.

To computationally evaluate the potential binding modes and affinities of these candidate peptides, we performed molecular docking simulations. The TRPML2 structure was docked against structure models of seven peptides predicted by AlphaFold3 using HDock. The docking scores and confidence scores confirmed interaction potential for all peptides (Figure 2A). Among them, four peptides, MMTX, BmP05, BmTX1 and BmKK12, consistently showed the most favorable binding profiles, suggesting a strong potential for stable interaction with the TRPML2 channel. At the same time, the three-dimensional structures of all seven peptides are shown in Figure 2B–H. Consistent with the canonical scaffold of scorpion toxins, each predicted model exhibits a compact fold comprising both α-helical and β-sheet secondary structure elements, a feature known to support stable architecture and diverse interaction surfaces. Based on this integrated experimental and computational screening, four peptides were selected for further functional characterization of their regulatory effects on TRPML2 activity.

2.3. Structural Basis of Four Candidate Scorpion Venom Peptides in Complex with TRPML2

To elucidate the potential binding mechanisms underlying the functional modulation of TRPML2 by these top candidate peptides, we performed an in-depth structural analysis of the predicted peptide-channel complexes. Non-covalent interactions were analyzed using Discovery Studio Visualizer 2024, and all structural visualizations were prepared using PyMOL 3.0. High-resolution docking models revealed that each peptide engages a distinct yet partially overlapping surface pocket on the extracellular side of TRPML2, primarily composed of loops and outer transmembrane helices. As shown in Figure 3A, MMTX engaged a broader surface via three hydrogen bonds with residues Met410, Thr409, and Ser413. In contrast, BmP05 adopts a compact orientation, forming a key hydrogen bond between its Lys6 residue and Tyr403 of TRPML2, which may serve as a stable and specific anchor point, potentially crucial for initial recognition (Figure 3B). Moreover, BmTX1 exhibited the most extensive polar network, forming three hydrogen bonds involving residues Met426, Asn461, and Glu454 (Figure 3C). Importantly, BmKK12 adopted a unique binding topology, positioning its N-terminal region near the channel vestibule through hydrogen bonds from Tyr11 and Lys12 to Gly345 and Gln411 of TRPML2 (Figure 2D). This strategic spatial arrangement implies a potential mechanism for directly influencing the channel’s gating. Collectively, these docking results not only validate the specific interactions suggested by our initial screening but also reveal a spectrum of binding modalities, supporting their selection for subsequent functional validation.

2.4. Chemical Synthesis and HPLC Analysis of the Four Candidate Scorpion Venom Peptides

The four top-ranking candidate scorpion venom peptides—MMTX, BmP05, BmTX1, and BmKK12—were chemically synthesized in their linear forms. To assess the purity of the synthetic products and establish a baseline for subsequent oxidative folding, each peptide was analyzed by analytical reversed-phase high-performance liquid chromatography (RP-HPLC). The HPLC analysis revealed that all four peptides eluted as predominantly single, well-defined peaks, indicating high synthetic purity suitable for downstream experiments (Figure 4A–D). The observed retention times were 18.1 min for MMTX, 22.3 min for BmP05, 19.6 min for BmTX1, and 19.8 min for BmKK12. The elution order generally correlated with the calculated hydrophobicity of the peptides. Notably, BmP05, with a hydrophobic ratio of 45.2%, exhibited the longest retention time, consistent with its strong interaction with the hydrophobic C18 stationary phase. In summary, we successfully obtained high-purity target peptides, and these baseline retention times serve as essential references for monitoring the conformational shift during oxidative folding.

2.5. Verification of Oxidative Folding for Four Candidate Peptides by HPLC and MS Analysis

The correct formation of disulfide bonds is essential for maintaining the structural integrity and biological activity of scorpion venom peptides [38,39]. Hence, the four synthetic peptides were oxidatively refolded to form their native, disulfide-bonded conformations, and then analyzed by HPLC. Compared to the single, well-defined peaks of the reduced forms, the chromatograms of the oxidized mixtures displayed increased complexity, with the appearance of multiple minor peaks (Figure 5A–D). Additionally, the dominant peak in each oxidized mixture exhibited a significant and consistent shift to an earlier retention time: 14.5 min for MMTX, 16.3 min for BmP05, 16.4 min for BmTX1, and 16.5 min for BmKK12. To unequivocally confirm the identity of these early-eluting dominant species, the corresponding fractions were collected and subjected to mass spectrometric analysis. The measured molecular weights for the main products were 3338.37 Da for MMTX, 3372.66 Da for BmP05, 4057.8 Da for BmTX1, and 3676.5 Da for BmKK12. Each mass was precisely 6 Da less than that of its fully reduced counterpart, consistent with the loss of six hydrogen atoms upon the formation of three disulfide bonds. Starting from 10 mg of each linear peptide, the final yield of purified, lyophilized, correctly folded peptide was approximately 3 mg, corresponding to a recovery of 30%, which is in line with typical refolding efficiencies reported for cysteine-rich scorpion toxins under air oxidation conditions. Collectively, these results confirm successful oxidative folding of all four candidate peptides, enabling subsequent functional studies.

2.6. Activation of TRPML2 by the Oxidized Scorpion Venom Peptides Using Calcium Imaging

TRPML2 is a calcium-permeable cation channel, and its activation leads to a rise in intracellular Ca^2+^ [8,40]. While direct electrophysiological recording is the gold standard for measuring channel activity, it poses technical challenges for high-throughput screening in live cells. Here, we employed ratiometric calcium imaging with Fura-2 AM as a sensitive and reliable alternative to monitor real-time TRPML2-mediated Ca^2+^ dynamics upon agonist stimulation. To assess activity, TRPML2-expressing HEK293 cells were treated with 10 µM of each oxidized peptide (MMTX, BmP05, BmTX1, or BmKK12), and the subsequent Ca^2+^ influx was monitored as an increase in the F340/F380 ratio, with the established TRPML2 agonist ML-SA1 (20 µM) as a positive control. Importantly, BmP05 and BmKK12 induced more robust and sustained Ca^2+^ responses compared to MMTX and BmTX1, with BmKK12 being the most efficacious activator (Figure 6A). Corresponding pseudocolor images visually confirmed this activation, showing a marked decrease in Fura-2 fluorescence upon peptide perfusion (yellow arrows), consistent with Ca^2+^ influx (Figure 6B). These results demonstrate that the refolded scorpion venom peptides function as TRPML2 agonists, with BmKK12 and BmP05 exhibiting the highest potency, aligning with their predicted favorable binding modes from molecular docking studies.

2.7. Differential Cytotoxicity of Four Oxidized Scorpion Venom Peptides Toward Antiviral Application in A549 Cells

ZIKV has caused significant outbreaks in tropical and subtropical regions, posing a serious public health threat due to its association with microcephaly in newborns and Guillain-Barré syndrome in adults [41,42]. Previous work has identified the TRPML2 channel agonist ML-SA1 as an effective inhibitor of ZIKV entry, highlighting TRPML2 as a promising host-directed antiviral target [10,43]. To identify safe and effective new TRPML2 activators, we first evaluated the cytotoxicity of the four oxidized peptides in A549 cells using the CCK-8 assay. The results revealed distinct cytotoxicity profiles for the four peptides. MMTX and BmKK12 showed minimal toxicity at concentrations up to 50 µM but induced a measurable decrease in viability at 100 µM (Figure 7A,D). In contrast, BmP05 was the most cytotoxic, eliciting a significant reduction in cell viability starting at 50 µM (Figure 7B). Meanwhile, BmTX1 exhibited the lowest toxicity, with no significant reduction in cell viability observed even at a concentration of 100 µM (Figure 7C). All together, these cytotoxicity profiles establish a critical safety window for each peptide in subsequent antiviral assays, ensuring that any observed viral inhibition is attributable to specific functional modulation rather than general cellular toxicity.

2.8. Assessment of Anti-ZIKV Activity for the Targeting TRPML2 Peptides In Vitro

To further test and directly link channel activation to antiviral function, we evaluated the four oxidized peptides for their ability to suppress ZIKV replication in A549 cells at their predefined non-toxic concentrations (0–10 µM). Consistent with their lower efficacy in calcium imaging assays, MMTX and BmTX1 did not exhibit significant, concentration-dependent inhibition of ZIKV replication at non-toxic concentrations up to 10 µM by both qRT-PCR and Western blotting (Figure 8A,C). In stark contrast, BmP05 and BmKK12, which were the most potent TRPML2 activators, demonstrating a clear, dose-dependent suppression of ZIKV at both viral RNA levels and E protein expression (Figure 8B,D). Obviously, BmKK12 emerged as the most effective antiviral agent, inhibiting viral RNA by 76.4% at 10 µM, compared to 50.7% for BmP05. This result establishes a positive correlation between TRPML2 activation potency and anti-ZIKV efficacy. Collectively, BmKK12 and BmP05 are identified as dual-function probes that both activate TRPML2 and inhibit ZIKV, offering new leads for studying channel biology and developing host-directed antiviral strategies.

3. Discussion

The TRPML channel family, particularly TRPML2, plays a critical role in regulating endo-lysosomal membrane trafficking and fusion—processes that are hijacked by many viruses, including ZIKV, for cellular entry and infection [44,45]. Despite its physiological importance, the pool of known pharmacological tools, especially specific agonists for TRPML2, remains limited, hindering mechanistic studies and therapeutic exploration. Scorpion venom peptides, with their innate propensity to evolve high-affinity and specific interactions with ion channels, represent a rich source of molecular probes and potential therapeutic leads [36,46,47]. This study identifies, for the first time, scorpion venom-derived peptides as potent and specific agonists of the TRPML2 channel, which subsequently confer host-directed inhibition against ZIKV infection. Our integrated strategy—spanning computational screening, chemical synthesis, and multidimensional functional assays—has not only yielded novel molecular probes for TRPML2 biology but also validated this endo-lysosomal channel as a druggable host target for antiviral intervention.

First, we screened the Mesobuthus martensii venom library against the channel using a co-immunoprecipitation-based affinity approach and identified seven candidate peptides as putative TRPML2-interacting venom components following bioinformatic analysis. Consistent with established features of scorpion venom peptides, these candidates, comprising 29–37 amino acids, adopt the canonical toxin scaffold stabilized by three intramolecular disulfide bonds—a structural motif critical for maintaining the bioactive conformation and mediating interactions with ion channels [34,35,48]. The functional hierarchy observed among the four candidate peptides—where BmKK12 and BmP05 emerged as superior TRPML2 activators and antiviral agents compared to MMTX and BmTX1—can be interpreted through a structural lens informed by recent mechanistic insights into the channel. The cryo-EM structure of TRPML2 reveals that its activation involves specific conformational changes in the extracellular pore region, a likely binding site for our peptides [13,16]. Our docking analyses suggest that BmP05 and BmKK12 achieve more effective engagement through a combination of favorable electrostatic complementarity (aligning basic residues like Lys12 in BmKK12 with the channel’s vestibule) and optimal presentation of hydrophobic patches, promoting stable agonist binding. In contrast, MMTX and particularly BmTX1—despite a high net positive charge—may exhibit less productive interactions due to suboptimal charge distribution or conformational flexibility that hinders precise docking. This structure–activity relationship not only explains the experimental results but also delineates a clear path for rational optimization: fine-tuning key cationic residues (e.g., in BmKK12) to enhance affinity, engineering the hydrophobic core of BmP05 to mitigate off-target cytotoxicity, and utilizing grafting strategies to transfer active motifs onto inactive scaffolds.

The strong positive correlation between TRPML2 activation and anti-ZIKV efficacy—where the potent agonists BmP05 and BmKK12 (50.7% and 76.4% inhibition at 10 µM, respectively) significantly outperformed the weak activators MMTX/BmTX1—strongly supports a host-directed mechanism. This aligns with established findings that TRPML2 activation, exemplified by the small-molecule agonist ML-SA1, impairs ZIKV replication by promoting endo-lysosomal acidification and degradative function [10]. Our results suggest that BmP05 and BmKK12 likely operate through a similar pathway mechanism, compromising endo-lysosomal trafficking and autophagy or perturbing the intracellular cholesterol distribution [12,43]. In addition, the antiviral potency of these two novel peptides at 10 µM surpasses that of some reported small-molecule TRPML2 agonists (e.g., ML-SA1), highlighting the efficacy of peptide-based activators. More importantly, the established maturity of peptide medicinal chemistry, encompassing precise residue substitution, backbone cyclization, and non-natural amino acid incorporation, offers a distinct advantage for rational optimization [49,50]. This allows for the systematic enhancement of key drug-like properties, including binding affinity, target specificity, and metabolic stability. Consequently, the lead peptides identified here, particularly BmKK12, present a highly tractable starting point for engineering next-generation TRPML2 agonists with superior activity and specificity, thereby accelerating their development toward viable host-directed antiviral therapeutics.

4. Conclusions

In this study, we identify BmP05 and BmKK12 as novel peptide agonists of the TRPML2 channel with potent anti-ZIKV activity, thereby expanding the pharmacological landscape of scorpion venoms and providing compelling proof-of-concept for TRPML2 as a viable host target against endocytosed viruses. While our multi-assay data strongly implicate TRPML2 as the primary target, future work employing genetic knockout models is warranted to definitively establish the channel as the essential mediator. Crucially, it remains to be determined whether the antiviral effect is specifically mediated through the TRPML2-dependent regulation of endo-lysosomal membrane trafficking and fusion—a key function of this channel that could critically disrupt viral entry. Further mechanistic elucidation, including electrophysiological characterization and binding site mapping, will be essential to translate these promising molecular probes into potential host-directed antiviral therapeutics.

5. Materials and Methods

5.1. Cells, Viruses and Peptides

The human alveolar epithelial cell line A549 (Cat#, GDC0063) and the human embryonic kidney HEK293T cell line (Cat#, GDC0187) were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China). Both cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco, Waltham, MA, USA). All cells were cultured at 37 °C in a humidified incubator with a 5% CO_2_ atmosphere and routinely passaged to maintain logarithmic growth phase. The full-length infectious cDNA clone of ZIKV Puerto Rico strain (PRVABC59) was kindly provided by Dr. Ren Sun and Dr. Danyang Gong (University of California, Los Angeles, CA, USA). Virus particles were harvested through a reverse genetic system, and viral titers were determined by plaque assay on Vero cells and expressed as plaque-forming units per milliliter (PFU/mL). The linear (reduced) forms of the scorpion venom peptides MMTX, BmP05, BmTX1, and BmKK12 were chemically synthesized by solid-phase peptide synthesis at QYAOBIO Co., Ltd. (Shanghai, China). The final products were purified to homogeneity by reversed-phase high-performance liquid chromatography (RP-HPLC), and their molecular weights and purities (>97%) were verified by electrospray ionization mass spectrometry (ESI-MS).

5.2. Co-Immunoprecipitation for Peptides Screening

To identify scorpion venom-derived peptides capable of binding to TRPML2, an in vitro co-immunoprecipitation (Co-IP)-based screening was performed. Crude venom was collected from approximately 100 live Mesobuthus martensii scorpions by electrical stimulation of the telson, immediately diluted in ice-cold phosphate-buffered saline (PBS), centrifuged to remove debris, aliquoted, and stored at −80 °C until use. All experimental procedures were approved by the Ethics Committee of Hubei University of Technology (Wuhan, Hubei, China). HEK293T cells were transfected with a plasmid encoding C-terminally 3 × Flag-tagged human TRPML2. Forty-eight hours post-transfection, cells were washed with PBS and lysed on ice using NHG lysis buffer supplemented with protease and phosphatase inhibitors. The clarified lysates were incubated with diluted crude venom for 1 h at 4 °C with gentle rotation to allow for binding. TRPML2–venom complexes were then immunoprecipitated by incubating the mixtures with anti-FLAG M2 affinity resin overnight at 4 °C. After extensive washing to remove nonspecific binders, the precipitated complexes were separated by SDS-PAGE. Protein bands uniquely present in venom-treated samples compared to controls were excised and identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Candidate peptides revealed by this screen were further subjected to molecular docking simulations to predict their interaction interfaces with TRPML2.

5.3. Molecular Docking

The AlphaFold-predicted structure of the human TRPML2 channel (AF-Q8IZK6-F1-model_v4.pdb) was obtained from the PDB database. The three-dimensional structures of the seven candidate scorpion venom peptides were constructed de novo using AlphaFold 3 [51]. Molecular docking was performed using the HDock server [52]. TRPML2 was set as the receptor, and each peptide was set as the ligand for global, blind docking simulations. The interactions were scored using the ITScorePP function. To assess the reliability of the docking poses, the confidence score provided by HDock was employed. This empirical metric estimates the likelihood of binding, where a score above 0.7 indicates a high probability of interaction. For each candidate peptide, the top-ranked pose based on the docking score, which also exhibited a high confidence score, was selected for subsequent analysis of the binding interface.

5.4. Oxidative Refolding and HPLC Analysis

The reduced linear peptides were subjected to oxidative refolding to form the native conformation with three intramolecular disulfide bonds [53,54]. Specifically, each peptide (10 mg) was dissolved in 20 mL of 0.1 M Tris-HCl buffer (pH 8.0) and incubated at 25 °C for 48 h with gentle shaking at 50 rpm to facilitate air oxidation. The reaction mixture was then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant containing the cyclized peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a Waters Breeze QS HPLC system (Waters Corporation, Milford, MA, USA) equipped with a semi-preparative C18 column (XBridge™ BEH C18, 250 × 10 mm, 5 μm particle size; Waters Corporation). Both the reduced (linear) and oxidized (cyclized) forms of each peptide were analyzed using the same HPLC system with a shallower gradient (5% to 95% acetonitrile over 40 min at 1 mL/min). Successful cyclization was confirmed by a measurable shift in the retention time between the reduced and oxidized peptides due to the change in hydrophobicity. The collected major peak from the semi-preparative run was lyophilized and stored at −20 °C for subsequent use.

5.5. Mass Spectrometry Analysis

The molecular mass and disulfide bond formation of the oxidized peptides were confirmed by LC-MS/MS using an Orbitrap Q-Exactive Plus mass spectrometer coupled to an EASY-nLC 1200 system (Thermo Fisher Scientific, Waltham, MA, USA) [55]. Peptides were separated on an Acclaim™ PepMap™ C18 analytical column (75 μm × 25 cm, Thermo Fisher Scientific) with a 60 min gradient from 2% to 35% acetonitrile (0.1% formic acid) at 300 nL/min. Full MS scans (m/z 200–1800) were acquired at 70,000 resolutions, followed by data-dependent HCD-MS/MS of the top 15 precursors at 17,500 resolutions with 28% collision energy. The observed monoisotopic masses matched the theoretical values, confirming successful cyclization. The acquired raw data were processed using Xcalibur software (Version 2.0.7 SP1, Thermo Fisher Scientific, Waltham, MA, USA). The observed monoisotopic masses of the intact peptides were compared against their theoretical molecular weights to confirm successful oxidative folding and disulfide bond formation.

5.6. Calcium Imaging

The functional activity of the candidate scorpion venom peptides on TRPML2 channels was assessed using calcium imaging [56]. HEK293T cells transiently expressing human TRPML2 were loaded in the dark for 30–45 min at room temperature with 10 μM Fura-2 AM (Yeasen Biotech Co., Ltd., Shanghai, China) and 0.01% Pluronic F-127 in a physiological Ringer’s solution (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM CaCl_2_, 2 mM MgCl_2_, 10 mM glucose, pH 7.4). Following dye loading and washing, cells were imaged on an inverted fluorescence microscope (Leica Stellaris 8, Leica Stellaris 8, Leica Microsystems, Wetzlar, Germany) equipped with a Lambda DG-4 high-speed wavelength switcher (Sutter Instrument, Novato, CA, USA) for rapid alternation between 340 nm and 380 nm excitation. The ratio of fluorescence intensity (F340/F380) was calculated to represent relative changes in intracellular free calcium concentration (Ca^2+^). A cell was defined as responsive if its Ca^2+^ increased by at least 30% for a minimum duration of 10 s following agonist application, clearly distinguishing specific activation from stochastic fluctuations. Each purified candidate peptide (10 μM) was applied to the bath, with the selective TRPML2 agonist ML-SA1 (20 μM) serving as a positive control. For the assay of calcium response traces, each colored line represents an individual HEK293T cell, and Dozens of cells analyzed per condition across a minimum of three independent experiments.

5.7. Cell Viability Assay

The cytotoxicity of four candidate scorpion venom peptides on A549 cells was assessed using the Cell Counting Kit-8 (CCK-8; YEASEN, Shanghai, China). Cells were seeded into 96-well plates at a density of 8 × 10^3^ cells per well in 100 µL of complete culture medium (DMEM supplemented with 10% FBS and 1% penicillin–streptomycin) and allowed to adhere for 12 h. When the cells reached approximately 80% confluency, the medium was replaced with fresh medium containing serially diluted peptides at the desired concentrations (From 0 to 100 µM). After incubating for 48 h at 37 °C in a humidified 5% CO_2_ incubator, 10 µL of the CCK-8 reagent was directly added to each well. The plates were then incubated for 1.5 h under the same conditions, and the absorbance of each well at 450 nm was measured using a microplate reader (ELx800™ Absorbance Microplate Reader, BioTek Instruments, Winooski, VT, USA). Each concentration was tested in at least three independent experiments with triplicate wells.

5.8. qRT-PCR

The antiviral activity of candidate scorpion venom peptides against ZIKV was evaluated by quantifying intracellular viral RNA copies using quantitative reverse transcription-PCR (qRT-PCR). A549 cells infected with virus were treated with different concentrations of scorpion venom peptides for 36 h at 37 °C; then, total RNA was extracted from the cells using TRIzol™ Reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions. After detection of RNA concentration and purity using a NanoDrop™ 2000 spectrophotometer, 1 µg of RNA was reverse transcribed to cDNA using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA). Quantitative PCR was performed on a Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Next, a quantitative fluorescence reaction with a volume of 20 µL reaction was performed, containing10 µL of Bestar^®^ SYBR Green qPCR Master Mix (DBI^®^ Bioscience, Ludwigshafen, Germany), 0.5 µL each of forward and reverse primers (10 µM), 1 µL of cDNA template, and 8 µL of double-distilled H_2_O. The qRT-PCR primers used for ZIKV were 5′-TTGTGGAAGGTATGTCAGGTG-3′ (sense) and 5′-ATCTTACCTCCGCCAT GTTG-3′ (antisense). GAPDH primers were 5′-TGATGACATCAAGAAGG TGGTGAAG-3′ (sense) and 5′-TCCTTGGAGGCCATGTGGGCCAT-3′. Finally, the gene copy numbers were determined by 7500 Real-Time PCR system (Applied Biosystems) and analyzed via the comparative method (∆∆CT). Relative viral RNA levels were normalized to GAPDH, and data from three independent experiments (each in duplicate) are presented as mean ± standard deviation (SD).

5.9. Statistics Analysis

All quantitative data were analyzed and visualized using GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA), and Graphical representation were performed using Adobe Photoshop (Version CS6, Adobe Systems Inc., San Jose, CA, USA). Data are presented as the mean ± SD from at least three independent experiments. For comparisons between two experimental groups, statistical significance was determined using an unpaired, two-tailed Student’s t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).