A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

An insight into the pharmacology of cysteine/methionine containing peptide drugs.

Since last century, peptides have emerged as potential drugs with >90 FDA approvals for various targets with several in the pipeline. Sulphur, in peptides is present either as thiol (-SH) from Cys or thioether from Met. In this review, all the peptides approved by FDA since 2000 containing sulphur have been included. Among them ∼50 % contains disulphide bridges. This clearly demonstrates the significance of disulphide bonds in peptide drugs. This can be achieved synthetically by using orthogonal protecting groups (PGs) for -SH. These PGs are compatible with Solid Phase Peptide Synthesis (SPPS), which is still the method of choice for peptide synthesis. The orthogonal PGs used for Cys thiol side chain protecting for disulphide bond formation have been included which are currently in use both by academia and industry from small scale to large scale synthesis. In addition, the details of the FDA approved drugs containing Cys and Met (or both) have also been discussed.

SARS-CoV-2 Mpro responds to oxidation by forming disulfide and NOS/SONOS bonds.

The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

Targeting the cysteine biosynthesis pathway in microorganisms: Mechanism, structure, and drug discovery.

Owing to the global health crisis of resistant pathogenic infections, researchers are emphasizing the importance of novel prevention and control strategies. Existing antimicrobial drugs predominantly target a few pathways, and their widespread use has pervasively increased drug resistance. Therefore, it is imperative to develop new antimicrobial drugs with novel targets and chemical structures. The de novo cysteine biosynthesis pathway, one of the microbial metabolic pathways, plays a crucial role in pathogenicity and drug resistance. This pathway notably differs from that in humans, thereby representing an unexplored target for developing antimicrobial drugs. Herein, we have presented an overview of cysteine biosynthesis pathways and their roles in the pathogenicity of various microorganisms. Additionally, we have investigated the structure and function of enzymes involved in these pathways as well as have discussed drug design strategies and structure-activity relationships of the enzyme inhibitors. This review provides valuable insights for developing novel antimicrobials and offers new avenues to combat drug resistance.

Reactivities of acrylamide warheads toward cysteine targets: a QM/ML approach to covalent inhibitor design.

Covalent inhibition offers many advantages over non-covalent inhibition, but covalent warhead reactivity must be carefully balanced to maintain potency while avoiding unwanted side effects. While warhead reactivities are commonly measured with assays, a computational model to predict warhead reactivities could be useful for several aspects of the covalent inhibitor design process. Studies have shown correlations between covalent warhead reactivities and quantum mechanic (QM) properties that describe important aspects of the covalent reaction mechanism. However, the models from these studies are often linear regression equations and can have limitations associated with their usage. Applications of machine learning (ML) models to predict covalent warhead reactivities with QM descriptors are not extensively seen in the literature. This study uses QM descriptors, calculated at different levels of theory, to train ML models to predict reactivities of covalent acrylamide warheads. The QM/ML models are compared with linear regression models built upon the same QM descriptors and with ML models trained on structure-based features like Morgan fingerprints and RDKit descriptors. Experiments show that the QM/ML models outperform the linear regression models and the structure-based ML models, and literature test sets demonstrate the power of the QM/ML models to predict reactivities of unseen acrylamide warhead scaffolds. Ultimately, these QM/ML models are effective, computationally feasible tools that can expedite the design of new covalent inhibitors.

Efficient turn-on fluorescent probe cooperated by cascade response for disclosing the fluctuation of cysteine in cells.

BACKGROUND: The research on cysteine (Cys) determination is deemed as a hot topic, since it has been reported to be connected with various physiological processes and disease prediction. However, existing Cys-responding probes may expose some defects such as long reaction time, disappointing photostability, and suboptimal sensitivity. Under such a circumstance, our team has proposed an efficient fluorescent probe with novel sensing mechanism to perfectly cope with the above-mentioned drawbacks. RESULTS: A novel cascade reaction-based probe 9-(2,2-dicyanovinyl)-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-8-yl acrylate (DPQA) has been synthesized for the first time. Undergoing addition-cleavage and cyclization-rearrangement processes, DPQA reacts with Cys to generate an iminocoumarin product with relucent green fluorescence, namely 11-imino-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonitrile (IMC-J), and the relative fluorescence quantum yield (Φf) soars from 0.007 to 0.793. Utilizing such a mechanism, DPQA shows a superb turn-on signal (172-fold), low detection limit (4.1 nM), and wide detection range (5-6000 nM) toward Cys detection. Encouraged by the admirable sensing performance of DPQA, bioimaging of endogenous Cys has been attempted in HeLa cells with satisfactory results. Moreover, cell model of H2O2-induced oxidative stress has been established and the Cys fluctuation during this process has been inspected, elucidating how living cells confront with the eruption of reactive oxygen species (ROS) storm. SIGNIFICANCE: The probe DPQA with such an intriguing cascade responding process for Cys detection has been endowed with many merits, such as fast reaction and superior sensitivity, conducive to improving responsiveness and rendering it more suitable for further applications. Thereby, we expect that the DPQA would be an efficient tool for detecting Cys fluctuation in living cells of different physiological processes.

Injectable Modified Sodium Alginate Microspheres for Enhanced Operative Efficiency and Safety in Endoscopic Submucosal Dissection.

Endoscopic submucosal dissection (ESD) is an effective method for resecting early-stage tumors in the digestive system. To achieve a low injection pressure of the injected fluid and continuous elevation of the mucosa following injection during the ESD technique, we introduced an innovative injectable sodium-alginate-based drug-loaded microsphere (Cipro-ThSA) for ESD surgery, which was generated through an emulsion reaction involving cysteine-modified sodium alginate (ThSA) and ciprofloxacin. Cipro-ThSA microspheres exhibited notable adhesiveness, antioxidant activity, and antimicrobial properties, providing a certain level of postoperative wound protection. In vitro cell assays confirmed the decent biocompatibility of the material. Lastly, according to animal experiments involving submucosal elevation of porcine colons, Cipro-ThSA microspheres ensure surgically removable lift height while maintaining the mucosa for approximately 246% longer than saline, which could effectively reduce surgical risks while providing sufficient time for operation. Consequently, the Cipro-ThSA microsphere holds great promise as a novel submucosal injection material, in terms of enhancing the operational safety and effectiveness of ESD surgery.

Light-triggered AND logic tetrapeptide dynamic covalent assembly.

We demonstrate light-triggered dynamic covalent assembly of a linear short tetrapeptide containing two terminal cysteine residues in an AND logic manner. A photobase generator is introduced to accomplish light-mediated pH regulation to increase the reduction potential of thiols in the tetrapeptide, which activates its oxidative polymerization through disulfide bonds. Interestingly, it is elucidated that under light irradiation, mere co-existence of photobase generator and the oxidizing agent permits the polymerization performance of this tetrapeptide. Hence, a light-triggered AND logic dynamic covalent assembly of a tetrapeptide is achieved. Further, upon redox response, the reversible aggregation and disaggregation can be transformed for numerous times due to the dynamic covalent feature of disulfide bond. As a comparison, no assembly occurs for a short peptide containing one terminal cysteine residue under the same stimuli condition. This work offers a new approach to remotely control programmable molecular assembly of short linear peptides based on dynamic covalent bond, holding great potential in wide bioapplications.

Computation-Guided Rational Design of Cysteine-Less Protein Variants in Engineered hCGL.

The engineered human cystathionine-γ-lyase (hCGL) resulting in enhanced activity toward both cysteine and cystine unveils a potential robust antitumor activity. However, the presence of cysteine residues has the potential to induce oligomerization or incorrect disulfide bonding, which may decrease the bioavailability of biopharmaceuticals. Through a meticulous design process targeting the cysteine residues within engineered hCGL, a set of potential beneficial mutants were obtained by virtual screening employing Rosetta and ABACUS. Experimental measurements have revealed that most of the mutants showed increased activity toward both substrates l-Cys and CSSC. Furthermore, mutants C109V and C229D demonstrated Tm value increases of 8.2 and 1.8 °C, respectively. After an 80 min incubation at 60 °C, mutant C229D still maintained high residual activity. Unexpectedly, mutant C109V, displaying activity approximately 2-fold higher than the activity of wild type (WT) for both substrates, showed disappointing instability in plasma, which suggests that computational design still requires further consideration. Analysis of their structure and molecular dynamics (MD) simulation revealed the impact of hydrophobic interaction, hydrogen bonds, and near-attack conformation (NAC) stability on activity and stability. This study acquired information about mutants that exhibit enhanced activity or thermal resistance and serve as valuable guidance for subsequent specific cysteine modifications.

In Solution Identification of the Lysine-Cysteine Redox Switch with a NOS Bridge in Transaldolase by Sulfur K-Edge X-ray Absorption Spectroscopy.

A novel covalent post-translational modification (lysine-NOS-cysteine) was discovered in proteins, initially in the enzyme transaldolase of Neisseria gonorrhoeae (NgTAL) [Nature 2021, 593, 460-464], acting as a redox switch. The identification of this novel linkage in solution was unprecedented until now. We present detection of the NOS redox switch in solution using sulfur K-edge X-ray absorption spectroscopy (XAS). The oxidized NgTAL spectrum shows a distinct shoulder on the low-energy side of the rising edge, corresponding to a dipole-allowed transition from the sulfur 1s core to the unoccupied σ* orbital of the S-O group in the NOS bridge. This feature is absent in the XAS spectrum of reduced NgTAL, where Lys-NOS-Cys is absent. Our experimental and calculated XAS data support the presence of a NOS bridge in solution, thus potentially facilitating future studies on enzyme activity regulation mediated by the NOS redox switches, drug discovery, biocatalytic applications, and protein design.

An AND-Gate Photoacoustic Probe for Cys and H2S Precise Photoacoustic Sensing in Localized Tumors.

Photoacoustic (PA) tomography has shown many promising aspects in noninvasive and precise imaging of deep-localized biomarkers. However, these traditional single-locked PA probes always face challenges in precise PA imaging with high specificity. Here, we report a novel AND-gate photoacoustic probe, BAE, to improve tumor imaging accuracy via the combination of two tumor-associated biomarkers, cysteine (Cys) and hydrogen sulfide (H2S). Only when Cys and H2S are concurrently introduced into the detection system does the absorption of BAE red-shift from the initial 680 to 810 nm, thereby showing a 5.29-fold enhancement in its PA signal at 810 nm. The good specificity of BAE is proven, since an obvious PA signal could be observed only in the solution containing both Cys and H2S and was not affected by other reactive sulfur species. After being taken up by tumors with the assistance of a nanomicelle, the AND-gate PA probe BAE was applied for dynamic real-time monitoring of Cys and H2S in vivo, achieving precise identification of tumors. This AND-gate PA probe provides a potential technical tool for precise sensing analysis of deep-seated diseases.

Peptide Mimics of the Cysteine-Rich Regions of HapX and SreA Bind a [2Fe-2S] Cluster In Vitro.

HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.

Acidic Derivatization of Thiols Using Diethyl 2-Methylenemalonate: Thiol-Michael Addition Click Reaction for Simultaneous Analysis of Cysteine and Cystine in Culture Media Using LC-MS/MS.

Cysteine (Cys) and its oxidized form, cystine (Cys2), play crucial roles in biological systems and have considerable applications in cell culture. However, Cys in cell culture media is easily oxidized to Cys2, leading to solubility issues. Traditional analytical methods struggle to maintain the oxidation states of Cys and Cys2 during analysis, posing a significant challenge to accurately measuring and controlling these compounds. To effectively control the Cys and Cys2 levels, a rapid and accurate analytical method is required. Here, we screened derivatizing reagents that can react with Cys even under acidic conditions to realize a novel analytical method for simultaneously determining Cys and Cys2 levels. Diethyl 2-methylenemalonate (EMM) was found to possess the desired traits. EMM, characterized by its dual electron-withdrawing attributes, allowed for a rapid reaction with Cys under acidic conditions, preserving intact information for understanding the functions of target compounds. Combined with LC-MS/MS and an internal standard, this method provided high analytical accuracy in a short analytical time of 9 min. Using the developed method, the rapid oxidation of Cys in cell culture media was observed with the headspace of the storage container considerably influencing Cys oxidation and Cys2 precipitation rates. The developed method enabled the direct and simplified analysis of Cys behavior in practical media samples and could be used in formulating new media compositions, ensuring quality assurance, and real-time analysis of Cys and Cys2 in cell culture supernatants. This novel approach holds the potential to further enhance the media performance by enabling the timely optimal addition of Cys.

Proteomic Ligandability Maps of Spirocycle Acrylamide Stereoprobes Identify Covalent ERCC3 Degraders.

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Here, we describe a set of stereo- and regiochemically defined spirocycle acrylamides and the analysis of these electrophilic "stereoprobes" in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamide stereoprobes, the spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound termed ZL-12A promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL-12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation but instead causes collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another's protein degradation profiles. Finally, we provide evidence that the antihypertension drug spironolactone─previously found to promote ERCC3 degradation through an enigmatic mechanism─also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.

Dual-functional porphyrinic zirconium-based metal-organic framework for the fluorescent sensing of histidine enantiomers and Hg2.

A novel fluorescence sensor based on a porphyrinic zirconium-based metal-organic framework, L-cysteine-modified PCN-222 (L-Cys/PCN-222), was developed to selectively recognize histidine enantiomers and sensitively detect Hg2+. The dual-functional sensor was successfully prepared via the solvent-assisted ligand incorporation method and characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), 1H nuclear magnetic resonance (1H NMR) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD), X-ray photoelectron spectroscopy (XPS), and nitrogen adsorption-desorption analyses. L-Cys/PCN-222 not only showed a higher quenching response for L-histidine than that for D-histidine with a fast fluorescent response rate of <40 s but also exhibited low detection limits for L- and D-histidine (2.48 µmol L-1 and 3.85 µmol L-1, respectively). Moreover, L-Cys/PCN-222 was employed as a fluorescent and visual sensor for the highly sensitive detection of Hg2+ in the linear range of 10-500 µmol L-1, and the detection limit was calculated to be 2.79 µmol L-1 in surface water. The specific and selective recognition of chiral compounds and metal ions by our probe make it suitable for real field applications.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

Investigations of Ni(II)Cysteine-Tyrosine Dithiocarbamate Complex: Synthesis, Characterization, Molecular Docking, Molecular Dynamic, and Anticancer Activity on MCF-7 Breast Cancer Cell Line.

OBJECTIVE: Breast cancer ranks second in terms of the highest number of cancer deaths for women worldwide and is one of the leading causes of death from cancer in women. The drug that is often used for chemotherapy is cisplatin. However, cisplatin drugs have a number of problems, including lack of selectivity, unwanted side effects, resistance, and toxicity in the body. In this work, we investigated Ni(II) cysteine-tyrosine dithiocarbamate complex against breast cancer. METHODS: Research on the new complex compound Ni(II) cysteine-tyrosine dithiocarbamate have several stages including synthesis, characterization, in-silico and in-vitro testing of MCF-7 cells for anticancer drugs. The synthesis involved reacting cysteine, CS2, KOH and tyrosine with Mn metal. The new complex compound Ni(II) cysteine-tyrosine dithiocarbamate has been synthesized, characterized, and tested in vitro MCF-7 cells for anticancer drugs. Characterization tests such as melting point, conductivity, SEM-EDS, UV Vis, XRD, and FT-IR spectroscopy have been carried out. RESULT: The synthesis yielded a 60,16%, conversion with a melting point of 216-218 oC and a conductivity value of 0.4 mS/cm. In vitro test results showed morphological changes (apoptosis) in MCF-7 cancer cells starting at a sample concentration of 250 µg/mL and an IC50 value of 618.40 µg/mL. Molecular docking study of Ni(II) cysteine-tyrosine dithiocarbamate complex identified with 4,4',4''-[(2R)-butane-1,1,2-triyl]triphenol - Estrogen α showing active site with acidic residue amino E323, M388, L387, G390 and I389. Hydrophobic and hydrophobic bonds are seen in Ni(II) cysteine-tyrosine dithiocarbamate - Estrogen α has a binding energy of -80.9429 kJ /mol. CONCLUSION: there were 5 residues responsible for maintaining the ligand binding stable. The compound had significant Hbond contact intensity, however, it was not strong enough to make a significant anticancer effect. Though the synthesized compound shows low bioactivity, this research is expected to give valuable insight into the effect of molecular structure on anticancer activity.

1H NMR based sulfonation reaction kinetics of wine relevant thiols in comparison with known carbonyls.

Sulfite addition is a common tool for ensuring wines' oxidative stability via the activity of its free and weakly bound molecular fraction. As a nucleophile, bisulfite forms covalent adducts with wine's most relevant electrophiles, such as carbonyls, polyphenols, and thiols. The equilibrium in these reactions is often represented as dissociation rather than formation. Recent studies from our laboratory demonstrate, first, the acetaldehyde sulfonate dissociation, and second, the chemical stability of cysteine and epicatechin sulfonates under wine aging conditions. Thus, the objective of this study was to monitor by 1H NMR the binding specificity of known carbonyl-derived SO2 binders (acetaldehyde and pyruvic acid) in the presence of S-containing compounds (cysteine and glutathione). We report that during simulated wine aging, the sulfur dioxide that is rapidly bound to carbonyl compounds will be released and will bind to cysteine and glutathione, demonstrating the long-term sulfur dioxide binding potential of S-containing compounds. These results are meant to serve as a complement to existing literature reviews focused on molecular markers related to wines' oxidative stability and emphasize once more the importance of S-containing compounds in wine aging chemical mechanisms.

Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Ligustilide covalently binds to Cys703 in the pre-S1 helix of TRPA1, blocking the opening of channel and relieving pain in rats with acute soft tissue injury.

ETHNOPHARMACOLOGICAL RELEVANCE: The natural anodyne Ligustilide (Lig), derived from Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort., has been traditionally employed for its analgesic properties in the treatment of dysmenorrhea and migraine, and rheumatoid arthritis pain. Despite the existing reports on the correlation between TRP channels and the analgesic effects of Lig, a comprehensive understanding of their underlying mechanisms of action remains elusive. AIM OF THE STUDY: The objective of this study is to elucidate the mechanism of action of Lig on the analgesic target TRPA1 channel. METHODS: The therapeutic effect of Lig was evaluated in a rat acute soft tissue injury model. The analgesic target was identified through competitive inhibition of TRP channel agonists at the animal level, followed by Fluo-4/Ca2+ imaging on live cells overexpressing TRP proteins. The potential target was verified through in-gel imaging, colocalization using a Lig-derived molecular probe, and a drug affinity response target stability assay. The binding site of Lig was identified through protein spectrometry and further analyzed using molecular docking, site-specific mutation, and multidisciplinary approaches. RESULTS: The administration of Lig effectively ameliorated pain and attenuated oxidative stress and inflammatory responses in rats with soft tissue injuries. Moreover, the analgesic effects of Lig were specifically attributed to TRPA1. Mechanistic studies have revealed that Lig directly activates TRPA1 by interacting with the linker domain in the pre-S1 region of TRPA1. Through metabolic transformation, 6,7-epoxyligustilide (EM-Lig) forms a covalent bond with Cys703 of TRPA1 at high concentrations and prolonged exposure time. This irreversible binding prevents endogenous electrophilic products from entering the cysteine active center of ligand-binding pocket of TRPA1, thereby inhibiting Ca2+ influx through the channel opening and ultimately relieving pain. CONCLUSIONS: Lig selectively modulates the TRPA1 channel in a bimodal manner via non-electrophilic/electrophilic metabolic conversion. The epoxidized metabolic intermediate EM-Lig exerts analgesic effects by irreversibly inhibiting the activation of TRPA1 on sensory neurons. These findings not only highlight the analgesic mechanism of Lig but also offer a novel nucleophilic attack site for the development of TRPA1 antagonists in the pre-S1 region.