Transient Receptor Potential Ankyrin 1 (TRPA1) is a non-selective cation channel involved in detecting harmful stimuli and endogenous ligands, primarily expressed in sensory neurons. Due to its role in pain and itch, TRPA1 is a potential drug target. We identified an oxindole core structure via high-throughput screening, modified it, and tested the modified compounds in vitro and in vivo. Calcium influx assays in primary dorsal root ganglion (DRG) cells and TRPA1-overexpressing HEK-293 T cells identified best compound ZQMT-10. ZQMT-10 demonstrated strong interaction with TRPA1 in the CETSA and MST assays. Oral administration of ZQMT-10 in C57BL/6J mice significantly reduced abnormal responses in the cold plate test. ZQMT-10 alleviated pain induced by AITC application on the mouse paw or by intracolonic administration, while also increasing the pain threshold and relieving persistent inflammatory pain. These results suggest ZQMT-10 as a promising TRPA1-targeted therapeutic agent.
Pain is a complex and debilitating sensation that affects millions of people worldwide, severely compromising their quality of life and imposing a significant burden on healthcare systems. Pain is commonly associated with various underlying conditions, including osteoarthritis, neuropathic pain, and fibromyalgia. Effective management of pain remains a serious challenge due to its multifactorial nature, involving both peripheral and central mechanisms. Neuropathic pain, resulting from nerve damage, often causes persistent burning or tingling sensations and can lead to long-term pain and functional impairments. Frostbite pain, due to extreme cold, can result in tissue necrosis, loss of function, or amputation, and may lead to chronic pain and sensory issues. Both conditions require immediate medical attention to prevent long-term damage.
The transient receptor potential ankyrin 1 (TRPA1) channel plays a crucial role in pain perception. As a member of the transient receptor potential (TRP) channel family, TRPA1 is predominantly expressed in sensory neurons and is essential for detecting noxious stimuli such as chemical irritants, extreme temperatures, and oxidative stress. TRPA1 is particularly significant in conditions where pain sensitivity to these stimuli is heightened. The TRPA1 channel is a homotetrameric structure, with each subunit containing six transmembrane α-helices, a series of ankyrin repeat domains, and intracellular amino (N-terminus) and carboxyl (C-terminus) termini. It is permeable to calcium and other monovalent cations, allowing inward cationic currents that regulate cellular functions primarily through intracellular calcium-dependent signaling pathways. TRPA1 functions as a sensor of cell damage signals and takes part in many diseases. TRPA1 is implicated in cold sensations related to painful stimuli. TRPA1 activity is often altered in neuropathic pain resulting from nerve injury or dysfunction, leading to increased pain sensitivity and inflammatory responses. In osteoarthritis, TRPA1 contributes to joint pain perception, which is aggravated by inflammatory mediators and irritants. In fibromyalgia, characterized by widespread musculoskeletal pain, TRPA1 responses to various stressors may further exacerbate pain.
Recent studies have provided valuable insights into TRPA1′s role in pain pathways and its potential as a therapeutic target. Studies have shown that TRPA1 can be activated by endogenous factors such as reactive oxygen species (ROS) and adenosine triphosphate (ATP), as well as exogenous factors, including chemical irritants like mustard oil and environmental pollutants such as diesel exhaust. This understanding elucidates TRPA1′s contribution to chronic pain states. Genetic studies have linked polymorphisms in the TRPA1 gene to altered pain perception and susceptibility to pain disorders, while functional assays have demonstrated that TRPA1 activation leads to elevated intracellular calcium levels and enhanced pain signaling.
The development of TRPA1 antagonists represents a promising research area, as these compounds have the potential to provide targeted pain relief. By specifically inhibiting TRPA1, antagonists can offer more precise pain control with fewer systemic side effects compared to conventional analgesics. In recent years, various chemical entities have been identified as TRPA1 antagonists (Fig. 1) in the literature. However, due to several recognized challenges in this drug discovery field, only a limited number of compounds have progressed to clinical development. Additionally, some existing compounds exhibit challenging physicochemical properties, such as poor solubility, low metabolic stability, and limited blood–brain barrier permeability. For example, A-967079 has poor selectivity, potentially affecting other TRP channels and causing side effects. It also has a short half-life, requiring high doses for efficacy. HC-030031 is rapidly metabolized, leading to a short duration of action and the need for frequent dosing.
In conclusion, studying TRPA1 and its role in nociception highlights significant progress in pain research. Targeting TRPA1 is expected to lead to the development of more effective and safer pain treatment strategies, ultimately improving outcomes for chronic pain patients.
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To identify potential TRPA1 antagonists, we utilized the Targetmol database, which includes approximately 160,000 compounds, and performed docking studies with the TRPA1 PDB structure 6WJ5. High-throughput virtual screening was conducted using Maestro, evaluating compounds based on their binding affinity to the receptor pocket and their interactions with key amino acids (such as Phe877, Leu881, Thr874, Leu880, Leu871, and Phe909) within the pocket.
We ranked the docking results from high to
The study aimed to identify potential TRPA1 antagonists through virtual screening, with oxyindoles emerging as promising scaffold compounds. The research focused on optimizing these compounds to enhance hydrogen bonding interactions at the target site, identifying critical amino acid residues, and significantly improving binding affinity. Through calcium ion influx analysis, the synthesized compound demonstrated strong TRPA1 antagonistic activity with low cytotoxicity. The most potent compound,
Commercially available reagents and solvents were used. Standard procedures were used to dry and purify the solvents. All reactions involving air, moisture-sensitive intermediates, or intermediates that were sensitive to air or moisture were carried out in nitrogen. 400 MHz, 101 MHz and 377 MHz were used to record 1 H, 13 C NMR and 19 F spectra in DMSO‑d 6 with tetramethylsilane (TMS) as the internal standard. On a Q-TOF X500R, high-resolution mass spectra (HR-MS) were collected. Flash column
All other authors contributed to the writing of the article. The final version of the paper has been approved by all authors.
Yiming Qi: Writing – original draft, Formal analysis, Data curation, Conceptualization. Hao Gong: Software, Resources, Data curation. Zhiya Wang: Validation, Supervision, Software. Xiaoxuan Song: Visualization, Resources. Zixian Shen: Formal analysis, Data curation. Limeng Wu: Writing – review & editing, Formal analysis. Yujia Gu: Supervision, Data curation. Weiyi Wang: Visualization, Resources. Xinyu Li: Data curation. Mingzuo Zhang: Methodology, Investigation. Zonghe Xu: Formal analysis.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
This work was supported by National major scientific research project (2022-JCJQ-ZD-095-00) and Scientific Technology Projects of Liao Ning province (2022JH2/101500035 and 2023-BS-031). We would like to express our gratitude to Prof. Mousheng Chen and Prof. Jian Wang of Shenyang Pharmaceutical University for providing the software and protocol required for molecular docking, and to Prof. Xiaolong Hu of China Pharmaceutical University for conducting the MST experiments and supplying the
