## 4 Conclusions  
In this study, to further understand the structural diversity of TRPA1 agonists, we integrated the image-based TRPA1 assay system with the chemical space clustering concept. To maximize the collection of data variation from the assay, the library compounds were classified into 127 clusters using 27 physicochemical molecular descriptors and subjected to the TRPA1 assay. We successfully obtained TRPA1 activity data of 197 compounds with physicochemical diversity. Various types of compounds, including alkyne, amide, and ester, showed TRPA1 activity and indicated the physicochemical diversity of TRPA1 agonists. Furthermore, structure A was proposed as a novel core for a potent TRPA1 agonist. The concept presented in this study would aid the understanding of the structural diversity of agonists and the development of various agonists. Our approach can be applied to other receptors and enzymes to investigate interaction mechanisms with ligands and substrates.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12031622/s1](https://www.mdpi.com/article/10.3390/app12031622/s1)]([https://www.mdpi.com/article/10.3390/app12031622/s1](https://www.mdpi.com/article/10.3390/app12031622/s1)), Table S1: List of molecular descriptors selected from the total descriptors; Table S2: List of Library707 compounds with their molecular descriptor profiles and selections for assays (indicated in the order of cluster number); Table S3: Profile of 127 cluster members; Table S4: Chemical structures and assayed results of 129 compounds in the first assay (a) and 70 compounds in the second assay (b); Figure S1: Concentration response-curves of P1 (high-activity) and P2 (medium-activity) compounds.  
Figure 6: Correlation between the TRPA1 agonist activity and representative physicochemical molecular descriptors. All P1s (48 compounds) and N2s (37 compounds) from both first and second assays were compared by representative physicochemical descriptors relevant for drug design: (**a**) Manhold LogP, (**b**) Largest Chain, (**c**) Bond Polarizabilities, (**d**) Bond Polarizabilities—Manhold LogP, and (**e**) Largest Chain—XLogP. Each dot indicates a single compound: P1, red; N2, blue. *_p_ < 0.05; **_p_ < 0.01; ***_p_ < 0.001 by Student’s _t_-test.  
**Author Contributions:** Conceptualization, Y.T., K.T., M.F., M.S., Y.Y., R.K. and K.I.; investigation, Y.T., K.T., M.M., M.F., M.S., R.K. and K.I.; writing--original draft, Y.T., K.T. and R.K.; writing--review and editing, R.K., M.S. and K.I. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This research was funded by the Japan Society for the Promotion of Science (KAKENHI grant JP 21H02144A0 and 21K19092 to K.I. and 19K15792 to Y.T.) and the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research: BINDS) from the Japan Agency for Medical Research and Development (AMED) under grant number JP21 am0101099 (Y.Y.).  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The data that support the findings of this study are available upon reasonable request to the corresponding author.  
**Conflicts of Interest:** The authors declare no potential conflict of interests.