## 3 Results and Discussion  
### Design of Efficient Assay Platform for Collecting TRPA1 Agonist Variations  
Although TRPA1 agonists have been widely studied for finding several interaction rules, further understanding of TRPA1 agonist interaction facilitates better agonist design. Therefore, in this study, we hypothesized that collecting various TRPA1 agonists would provide novel insights for understanding the receptor-agonist interaction and the design of agonist structures. We combined the image-based TRPA1 assay system with the in silico chemical space clustering approach to perform such an assay to efficiently collect agonist variations.  
Our image-based assay can analyze the kinetics of TRPA1 agonists by monitoring the intracellular Ca\({}^{2+}\) concentration changes induced by TRPA1 activation using Ca\({}^{2+}\)-sensitive fluorescent dye, Fluo-8. This system enabled the collection of kinetics data from the 96-well plate platform of TRPA1 agonists within 30 min (assay scheme illustrated in Figure 1).  
For our assay target, we set Library707 for our TRPA1 agonist data collection (Table S2). There are two main reasons why we set this library for our study: one, it consists of our original compounds, all of which have been chemically synthesized, so intermediates or compound variants could be easily synthesized for further structure-activity relation studies, and two, it provides the opportunity to explore novel structural rules for further TRPA1 agonist study since this library consists of compounds with rare structural characteristics, which are not frequently detected in natural compounds.  
To maximize the collection of data variation from the assay of Library707, we challenged ourselves to avoid making assays that analyze structurally similar compounds. For this objective, Library707 was clustered into 127 independent clusters using 27 physicochemical molecular descriptors (Figures 2 and 3a). Physicochemically similar compounds can be grouped objectively into clusters using multiple molecular descriptors. To obtain more compound variants in an assay, a single compound was selected from each cluster, and 127 representative compounds were evaluated in the first assay (Table S2). The clustering revealed that Library707 did not exhibit a strong bias on certain structures, as the largest cluster had 47 compounds and the average member number was 5.6 (Table S3).  
Figure 1: Schematic illustration of the image-based TRPA1 assay platform. Human TRPA1 was stably expressed in the T-Rex293 cells. Activation of TRPA1 was quantitatively evaluated by measuring the intracellular Ca\({}^{2+}\) concentration changes. TRPA1 activity was calculated using the following equation: TRPA1 activity = [F (maximum fluorescence intensity) \(-\) F\({}_{0}\) (baseline fluorescence)]/F\({}_{0}\).  
Figure 2: Schematic chart of our TRPA1 agonist data collection. Starting from Library707, first and second assays were conducted covering 197 compounds. To collect interaction data from diverse candidates, 707 compounds in the library were clustered using physicochemical molecular descriptors to select 127 representative compounds for the first assay. From the first assay results, several clusters were selected for the second assay by two concepts: the clusters with a high rate of positive compounds, and the clusters with compounds carrying chemical groups that are associated with the previously known agonist mechanism. P1, P2, N1, and N2 show the categories of assay results; P1: high-activity agonists, P2: medium-activity agonists, N1: difficult to evaluate activity due to its low solubility, N2: no activity.