# Harnessing Odorant Receptor Activation to Suppress Real Malodor
## 2 Results and Discussions  
### Identification of Cat-Urine-Responsive ORs  
To identify the specific ORs responding to cat urine, we used the urine of a 7-year-old neutered male Scottish Fold cat, one of the most commonly maintained breeds in Japan [28]. Cat urine was collected from a tray under the layer of sand that does not absorb liquid in the system toilet box for more than 12 h and less than 24 h (Figure 1A). Evaluation of odor intensity of the collected urine showed that some samples had a strong, pungent odor, whereas others did not (Figure 1B). To minimize variability in the odor intensity and components, we used a mixture of urine samples collected on different days for the initial screening of ORs. The mixture was prepared with a ratio of 30% urine with a mild pungent odor and 5% urine with a strong pungent odor. The presence of a pungent odor was confirmed after adjusting the mixed solution. For stimulating receptor-expressing cells, we added cat urine to the wells of assay plates containing Hana3A cells transfected with different ORs and then sealed the plates with lids to allow the volatilized urine odor to stimulate the cells within the plates (Figure 1C,D). In the initial screening, we identified receptors responsive to cat urine based on the threshold defined as the 3\(\sigma\) value (SD = 0.78, 3\(\sigma\) value = 2.35) calculated from the response data of all receptor-expressing cells (Figure 1E). This process resulted in the selection of mouse TAAR5 (average = 10.36) and human OR5H14 (average score = 2.35) (Figure 1F). Because mouse TAAR5 demonstrated a clearly higher response to cat urine than OR5H14, we performed a second screening to evaluate the concentration-dependent responses of the mouse TAAR5-expressing cells to varying dilutions of strong odor urine. Human TAAR5,the ortholog of mouse TAAR5, might not have reached the response threshold in the first screening due to differences in expression levels in Hana3A cells (Figure S1). Human TAAR5 is known as a trimethylamine (TMA) receptor [10; 29]. We further investigated the responses of both mouse and human TAAR5 to urine samples with varying pungent odors and found that both receptors responded robustly to the strongly pungent urine (Figure 1H,I). We also examined whether TAAR5-expressing cells responded to urine from cats of different types, sexes, and ages; TAAR5 showed a significant response to all tested samples (Figure S2). These findings suggest that the molecules acting as agonists for TAAR5 are responsible for the pungent odor of cat urine. Another OR that surpassed the 3\(\sigma\) value, OR10H2, responds to relatively pleasant odor components such as cinnamaldehyde and geranyl acetate as known agonists [23]. Because the purpose of this study was to suppress malodorous and irritating odors from  
Figure 1: Identification of cat-urine-responsive odorant receptors. (**A**) Schematic of the method for collecting cat urine using a systematic toilet for cats. (**B**) Malodor intensity ratings of cat urine collected on different days as evaluated by six subjects. Nonparametric multiple comparisons were conducted using one-way analysis of variance (ANOVA) followed by [PERSON]’s multiple comparison test (\(p<0.05\)). Different letters (a, ab, b) indicate significant differences between groups. (**C**) Schematic of the Glosensor assay showing the OR signal transduction pathway. Abbreviations: AC, adenylyl cyclase; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; RTP15/2, receptor transporting protein 1 short and 2. When odor molecules volatilized from the solution activate the ORs, the enzymatic reaction of Glosensor is triggered by the increase in cAMP concentration (red arrows). (**D**) Schematic of vapor stimulation using cat urine. (**E**) Screening strategy for OR selection. (**F**) Responses of selected human ORs, human TAARs, and mouse TAARs to the vapor phase from the cat urine mixture. The dotted line indicates the 3\(\sigma\) value calculated from all sample data.