**3. Discussion**

During the HCS we assessed dolabellanes-based clavinflol C (**1**), stolonidiol (**2**), and stolonidiol-17-acetate (**3**) and clavinflol B (**4**) with proteasome inhibition activities. Several groups demonstrated that stolonidiol (**2**) and stolonidiol-17-acetate (**3**) enhance the activities of choline acetyl transferase (ChAT) [8], which likely is mediated by protein kinase C [9]. ChAT catalyzes the production of acetylcholine which is an essential neurotransmitter. Moreover, proteasome inhibitor MG132 stabilizes ChAT steady-state protein levels and increases the enzyme activity [10]. Therefore, we reason that stolonidiol may mediate the elevation of ChAT activity via both pathways in the inhibition of proteasome and the activation of protein kinase C [9–11]. Among the four dolabellanes-based compounds with stolonidiol (**2**) 5 μg/mL treatment, the EGFP-UL76 displayed the highest increase ratios of 2.12 and 1.75 of integrated and average intensity, respectively (Figure 2). Consistent to this result, the tolonidiol (**2**) potentiated the highest ratios of aggresome pit and vesicle integrated and average intensities (Figure 4). For the remaining three, increased ratios were moderate, greater than 10% increase, for all the high-content measurements. Compared to compounds **1** and **4**, compound **2** and **3** show a lack of chloride at C-6, which may contribute to a less cytotoxic effect for HEK293T cell and increased efficacy for proteasome inhibition. Acetylation at C-17 of compound **3** may reduce activity in proteasome inhibition.

Overviewing the known proteasome inhibitors with steroid structure were polyhydroxylated sterolbased agosterols [12] and secosteroid-based physalin B and C [13,14]. Endogenous 25-hydroxyvitamin D (25OHDO) was identified to impair sterol regulatory element-binding proteins (SREBPs) activation by inducing proteolytic processing and ubiquitin-mediated degradation of SREBP cleavage-activating

protein (SCAP) [15]. In this study, secosteroid-based compound **6** showed the highest inhibitory measurements of EGFP-UL76 integrated and average intensities and at 1 μg/mL (Figure 3), whereas with 5 μg/mL treatment the EGFP-UL76 exhibited the highest increase ratios of pit and vesicle measurements (Figure 5). As for the secosteroid related compound **5**, it showed less potent of proteasome inhibition with 25 μg/mL treatment which exhibited comparable activities to those of compound **6**. Compound **6** with a conjugated double bond may contribute cytotoxicity to HEK293T cells and display a significant reduction of all high-content measurements at 25 μg/mL.

### **4. Materials and Methods**
