Ubiquitin-Proteasome Modulating Dolabellanes and Secosteroids from Soft Coral Clavularia flava

We performed a high-content screening (HCS) assay aiming to discover bioactive molecules with proteasome inhibitory activity. By structural elucidation, we identified six compounds purified from soft coral Clavularia flava, which potentiates proteasome inhibition. Chemical structure elucidation revealed they are dolabellane- and secosteroid-based compounds including a new dolabellane, clavinflol C (1), three known dolabellanes, stolonidiol (2), stolonidiol-17-acetate (3), and clavinflol B (4) as well as two new secosteroids, 3β,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9,23-dione (5) and 3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9,23-dione (6). All six compounds show less cytotoxicity than those of known proteasome inhibitors, bortezomib and MG132. In summary, the high-content measurements of control inhibitors, bortezomib and MG132, manifest the highest ratio >2 in high-content measurement. Of the isolated compounds, 2 and 5 showed higher activity, followed by 3 and 6, and then 1 and 4 exhibited moderate inhibition.


Compound Purification and Structure Elucidation
Chromatographic fractionation of ethyl acetate solubles from C. flava afforded four dolabellane diterpenes, 1-4, as well as two secosteroids, 5 and 6. The three known dolabellane diterpenes, 2-4 were identified by comparison of their spectral data with those of reported literatures [5,6]. The structures of new compounds, 1, 5, and 6 were elucidated by analysis of 1D and 2D spectral data (Figures S1-S24).

Compound Purification and Structure Elucidation
Chromatographic fractionation of ethyl acetate solubles from C. flava afforded four dolabellane diterpenes, 1-4, as well as two secosteroids, 5 and 6. The three known dolabellane diterpenes, 2-4 were identified by comparison of their spectral data with those of reported literatures [5,6]. The structures of new compounds, 1, 5, and 6 were elucidated by analysis of 1D and 2D spectral data (Figures S1-S24).
Compound 6 appeared as a white amorphous powder like 5. Careful inspection of the 2D NMR spectroscopic data (Figures S21-23) of 6 led to the establishment of the same nucleus as that of 5. The NMR spectroscopic data (Figures S19 and S20) of 6 were analogous to those of 5, except for NMR signals due to the conjugated enone in 6. The location of the conjugated enone was identified by the HMBC correlations ( Figure S23) from the methylene protons (H 2 -22) to the carbonyl carbon (C-23) and from H 3 -26, 27 to C-24, securing the structure of 6, which was shown as 3β,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9,23-dione.
Consequently after the assay procedure, we performed Western blotting analysis and q-PCR experiments to examine the levels of EGFP-UL76 protein and mRNA transcript under the same experimental conditions ( Figure 2B,C and Figure 3B,C). In these two experiments, cells treated with bortezomib 25 nM and MG132 1 µM were used in parallel as proteasome inhibitory controls. We obtained similar results that the ratios of EGFP-UL76/tubulin under treatment with bortezomib and MG132 showed no difference from the control level, which was consistent with a previous report [4].
Mar. Drugs 2019, 17, x FOR PEER REVIEW 4 of 9 bortezomib 25 nM and MG132 1 μM were used in parallel as proteasome inhibitory controls. We obtained similar results that the ratios of EGFP-UL76/tubulin under treatment with bortezomib and MG132 showed no difference from the control level, which was consistent with a previous report [4].   The assessment of proteasome inhibitory activity of marine secosteroid-based compounds (5 and 6) using a standard operation protocol of high-content EGFP-UL76 aggresomes screening assay as described in Figure 2 legend.

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][10][11]. Among the four dolabellanesbased compounds with stolonidiol (2) 5 g/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 sterol-based agosterols [12] and secosteroid-based physalin B and C [13,14]. Endogenous 25hydroxyvitamin 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 g/mL Figure 5. Classification of the high-content measurements of EGFP-UL76 aggresomes by size with marine secosteroid-based compounds (5 and 6). Treatment as described in Figure 4 legend.

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][10][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.

High-Content Screening (HCS) Assay for Proteasome Inhibition
We followed the HCS assay routinely conducted in the lab. In brief, the complete assay includes a series of experiments [4]. Experiments were sequentially performed: (1) the DNA transfection into cell culture with compound treatment, (2) cells fixation, (3) image acquisition using an ImageXpress Micro Widefield HCS system (Molecular Device, San Jose, CA, USA), (4) high-content measurements analyzed by modules of MetaExpress, Cell Scoring and Multi-Wavelength Cell Scoring, (5) Western blotting imaging and densitometric analysis, and (6) RNA purification and quantitative PCR.