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, 3,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9,23-dione (5) and 3,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.

A High-Content Screening Assay for the Discovery of Novel Proteasome Inhibitors from Formosan Soft Corals.

The ubiquitin-proteasome system (UPS) is a major proteolytic pathway that safeguards protein homeostasis. The main 26S proteasome consists of a 20S catalytic core proteasome and a 19S substrate recognition proteasome. UPS dysfunction underlies many important clinical diseases involving inflammation, tumors, and neurodegeneration. Currently, three 20S proteasome inhibitors, bortezomib, carfilzomib, and ixazomib, have been approved for the treatment of multiple myeloma. We aim to screen UPS inhibitors for biomedical purposes. The protein interaction network of human cytomegalovirus UL76 targets UPS, resulting in aggregations of ubiquitinated proteins termed aggresomes. In this study, we demonstrated that cell-based high-content measurements of EGFP-UL76 aggresomes responded to bortezomib and MG132 treatment in a dose-dependent manner. Employing this high-content screening (HCS) assay, we screened natural compounds purified from Formosan soft corals. Four cembrane-based compounds, sarcophytonin A (1), sarcophytoxide (2), sarcophine (3), and laevigatol A (4), were found to enhance the high-content profiles of EGFP-UL76 aggresomes with relative ratios of 0.2. By comparison to the mechanistic action of proteasome inhibitors, compounds 1 and 3 modulated the accumulation of ubiquitinated proteins, with a unique pattern likely targeting 19S proteasome. We confirmed that the EGFP-UL76 aggresome-based HCS system greatly improves the efficacy and sensitivity of the identification of proteasome inhibitors.

Xeniaphyllane-Derived Terpenoids from Soft Coral Sinularia nanolobata.

A novel tetranorditerpenoid, sinubatin A (1) (having an unprecedented carbon skeleton), a new norditerpenoid, sinubatin B (2) (a 4,5-epoxycaryophyllene possessing an unusual methylfuran moiety side chain), and a known diterpenoid, gibberosin J (3) were isolated from soft coral Sinulariananolobata. The structures of the new compounds were elucidated by extensive analysis of spectroscopic data.

New Cytotoxic Terpenoids from Soft Corals Nephthea chabroli and Paralemnalia thyrsoides.

A novel cytotoxic diterpenoid, chabrolin A (1) (possessing an unprecedented terpenoid skeleton), as well as three new cytotoxic sesquiterpenoids, parathyrsoidins E-G (2-4), were isolated by cytotoxicity-guided fractionation from soft corals Nephtheachabroli and Paralemnalia thyrsoides. The structures of the new compounds were determined by extensive analysis of spectroscopic data.

Mollisolactones A and B, novel dinormonoterpenes from the soft coral Sinularia mollis.

Two novel dinormonoterpenes, designated as mollisolactones A and B, were discovered from the soft coral Sinularia mollis on the basis of a chromatographic and NMR spectroscopy-based fractionation. Their structures were solved through analysis of comprehensive 1D and 2D NMR spectroscopic data and HRESIMS experiments. The biological activities of the obtained metabolites were evaluated for cytotoxicity against A-459 (human lung carcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cancer cell lines as well as antiviral activity against HCMV (human cytomegalovirus).

Sandensone A, a novel sesquiterpenoid from the Formosan soft coral Sinularia sandensis.

A chromatographic and NMR spectroscopy-based fractionation on the acetone extracts of the soft coral Sinularia sandensis led to the isolation of a novel sesquiterpenoid, sandensone A (1). The structure of 1 was elucidated based on comprehensive 1D and 2D NMR spectroscopic data as well as HRESIMS spectrometry. The absolute configuration at C-12 of 1 was determined as R using a modified Mosher's method. The cytotoxicity against A549 (human lung carcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cancer cell lines as well as antiviral activity against HCMV (human cytomegalovirus) were evaluated in vitro.

Polyoxygenated cembrane diterpenoids from the soft coral Sarcophyton ehrenbergi.

Five new polyoxygenated cembranoids, named (+)-1,15-epoxy-2-methoxy-12-methoxycarbonyl-11E-sarcophytoxide (1), (+)-2-epi-12-methoxycarbonyl-11E-sarcophine (2), 3,4-epoxyehrenberoxide A (3), ehrenbergol D (4) and ehrenbergol E (5), were obtained from the soft coral Sarcophyton ehrenbergi. The structures of 1-5 were established on the basis of comprehensive NMR and HR-ESI-MS analyses and by comparison with reported data in the literature. Compounds 4 and 5 showed moderate cytotoxicity against P-388 (mouse lymphocytic leukemia) cancer cell line with EC50 values of 2.0 and 3.0 µM, respectively. Compound 2 exhibited slight antiviral activity against HCMV (human cytomegalovirus) with IC50 values of 25.0 µg/mL.

Calpains mediate the proteolytic modification of human cytomegalovirus UL112-113 proteins.

The human cytomegalovirus (HCMV) UL112-113 gene is implicated in lytic viral replication. The UL112-113 proteins p34, p43, p50 and p84 are expressed via alternative splicing. However, the mechanism for the generation of three additional virus-associated proteins (p20, p26 and p28), which share the UL112 reading frame, remains unknown. Bioinformatic analyses indicated that p34, p43, p50 and p84 contain potential PEST-like degradation motifs. In this study, inhibitors of calpains, lysosomes and proteasomes reduced p20, p26 and p28 levels in virus-infected cells, suggesting the involvement of proteolytic modification. Moreover, maitotoxin, which increases intracellular calcium levels and activates calpain activity, induced the intracellular proteolysis of p34 into p20, p26 and p28 and the cleavage of p43, p50 and p84 into p38 and a novel protein, p34c. Proteolytic assays further indicated that p34, p43, p50 and p84 were substrates of calpain-1 and calpain-2 and that they generated proteolytic products that corresponded to those detected during the HCMV infectious period. Furthermore, substitution mutations in the putative calpain cleavage sites of p34 reduced accumulation of proteolytic products. The knockdown of endogenous calpain-1 and calpain-2 by RNA interference reduced accumulation of p20, p26 and p28 and concurrently increased levels of nascent p43, p50 and p84 during the infectious cycle. Intriguingly, calpain depletion enhanced viral genome synthesis. Moreover, HCMV-permissive cells that stably expressed p20, p26 or p28 exhibited reduced viral genome synthesis and mature virus production. Our findings suggest that cognate UL112-113 proteins derived from calpain-catalysed proteolysis are involved in the HCMV replication process.

Secocrassumol, a seco-cembranoid from the Dongsha Atoll soft coral Lobophytum crassum.

Chemical investigations on the Dongsha Atoll soft coral Lobophytum crassum led to the purification of a new seco-cembranoid, secocrassumol. The structural elucidation was established by extensive NMR, HRESIMS and CD data. The absolute configuration at C-12 was determined as S using a modified Mosher's acylation. Secocrassumol differs from previously known marine seco-cembranoid in that it possesses an unprecedented skeleton functionalized at C11-C12 bond cleavage. Secocrassumol showed antiviral activity against human cytomegalovirus (HCMV) with an IC50 value of 5.0 µg/mL.

Numerosol A-D, new cembranoid diterpenes from the soft coral Sinularia numerosa.

Four new cembrane-type diterpenes; numerosol A-D (1-4); along with a known steroid; gibberoketosterol (5); were isolated from the Taiwanese soft coral Sinularia numerosa. The structures of these metabolites were determined by extensive analysis of spectroscopic data. Gibberoketosterol (5) exhibited cytotoxicity against P-388 (mouse lymphocytic leukemia) cell line with an ED50 of 6.9 µM.

Sinugyrosanolide A, an unprecedented C-4 norcembranoid, from the Formosan soft coral Sinularia gyrosa.

Chemical investigations on the acetone extract of the Formosan soft coral Sinularia gyrosa have obtained a novel C-4 norcembranoid possessing an unprecedented tricyclo[9.3.0.0(3,8)]tetradecane skeleton, namely sinugyrosanolide A. The NMR spectroscopic data of the novel norcembranoid were completely assigned by using a combination of 2D NMR experiments including (1)H-(1)H COSY, HSQC, HMBC, and NOESY. The cytotoxicities, anti-HCMV (human cytomegalovirus) endonuclease activities and antibacterial activities were evaluated in vitro. It showed moderate cytotoxicity against P-388 (mouse lymphocytic leukemia) cancer cell line with an EC50 of 11.8µM.

New diterpenoids from soft coral Sarcophyton ehrenbergi.

Continuing chemical investigation on the acetone extracts of the soft coral Sarcophyton ehrenbergi collected off the coast of San-hsian-tai, Taitong County, Taiwan led to the isolation of two new diterpenoids, ehrenbergol C and acetyl ehrenberoxide B (1 and 2). The structures of these isolated metabolites were elucidated through extensive spectroscopic analyses. Moreover, in vitro tests show that compounds 1 and 2 displayed antiviral activity towards human cytomegalovirus, with EC50 of 20 and 8.0 µg/mL, respectively.

Secosteroids and norcembranoids from the soft coral Sinularia nanolobata.

Two new 9,11-secosteroids, 22α-acetoxy-24-methylene-3ß,6α,11-trihydroxy-9, 11-seco-cholest-7-en-9-one (1) and 11-acetoxy-24-methylene-1ß,3ß,6α-trihydroxy-9, 11-seco-cholest-7-en-9-one (2), as well as two known norcembranoids, 5-epi-sinuleptolide (3) and sinuleptolide (4), were isolated from the soft coral Sinularia nanolobata. The structures of these metabolites were elucidated on the basis of extensive spectroscopic analysis. The anti-HCMV (human cytomegalovirus) activity of 1-4 and its cytotoxicity against selected cell lines were evaluated.

Human cytomegalovirus UL76 elicits novel aggresome formation via interaction with S5a of the ubiquitin proteasome system.

HCMV UL76 is a member of a conserved Herpesviridae protein family (Herpes_UL24) that is involved in viral production, latency, and reactivation. UL76 presents as globular aggresomes in the nuclei of transiently transfected cells. Bioinformatic analyses predict that UL76 has a propensity for aggregation and targets cellular proteins implicated in protein folding and ubiquitin-proteasome systems (UPS). Furthermore, fluorescence recovery after photobleaching experiments suggests that UL76 reduces protein mobility in the aggresome, which indicates that UL76 elicits the aggregation of misfolded proteins. Moreover, in the absence of other viral proteins, UL76 interacts with S5a, which is a major receptor of polyubiquitinated proteins for UPS proteolysis via its conserved region and the von Willebrand factor type A (VWA) domain of S5a. We demonstrate that UL76 sequesters polyubiquitinated proteins and S5a to nuclear aggresomes in biological proximity. After knockdown of endogenous S5a by RNA interference techniques, the UL76 level was only minimally affected in transiently expressing cells. However, a significant reduction in the number of cells containing UL76 nuclear aggresomes was observed, which suggests that S5a may play a key role in aggresome formation. Moreover, we show that UL76 interacts with S5a in the late phase of viral infection and that knockdown of S5a hinders the development of both the replication compartment and the aggresome. In this study, we demonstrate that UL76 induces a novel nuclear aggresome, likely by subverting S5a of the UPS. Given that UL76 belongs to a conserved family, this underlying mechanism may be shared by all members of the Herpesviridae.

Parathyrsoidins A-D, four new sesquiterpenoids from the soft coral Paralemnalia thyrsoides.

Four new nardosinane-type sesquiterpenoids, parathyrsoidins A-D (1-4) were isolated from the soft coral Paralemnalia thyrsoides. The structures of parathyrsoidins A-D (1-4) were determined by extensive spectral analysis and their cytotoxicity against selected cancer cell lines as well as antiviral activity against human cytomegalovirus (HCMV) were evaluated in vitro.

New steroids from the soft coral Nephthea chabrolii.

A new cytotoxic 19-oxygenated steroid, nebrosteroid Q(1) and two new cytotoxic 19-norergosterols, nebrosteroids R and S (2 and 3) were isolated from the soft coral Nephthea chabrolii collected at San-Hsian-Tai. The structures of nebrosteroids Q-S (1-3) were elucidated by spectral analysis, and their cytotoxicity against selected cancer cells as well as antiviral activity against human cytomegalovirus (HCMV) were measured in vitro.

Briacavatolides D-F, new briaranes from the Taiwanese octocoral Briareum excavatum.

In the continued search for novel bioactive substances from the Taiwanese octocoral Briareum excavatum collected at Orchid Island, three new briarane-type diterpenoids, briacavatolides D-F (1-3) were isolated from the acetone extract. The structures of these compounds were elucidated by extensive NMR spectroscopic analysis and physical data. The anti-HCMV (human cytomegalovirus) activity of 1-3 and their cytotoxicity against selected cancer cell lines were evaluated.

Three new cembranoids from the Taiwanese soft coral Sarcophyton ehrenbergi.

In order to search for new bioactive substances from marine organisms, we have investigated the acetone extracts of the soft coral Sarcophyton ehrenbergi collected at San-Hsian-Tai, Taitong County, Taiwan. Chromatographic fractionation of the extracts of the octocoral S. ehrenbergi led to the isolation of three new cembranoids, (+)-12-ethoxycarbonyl-11Z-sarcophine (1), ehrenbergol A and B (2 and 3). The structures of these isolated metabolites were elucidated through extensive spectroscopic analyses. Moreover, metabolites 1-3 were evaluated in vitro for their cytotoxicity towards selected cancer cell lines and antiviral activity against human cytomegalovirus (HCMV).

Paralemnolide A, an unprecedented bisnorsesquiterpene from the Taiwanese soft coral Paralemnalia thyrsoides.

Paralemnolide A (1), possessing an unprecedented bisnorsesquiterpene skeleton, was isolated from the soft coral Paralemnalia thyrsoides. The structure of paralemnolide A was elucidated by extensive analysis of spectroscopic data. The anti-HCMV (human cytomegalovirus) activity of 1 and its cytotoxicity against selected cell lines were evaluated.

Briacavatolides A-C, new briaranes from the Taiwanese octocoral Briareum excavatum.

In order to search for novel bioactive substances from marine organisms, we have investigated the organic extracts of the Taiwanese octocoral Briareumexcavatum collected at Orchid Island. Three new briarane-type diterpenoids, briacavatolides A-C (1-3) as well as two known briaranes, briaexcavatolide U (4) and briaexcavatin L (5) were isolated from the acetone extract. The structures of these compounds were elucidated by extensive NMR spectroscopic analysis and physical data. The anti-HCMV (human cytomegalovirus) activity of 1-5 and their cytotoxicity against selected cancer cell lines were evaluated.