Secomycalolide A: A New Proteasome Inhibitor Isolated from a Marine Sponge of the Genus Mycale

A new oxazole-containing proteasome inhibitor, secomycalolide A, together with known mycalolide A and 30-hydroxymycalolide A, was isolated from a marine sponge of the genus Mycale. They showed proteasome inhibitory activities with IC50 values of 11–45 μg/mL.


Introduction
Mycalolides have been isolated from marine sponges of the genus of Mycale [1][2][3] and the hard coral Tubastrea faulkneri [4], and their structures are elucidated to be macrocyclic lactones characterized with an unusual tris-oxazole moiety and N-methylformyl terminus. So far, kabiramides [5], ulapualides [6], halichondramides [7], and jaspamides [8] are known as structurally related metabolites. Mycalolides A-C exhibited significant cytotoxicity against B-16 melanoma cells [1] and potent actin depolymerizing activity [9]. The ubiquitin-proteasome proteolytic pathway plays a major role in selective protein degradation and regulates various cellular events including cell growth and apoptosis [10][11][12][13]. Ubiquitin, composed of 76 amino acids, attaches to a target protein prior to degradation. The polyubiquitin chains are recognized by the 26S proteasome, an intracellular highmolecular mass protease subunit complex, and the protein portion is degraded by the proteolytic active sites in a cavity in the 26S proteasome [10,11]. Several proteasome inhibitors show anti-tumor activity against various tumor cells that are resistant to conventional chemotherapeutic agents. Proteasome inhibitors have been reported to inhibit the degradation of Iκ-B followed by suppression of NF-κB transcriptional activity to induce apoptosis [14]. To date, synthetic peptides, such as MG132 [15] and PS-341 (bortezomib, Velcade ® ) [16], and natural products, including lactacystin [17][18][19], epoxomycin [20,21], and salinosporamide A [22], have been reported to inhibit proteasome activity. Among them, PS-341 has been recently approved by FDA for multiple myeloma treatment [23]. Interestingly, different classes of proteasome inhibitors can differentially affect the degradation of various proteasome substrates, resulting in diverse cellular responses [14]. Proteasome inhibitors are now under intensive investigation not only for anti-cancer drugs but also for drugs to treat inflammatory and immune deficiency diseases [24]. In the course of our search for inhibitors against the ubiquitin-proteasome pathway, we succeeded in isolating new agosterol derivatives [25] and a pyrone derivative named himeic acid A [26] as proteasome inhibitors and a ubiquitin-activating enzyme (E1) inhibitor, respectively. In addition, we found that girolline, an anti-cancer compound, is the first agent inhibiting the recruitment of polyubiquitinated p53 to the proteasome [27]. In this paper, we describe the isolation, structure elucidation, and proteasome inhibitory activity of three mycalolides.

Results and Discussion
The MeOH extract of a sponge collected from shallow waters off Sugashima Island 130 km southeast of Osaka was subjected to solvent partitioning between EtOAc and water. The active EtOAc fraction was further partitioned between n-hexane and 90% MeOH/H 2 O, and the latter fraction was purified by ODS chromatography and ODS HPLC to afford a new mycalolide derivative, secomycalolide A (1) together with known mycalolide A (2) [1] and 30-hydroxymycalolide A (3) [2].
The proteasome displays multicatalytic activities, e.g. chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities. Inhibitory activities of three mycalolides were tested using a chymotrypsin-like substrate, and the IC 50 values of 1, 2, and 3 were found to be 11, 30, and 45 µg/mL, respectively.

Conclusion
We have isolated a new mycalolide derivative secomycalolide A (1) together with known mycalolide A (2) and 30-hydroxymycalolide A (3). They showed moderate inhibitory activities against chymotrypsin-like activity of the proteasome. Among three mycalolides, seco-oxyazole derivative 1 showed the most potent inhibitory activity. So far, cytotoxicity [1] and potent actin depolymerizing activity [9] of mycalolides have been reported; however, this is the first report of proteasome inhibitory activity of mycalolides.

Experimental
General NMR spectra were recorded on a JEOL GSX500 in CDCl 3 . All chemical shifts were reported with respect to the residual solvent peaks (δ H 7.26). Mass spectra were measured on a JEOL SX-102 mass spectrometer. The fluorescence intensity (excitation, 360 nm; emission, 460 nm) was measured using a BIO-RAD VersaFluor Fluorometer.

Extraction and Isolation
A marine sponge of the genus Mycale was collected from shallow waters off Sugashima Island 130 km southeast of Osaka. The frozen material (4.2 kg, wet wt) was extracted with MeOH (3 L × 3). The extract was concentrated under reduced pressure and extracted with EtOAc.

Preparation of Proteasome-enriched Fraction
Proteasome used in this study was partially purified from rat liver. The liver was dissected and homogenized in ice-cold lysis buffer consisting of 20 mM HEPES, pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM dithiothreitol, 2 mM ATP, and 10% glycerol at 4 o C for 5 min.
The extract was filtered through cheese cloth, and the filtrate was immediately centrifuged at 10,000 rpm for 5 min. The supernatant was centrifuged at 105,000 × g for 20 min, and the resultant supernatant was further centrifuged at 300,000 × g for 2 h. The precipitates thus obtained were suspended in lysis buffer containing 50% glycerol and used as the crude proteasome-enriched preparation.

Assay for Proteasome Activity
The fluorogenic substrate succinyl-leucyl-leucyl-valyl-tyrosine 4-methylcoumaryl-7-amide (MCA) (Peptide Institute, Inc., Osaka) was used as a substrate for chymotrypsin-like activity of the proteasome. The proteasome-enriched fraction in a mixture (0.1 mL) that contained 50 mM Tris-HCl, pH 7.8, 1 mM dithiothreitol, and 5 mM EDTA was pre-incubated with each inhibitor at 30 o C for 10 min. Then, 0.05 mM substrate was added to the mixture and the mixture was further incubated at 30 o C for 1 h. The reaction was stopped by adding 0.1 mL of 10% SDS and the fluorescence intensity owing to 7-amino-4-methylcoumarin (AMC) was measured (excitation, 360 nm; emission, 460 nm). The value of IC 50 , the concentration required for 50% inhibition of proteasome inhibitory activity, was calculated from the data of duplicate measurements.