DOI: 10.1002/cbic.201200113 Modulation of Proteasome Machinery by Natural and Synthetic Analogues of the Marine Bioactive Compound Petrosaspongiolide M Luigi Margarucci, Alessandra Tosco, Rosa De Simone, Raffaele Riccio, Maria Chiara Monti,* and Agostino Casapullo* [a] The ubiquitin (Ub)-proteasome pathway is a nonlysosomal pro- tein-degradation system responsible for degrading both dam- aged/unfolded proteins, dangerous for normal cell growth and metabolism, [1, 7] and critical regulatory proteins. It is composed of the catalytic barrel 20S and different regulatory subunits, such as 19S and 11S. The central core of this system is the 20S proteasome, a cylindrical structure with an internal cavity com- posed of four rings, each containing seven different a and b subunits; only three of the seven b subunits, b1, b2, and b5, located inside the main chamber are responsible for the proteolytic activity. [3–4] In particular, b1 is associated with the peptidyl-glutamyl peptide-hydrolyzing (PGPH) activity, turned towards limited branched-chain amino acids, while b2 and b5 are responsible for the trypsin-like (T-L) and chymotrypsin-like (ChTL) activities, respectively. [2] Nevertheless, it is widely as- sumed that inhibition of the ChTL activity, which is the primary target of the proteasome inhibitors used in research and cancer therapy, reflects the degree of inhibition of protein breakdown. [3, 7] Moreover, it is also acknowledged that the 19S cap and the 11S activator allosterically regulate the gate status of the 20S barrel. [4] In more detail, 11S stimulates the peptidase activity of the 20S proteasome in vitro and can facilitate prod- uct release in vivo. [8] Due to their ability to induce apoptosis and interfere with cell cycle regulation, [9] gene expression, [10] carcinogenesis and DNA repair, [11, 12] proteasome inhibitors have recently been gaining greater attention as novel pharmacological tools in cancer drug discovery. Several natural and synthetic protea- some inhibitors have been discovered and investigated, with bortezomib being the first molecule to enter clinical trials and receive approval for the treatment of patients with multiple myeloma (MM). [13, 14] In a previous study, we reported petrosas- pongiolide M (PM), a marine sesterterpene containing a g-hy- droxybutenolide ring, as a new inhibitor of the proteasome machinery, able to strongly modulate the ChTL and PGPH ac- tivities. [15] Thus, in an attempt to broaden our knowledge on the structural moieties responsible for the activity of PM, and to find new chemical scaffolds able to modulate the protea- some activity, we tested other natural compounds belonging to the petrosaspongiolide family, [16] and a small library of sim- plified PM analogues (see Scheme 1), which were previously synthesized by our research group as modulators of microbial prostaglandin E synthase-1 (mPGES-1) expression (for further details on the syntheses of compounds 4 and 4a, see the Sup- porting Information). [17–20] The proteasome inhibition was measured for each catalytic subunit by applying a fluorimetric assay, using three different peptide substrates. [21, 22] The 11S activator complex was added to the 20S catalytic system to increase its intrinsic activity. As reported in Table 1, the natural analogues of PM bearing acetyl groups at positions C21 and C24 (PN) or a hydroxy group at position C24 (PP) were almost inactive, since only a small degree of inhibition of the ChTL activity was detected, even though PP did show a higher effect on the PGPH activity than PM. Furthermore, the low inhibitory effect reported for acetyl-PM (a semisynthetic analogue of PM) is a consequence of the acetylation of the masked aldehyde function at position C25, a potential reactive site for covalent binding to the pro- teasome complex. Among the synthetic PM analogues, we selected a first gen- eration of molecules characterized by a 3-bromo-substituted g- hydroxybutenolide ring bearing different chemical scaffolds at C4 instead of the terpenoid portion, such as b-naphthyl (1), Table 1. IC 50 values [mm] of PM analogues on the chymotrypsin-like (ChTL), trypsin-like (T-L) and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities of the 20S–PA28 proteasome complex. [a] Compd ChTL PGPH T-L PM 0.085 Æ 0.1 1.05 Æ 0.1 > 10 PP 3.26 Æ 0.4 0.05 Æ 0.01 > 10 PN 5 Æ 0.7 5 Æ 0.7 > 10 Acetyl-PM 3.2 Æ 0.4 2.9 Æ 0.4 > 10 1 2.4 Æ 0.3 n.i. > 10 2 n.i. n.i. > 10 2a n.i. n.i. > 10 3 n.i. 6.9 Æ 1.3 > 10 3a n.i. n.i. > 10 4 n.i. 1.4 Æ 0.2 > 10 4a n.i. n.i. > 10 5 n.i. 1.05 Æ 0.1 > 10 6 5.9 Æ 0.8 n.i. > 10 1a 1.8 Æ 0.27 n.i. > 10 1b 0.06 Æ 0.009 0.22 Æ 0.03 > 10 1c n.i. n.i. > 10 1d 0.07 Æ 0.01 0.07 Æ 0.01 > 10 1e 3.7 Æ 0.6 1.1 Æ 0.1 > 10 [a] The values reported are the average of three independent determina- tions; n.i.: no inhibition at the highest concentration tested. [a] Dr. L. Margarucci, Dr. A. Tosco, Dr. R. De Simone, Prof. R. Riccio, Dr. M. C. Monti, Prof. A. Casapullo Department of Pharmaceutical and Biomedical Sciences University of Salerno Via Ponte don Melillo, 84084 Fisciano (Italy) E-mail : casapullo@unisa.it mcmonti@unisa.it Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201200113. 982  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2012, 13, 982 – 986