This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 385–387 385 The natural product hybrid of Syringolin A and Glidobactin A synergizes proteasome inhibition potency with subsite selectivitywz Je´roˆ me Clerc, a Nan Li, b Daniel Krahn, a Michael Groll, c Andre´ S. Bachmann, d Bogdan I. Florea, b Herman S. Overkleeft* b and Markus Kaiser* a Received 30th June 2010, Accepted 17th August 2010 DOI: 10.1039/c0cc02238a The preparation of a Syringolin A/Glidobactin A hybrid (SylA–GlbA) consisting of a SylA macrocycle connected to the GlbA side chain and its potent proteasome targeting of all three proteasomal subsites is reported. The influence of the syrbactin macrocycle moiety on subsite selectivity is demonstrated. Proteasomal protein degradation plays a critical role in innumerous biological processes such as cell division, immune responses, apoptosis and gene expression. 1 Proteolysis takes place in the 20S core particle (CP), a 670 kDa cylinder built up from four stacked heptameric rings, in which only the b1, b2 and b5 subunits harbor distinct proteolytic activities with different substrate specificities. 2 Besides the constitutive 20S CP, alternative 20S proteasome species are expressed in a tissue-specific manner. In the immunoproteasome, the constitutive b1, b2 and b5 subunits are replaced by the b1i, b2i and b5i subunits while the thymoproteasome encompasses the proteolytically active b1i, b2i and b5t subunits. 3,4 These subunits display alternative cleavage activities, thereby modulating the overall trimming of proteins during degradation. Active-site directed proteasome inhibitors are valuable anticancer agents as well as versatile research tools. 5 Consequently, various rationally-designed small molecules and natural products have been explored in recent years. 6 These inhibitors usually target the proteasomal proteolytic subsites with different potencies. To date, the physiological consequences of such subsite specific inhibition are not well understood although distinct roles of individual proteasomal subunits for certain biological processes have already been observed. 7 This pinpoints that proteasome inhibitors with different subsite potencies could induce diverse pharmacological effects and consequently demands for a deeper understanding of the structural determinants governing subsite selectivity of proteasome inhibitor classes. We recently elucidated the syrbactin natural products syringolin A (SylA), syringolin B (SylB) and glidobactin A (GlbA) as mechanism-based irreversible proteasome inhibitors (for the chemical structures of the syrbactins used during this study, see ESIz). 8 These natural products exert distinct proteasomal subsite selectivities in biochemical activity assays as well as in structural studies and selectively target the proteasome in complex proteomes. 9 SylA targets all three proteolytic activities, albeit with different potencies. SylB and GlbA on the other hand selectively inhibit b2 and b5 even at high concentrations, inviting the hypothesis that the macro- cyclic residue critically influences subsite selectivity while the N-terminal lipophilic tail strongly enhances inhibition potency. This hypothesis is supported by the finding that model compound SylA–LIP, consisting of a SylA core structure attached to a lipophilic side chain, inhibits all three proteolytic sites, thereby displaying the most potent inhibition of all syrbactins reported so far. 10 While SylA, GlbA and SylA–LIP showed promising anticancer activities in cell-based assays, the order of activity was somewhat surprising, with GlbA being more potent than SylA–LIP. 11 To gain further insights into syrbactins’ structure–activity relationships, we decided to put to the test two additional derivatives, being SylA–GlbA as a ‘true’ hybrid of Syringolin A and Glidobactin A, together with sat-SylA as a negative control. The synthesis of SylA–GlbA started with a Horner– Wadsworth–Emmons (HWE) olefination of octanal (1) using (E)-triethyl-4-phosphonocrotonate and LDA (Scheme 1). The resulting ethyl ester 2 was hydrolyzed with LiOH to yield the free acid 3. Peptide coupling of Boc–Thr(OtBu)–OH to the previously reported SylA macrocycle 4 12 led to intermediate 5 which upon deprotection and PyBOP-mediated coupling of 3 yielded the desired SylA–GlbA hybrid (6). The synthesis of sat-SylA is reported in the ESI.z With these additional derivatives in hand, their inhibition potency and subsite selectivity was established using competitive activity-based protein profiling (ABPP) in cell lysates and living cells in comparison with known syrbactins. 13 To this end, HEK cell lysates were preincubated with varying concentrations of the syrbactins and profiled with the proteasomal activity based probe (ABP) MV151 (Fig. 1, for chemical structures of the ABPs, see ESIz). 14 The results from these experiments were generally in good agreement with the results from the previous biochemical inhibition studies with a Zentrum fu ¨r Medizinische Biotechnologie, Fakulta ¨t fu ¨r Biologie und Geographie, Universita ¨t Duisburg-Essen, 45117 Essen, Germany. E-mail: markus.kaiser@uni-due.de; Fax: +49 201 1833135; Tel: +49 201 1832949 b Leiden Institute of Chemistry and Netherlands Proteomics Centre, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands. E-mail: h.s.overkleeft@chem.leidenuniv.nl; Fax: +31 71 5274307; Tel: +31 71 5274342 c Center for Integrated Protein Science at the Department Chemie, Technische Universita ¨t Mu ¨nchen, Lichtenbergstr. 4, 85747 Garching, Germany d Cancer Research Center of Hawaii, University of Hawaii at Manao, 1236 Lauhala Street, Honolulu, Hawaii 96813, USA w This article is part of the ‘Emerging Investigators’ themed issue for ChemComm. z Electronic supplementary information (ESI) available: Experimental details of synthesis of SylA–GlbA, synthesis of sat-SylA, chemical structures of syrbactins and ABPs used during these studies, and supplementary figures and experimental procedures for competitive ABPP studies. See DOI: 10.1039/c0cc02238a COMMUNICATION www.rsc.org/chemcomm | ChemComm Downloaded on 10 August 2012 Published on 07 September 2010 on http://pubs.rsc.org | doi:10.1039/C0CC02238A View Online / Journal Homepage / Table of Contents for this issue