Dimeric analogs of immunosuppressive decapeptide fragment of ubiquitin { Alicja Kluczyk, a Marzena Cydzik, a Monika Biernat, a Remigiusz Bąchor, a Paweł Pasikowski, a Piotr Stefanowicz, a Jolanta Artym, b Michał Zimecki b and Zbigniew Szewczuk a * Our previous studies revealed that ubiquitin and its decapeptide fragment with the LEDGRTLSDY sequence, located on the exposed molecule loop, strongly suppressed the immune response. This suggested that the loop may serve as a functional epitope of ubiquitin molecule and that a possible mechanism of biological action of the synthesized peptides is associated with interfering in interactions of ubiquitin with other molecules. Ubiquitin is known to exist in oligomeric forms, which can interact with various oligomeric receptors. We designed and synthesized new dimeric analogs of the ubiquitin fragment, to probe whether dimeric peptides may have higher affinity towards the ubiquitin receptors responsible for immunosuppres- sion, which are believed to form oligomeric structures. Three dimerization strategies, N-terminus to N-terminus, C-terminus to C-terminus, and N-terminus to C-terminus (head-to-tail) via PEG derivatives were used to synthesize the dimeric peptides on solid support. In the course of our research, we developed a new and straightforward procedure of dimerization where a-amino groups of the C-terminal lysine residues of two peptide fragments were linked by PEG spacer directly on solid support. The effect of dimeric analogs on the immunological response was tested in the AFC in vitro experiment. The immunological tests showed that the head-to-tail dimerization caused a more profound increase in the biological activity than other tested dimerization methods. Our results suggest that such orientation of peptide components may correspond to orientation of the hypothetic ubiquitin receptors responsible for the immunomodulatory activity. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. Keywords: dimerization strategies; dimerization on solid support; PEG linker; linker length; ubiquitin fragments Introduction Dimerization or oligomerization of receptors is an essential step in various cellular signal transduction processes. Understanding of the multivalent character of some bioreceptors may be helpful in designing new compounds with high affinity and enhanced selectivity for a given receptor. Substances that are able to mod- ulate the receptor dimerization may control such a process and therefore affect the biological outcome. Dimerization of an active biomodulator often results in enhanced binding and improved pharmacological properties. Compounds consisting of two ligands connected by a proper spacer have a potential for bridging vicinal receptors. Therefore, the synthesis and biological evaluation of dimeric analogs of biologically active compounds is of great interest in biomedical applications [1]. Many biological ligands are also composed of clustered binding epitopes [2]. Properly designed dimeric analogs of bioligands should enhance interactions between neighboring receptors, possibly by reducing unfavorable entropic effects. The dimerization strategy was successfully applied to antagonists of chemokine receptor CXCR4, responsible for regulation of inflamma- tion and immune response, with linkers composed of oligoglycine and dicarboxylic acids [3]. Multimeric forms of cyclic RGD analogs attached to rigid scaffolds presented increased binding avidity towards avb3 integrin, although addition of oligo(ethylene glycol) spacers reduced the effect, presumably by enthropic effect [4]. On the other hand, the 18 F-labeled PEG 4 -E[PEG 4 -c(RGDfK)] 2 construct showed increased in vitro receptor binding affinity and significantly enhanced tumor uptake in vivo compared with PEG 4 - E[c(RGDfK)] 2 without the two PEG 4 spacers between the two RGD motifs [5]. The influence of dimerization and linker length on the activity of Smac fragments, binding to XIAP, an important regulator of apoptosis, was analyzed by biological and physicochemical methods [6,7]. A combinatorial approach based on dipeptide linkers attached to a central scaffold (bis-3,5-aminomethyl benzoic acid) was applied to the design of bivalent glutathione S-transferase inhibitors, resulting in compounds with increased affinity and selectivity, and better sol- ubility [8]. Two peptide fragments of discontinuous HLA-DQ epitope were linked by g-Abu residue to increase their immunosuppressory * Correspondence to: Zbigniew Szewczuk, Wydzial Chemii, Uniwersytet Wroclawski, ul. F. Joliot-Curie 14, 50-383 Wroclaw, Poland. E-mail: zbigniew. szewczuk@chem.uni.wroc.pl Dedicated to Prof. Ignacy Z. Siemion on the occasion of his 80th birthday. { A preliminary report of this work ‘Dimerization strategies of the immunosup- pressory decapeptide ubiquitin fragment’ was presented at the European Peptide Symposium in 2010. a Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland b Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland Abbreviations used: HLA, human leukocyte antigen; AFC, antibody-forming cells; SRBC, sheep red blood cells J. Pept. Sci. 2012; 18: 456–465 Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. Research Article Received: 21 December 2011 Revised: 30 March 2012 Accepted: 30 March 2012 Published online in Wiley Online Library: 25 May 2012 (wileyonlinelibrary.com) DOI 10.1002/psc.2416 456