Research paper The cell-penetrating peptide octa-arginine is a potent inhibitor of proteasome activities Alexander Kloß a , Peter Henklein a , Dagmar Siele a , Marion Schmolke b , Sébastien Apcher c , Lothar Kuehn d , Paul W. Sheppard e , Burkhardt Dahlmann a, * a Institut für Biochemie/CCM, Charité-Universitätsmedizin-Berlin, Berlin, Germany b Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhems-Universität, Münster, Germany c Laboratoire de pharmacologie experimentale et clinique, INSERM u716, Paris, France d Klinische Biochemie und Pathobiochemie, Deutsches-Diabetes-Forschungsinstitut University, Düsseldorf, Germany e BIOMOL International LP, Exeter, UK article info Article history: Received 16 July 2008 Accepted in revised form 31 October 2008 Available online 7 November 2008 Keywords: Cell-penetrating peptides Octa-arginine Proteasome Inhibitor Ubiquitin-conjugated proteins HeLa cells abstract Oligo-arginines are cell-penetrating peptides and find use as carriers for transportation of various mem- brane-impermeable biopharmaceuticals into target cells. We have found that oligo-arginines of a length of 4–10 amino acids, but especially (Arg) 8 , are able to inhibit the major intracellular proteolytic system, the proteasome, with mixed-type inhibition characteristics. The IC 50 values of (Arg) 8 for the proteasomal chymotrypsin-like and caspase-like activities are approximately 100 and 200 nM, respectively. The inhi- bition of the trypsin-like activity never exceeds 50% even at micromolar concentrations. (Arg) 8 also inhib- its 20S proteasome/PA28 complexes as well as 26S proteasomes, although with a decreased efficiency. Due to its cell membrane-penetrating capability, incubation of HeLa cells in the presence of (Arg) 8 resulted in an impaired activity of proteasomes going along with an accumulation of high-molecular mass ubiquitin-conjugated proteins, the preferred substrates of 26S proteasomes. The in vivo susceptibil- ity of the three proteasome activities resembles that found in vitro with chymotrypsin-like > caspase- like > trypsin-like activities. Since inhibition of the proteasome system might affect fundamental basic cellular processes but on the other side might also prevent the degradation of a proteinacous cargo, we suggest that this proteasome inhibitory activity should be taken into account when oligo-arginines are being considered for use as vectors for the intracellular delivery of pharmaceuticals. Ó 2008 Elsevier B.V. All rights reserved. 1. Introduction One obstacle to the successful delivery of biopharmaceuticals in the drug-based therapeutic interventions is the impermeability of biological membranes. A great step forward in this area came with the discovery and development of cell-penetrating peptides (CPPs) [1,2]. One important group of CPPs is oligo-arginine vectors, the membrane permeability of which was identified fortuitously in the arginine-rich sequence responsible for the internalization of the HIV Tat protein [3]. Oligo-arginines have been used to transfer various compounds and proteins into cells and tissues [4]. Although the mechanism of uptake of oligo-arginines into the cell is not entirely clear, interaction with negatively charged mem- brane proteoglycans, macropinocytosis, and actin rearrangement seems to be involved [5,6]. Even less well understood is the fate of oligo-arginine peptides within the cell. Whilst one fraction might enter lysosomes, another seems to escape from endosomes via the Golgi by retrograde transport, or by other undefined mech- anisms, to end up in the cytosol [7]. Membrane transduction of oli- go-arginines directly into the cytosol and not mediated by macropinocytosis was observed in certain cases [8,9]. On its tran- sition through the various cell compartments, oligo-arginine is ex- posed to many proteases and peptidases and, thus, may constantly be subject to degradation. In contrast, oligo-arginines have been found to be potent inhibitors of proteases located within the endo- somal-lysosomal compartment, with examples including furin [10,11] and cathepsin C [12]. This property may ensure that con- siderable amounts of oligo-arginine peptides survive administra- tion and, when applied intravascularly, allow them to penetrate deep into the tissue [13]. Oligo-arginine-mediated inhibition of furin has been used to suppress productive infection of T-cell lines by HIV-1, since furin is essential for the correct processing of the HIV envelope protein gp160 [14]. 0939-6411/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2008.10.016 Abbreviations: Arg, arginine; Bz-VGR, benzoyl-Val-Gly-Arg; CPP, cell-penetrat- ing peptide; AMC, 7-amino-4-methylcoumarin; Suc-LLVY, succinyl-Leu-Leu-Val- Tyr; Z-LLE, benzyloxycarbonyl-Leu-Leu-Glu. * Corresponding author. Institut für Biochemie/CCM, Charité-Universitätsmedi- zin-Berlin, Monbijoustr 2, 10117 Berlin, Germany. Tel.: +49 (0)30 450528309; fax: +49 (0)30 450528916. E-mail address: burkhardt.dahlmann@charite.de (B. Dahlmann). European Journal of Pharmaceutics and Biopharmaceutics 72 (2009) 219–225 Contents lists available at ScienceDirect European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb