Article pubs.acs.org/jmc 3-Aminopiperidine-Based Peptide Analogues as the First Selective Noncovalent Inhibitors of the Bacterial Cysteine Protease IdeS Kristina Berggren,†,§ Reine Vindebro,‡ Claes Bergström,† Christian Spoerry,‡ Helena Persson, Tomas Fex,§ Jan Kihlberg,§ Ulrich von Pawel-Rammingen,‡ and Kristina Luthman*,† ‡ †Department of Chemistry, Medicinal Chemistry, University of Gothenburg, SE-412 96 Göteborg, Sweden ‡Department of Molecular Biology and Umeå Centre for Microbial Research, Umeå University, SE-901 87 Umea,̊ Sweden §AstraZeneca R&D Mölndal, SE-431 83 Mölndal, Sweden *S Supporting Information ABSTRACT: A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies. ■ INTRODUCTION Proteolytic activities play a decisive role in all aspects of cellular life, including cell division processes, metabolism and catabolism, protein translocation, immune defense mechanisms, and more.1 The human bacterial pathogen Streptococcus pyogenes (or group A streptococcus) is the causative agent of a great variety of infections, ranging from mucocutaneous infections of the throat and skin to life threatening conditions including necrotizing fasciitis and streptococcal toxic shock syndrome.2 More than 600 million people are estimated to suffer from streptococcal pharyngitis alone, and mucocutaneous infections cause substantial morbidity and considerable economic loss to society.3 Postinfectious sequelae include serious inflammatory diseases such as acute rheumatic fever (ARF), rheumatic heart disease (RHD), and poststreptococcal glomerulonephritis (PSGN). Inflammatory autoimmune dis- eases such as guttate psoriasis have also been associated with streptococcal infections4 although the underlying molecular mechanisms still remain to be solved. S. pyogenes employs two papain-like cysteine proteases to adapt to the dynamic environment in its human host and to evade the human immune response: the classical streptococcal cysteine protease SpeB and the immunoglobulin G (IgG) degrading protease, IdeS.5,6 Both enzymes adopt a canonical papain-like structural fold and show, despite the lack of sequence similarity, large structural similarities.7−10 Besides IdeS, also SpeB and papain have the ability to cleave the IgG heavy chain. The SpeB cleavage site is identical to IdeS cleavage at a defined site between glycine residues 236 and 237, creating one F(ab′)2 fragment and two identical 1/2Fc fragments.6,11,12 Papain cleavage occurs at the peptide bond between histidine in position 224 and threonine in position 225 of the hinge region of IgG, thereby generating two Fab fragments and one Fc fragment.13 However, the proteases have distinguished substrate recognition properties: SpeB and papain exhibit a broad proteolytic activity and degrade or activate a wide variety of substrates.1,14 IdeS, on the other side, is highly specific and recognizes only IgG as substrate.6,12,15 Furthermore, IdeS, in contrast to papain and other prokaryotic cysteine proteases, including SpeB and the staphylococcal cysteine protease StpA,16 is not inhibited by the classical cysteine protease inhibitor E64.6,12 This interesting property is explained by an unusually narrow active site cleft that does not offer enough space to accommodate the P3 residue of E64 and thus points to distinct substrate recognition properties.7 Given the essential role of IdeS in the evasion of IgG mediated immune responses, there is a high medical interest to identify specific inhibitors for prokaryotic cysteine proteases. Furthermore, IdeS is currently evaluated as a therapeutic agent to treat conditions in which antibodies reacting against human antigens misdirect the human immune response toward the Received: June 27, 2011 Published: February 27, 2012 © 2012 American Chemical Society 2549 dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560