Journal of Medicinal Chemistry Determination of Inhibition Constants. Ki values were determined by accessing velocity curves at different substrate concentrations in the presence or absence of inhibitor. Inhibitor concentrations were 5, 2, and 0.5 mM for (R)-LpipG ((R)-7) and 5, 2.5, and 1 mM for (S)- pipG ((S)-3). For IdeS, repurified polyclonal IgG at concentrations of 150, 100, 50, 20, or 10 μM, was used in reactions with 2 μM of IdeS. Reactions were analyzed by non- reducing SDS page gel analysis to allow measurements of the initial substrate cleavage. The enzyme velocity v was determined according to the following equation: For papain, Nα-benzoyl-DL-arginine 4-nitroanilide hydrochlor- ide (Sigma) at concentrations of 6, 3, 1, 0.5, or 0.25 mM was used with 5 μM of papain and inhibitor. Ki nonlinear regression (curve fit) for competitive inhibition and enzymatic kinetics were calculated using GraphPad Prism version 5.04 for Windows, GraphPad Software, San Diego California, www. graphpad.com. Surface Plasmon Resonance Spectroscopy Assays. Surface plasmon resonance spectroscopy assays were performed with 1042.5 RU of Protein A (Abcam) covalently bound to a CM5 chip using NHS/EDC coupling according to the manufacturer’s instructions. One part of the IgG1/IdeS reaction was mixed with two parts water before applying the sample on the Protein A-CM5 chip. Run conditions on the Biacore X100 machine were 5 μL/min flow rate, a 90 s sample injection time, HBS-EP+ (Biacore) as running buffer, and 20 mM glycine pH 2.0 as regeneration solution of the chip. Report point for amount of protein bound to the chip surface was retrieved at 190 s after sample injection stop. Amount of IgG1 cleavage was first plotted as percentage decrease between PBS-control (uncleaved IgG1) and IdeS/IgG1 samples. A standard curve for IdeS concentration was made without the presence of peptide or peptide analogue. The experimental data obtained from assays with IdeS together with peptide/peptide analogue experiments were plotted to the standard curve and presented as approximate fraction of active IdeS at any given time point. Papain Activity Assay. Inhibition of the enzymatic activity of papain by peptides and peptide analogues was determined by a colorimetric assay. Papain (Fluka 76218) at a concentration of 1.23 μM was diluted in assay buffer (81 μL) (123 mM phosphate buffer pH 6.2, 1.23 mM EDTA, 1.23 mM DTT), mixed with peptides or peptide analogues (16 μL) (30 mM in 50% DMSO, 50 mM phosphate buffer pH 6.2) and incubated for 10 min at room temperature. The final concentration of papain during the experiment was 0.9 μM. The chromogenic papain substrate Nα-benzoyl-DL-arginine-4-nitroanilide hydrochloride (3 μL) (Sigma B4875, 100 mM in DMSO) was added, and the reaction was incubated for 2 h at 37 °C before termination by the addition of glacial acetic acid (10 μL). Abs405 was measured to determine the amount of cleaved substrate. Samples were run in duplicates on two separate occasions. Purification of SpeB. For purification of SpeB, S. pyogenes strain 5448 was grown for 16 h in Todd−Hewitt broth (TH) (BD Biosciences) at 37 °C; 5% (v/v) CO2. The growth media was cleared by centrifugation and subjected to ammonium sulfate precipitation (50−80% (w/v)). The resulting pellet was dissolved in 1× PBS, dialyzed o/n at 4 °C against 20 mM sodium acetate buffer, pH 5. The sample was further purified on a HiTrap SP FF anion exchange column (GE Healthcare), equilibrated in 20 mM sodium acetate, pH 5. Proteins were eluted by a linear NaCl gradient and SpeB eluted at approximately 0.2 M NaCl. The eluted protein fraction was dialyzed o/n against 1× PBS and kept at −20 °C. SpeB Activity Assay. SpeB activity was measured essentially as previously described.30 Briefly, SpeB was activated with 2 mM dithiothreitol (DTT) for 30 min at 37 °C and mixed with 150 μL of 1 mM substrate solution, n-benzoyl-proline-phenylalanine-arginine-p- nitroanilide hydrochloride (BPFA) (pH 4) (Sigma), in 0.1 M phosphate buffer (pH 6). The change in absorbance at OD405 was ■ ■ ■ ■ recorded. The final concentration of SpeB during the experiment was 0.28 μM The amount of active SpeB was determined by active-site titration using various amount of the cysteine protease inhibitor E-64 as previously described.31 For inhibition assays, 11 μL of activated SpeB (1.12 μM in PBS) was mixed with 2 μL of peptide or peptide analogue (30 mM in 50% DMSO, 50 mM phosphate buffer pH 7.4) and incubated for 15 min at room temperature prior to the addition of substrate solution. All assays were performed in triplicate. ■ ASSOCIATED CONTENT *S Supporting Information Synthetic procedures and characterization of compounds shown in Schemes 3−6; NMR spectral assignments of (R)-1, (R)-2, (R)-3, (R)-4, (R)-7, and (R)-10; 1H and 13C NMR spectra of tested compounds. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Phone: +46-31-786 9031. Fax: +46-31-772 1394. E-mail: luthman@chem.gu.se. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the Swedish Research Council (project numbers 2005-3145 (K.L.) and 2009-4997 (UvPR) and Insamlingsstiftelsen at Umeå University. We thank AstraZeneca R&D Mölndal, Sweden, for financial support of the graduate student fellowship for KB. We also thank Madeleine Åhman, AstraZeneca, Sweden, for help with the pKa measurements. ABBREVIATIONS USED Boc, tert-butyloxycarbonyl; Cbz, benzyloxycarbonyl; Cbz-OSu, N-(benzyloxycarbonyloxy)succinimide; DMSO, dimethylsulf- oxide; E64, 1-[N-[(3-trans-carboxyoxirane-2-carbonyl)-L- leucyl]amino]-4-guanidinobutane; EDC, N-(3-methylamino- propyl)-N′-ethylcarbodiimide; ee, enantiomeric excess; HOBt, hydroxybenzotriazole; HPLC, high pressure liquid chromatog- raphy; IdeS, immunoglobulin G-degrading enzyme of Strepto- coccus pyogenes; NMR, nuclear magnetic resonance; pip, the piperidine moiety replacing glycine residues; rac, racemic; SDS- PAGE, sodium dodecyl sulfate polyacrylamide gel electro- phoresis; SpeB, streptococcal pyogenic exotoxin B; TFA, trifluoroacetic acid; TLCK, tosyl lysyl chloromethyl ketone; TPCK, tosyl phenylalanyl chloromethyl ketone; Z-LVG CHN2, N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethylketone Article REFERENCES (1) Rawlings, N. D.; Tolle, D. P.; Barrett, A. J. MEROPS: the peptidase database. Nucleic Acids Res. 2004, 32, D160−D164. 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