Journal of Medicinal Chemistry Figure 4. IdeS activity in presence of peptides and peptide analogues using IgG1 as substrate. IdeS activity is expressed relative to a standard curve obtained in absence of inhibitors. (R) and (S) indicate the different stereoisomers of each analogue; “pip” represents the piperidine moiety replacing glycine residues. The enzyme activity was determined for IdeS at a final concentration of 0.8 μM in the presence of 4.8 × 103 molar excess of peptides/peptide analogues. indicates that the extra amino acids are not contributing to more favorable interactions to accomplish more efficient binding or are too flexible to gain favorable binding energies. Thus, when evaluating ligand efficiency (LE), where binding energy per atom is considered, (S)-pipG ((S)-3) is the most favorable peptide analogue in the series.26 The pKa values for (R)- and (S)-pipG (3) and (R)- and (S)- LpipGP (18) have been determined (Table 1). Interestingly, the two pKa values differ considerably. For (R)- and (S)-pipG (3), the acyclic amine bears the electron withdrawing acetamide group, thus the piperidine nitrogen would be expected to be protonated first. For (R)- and (S)-LpipGP (18), the primary amine would be expected to have the pKa of an amino acid amide, i.e., between 8 and 9, which is what is found. The acyclic secondary amines will not be protonated at physiological pH, and the pip moiety is therefore able to act as a glycine isostere. Specificity of Peptide Analogues. IdeS exhibits pro- nounced substrate specificity in comparison to other IgG cleaving cysteine proteases of the papain superfamily.12 Still, the Table 1. pKa Values for the pipG and LpipGP Analoguesa Article extended structural similarity of papain-like proteases raised the question whether piperidine containing peptide analogues could have inhibitory capacity toward other IgG cleaving proteases or in fact harbor a potential specificity toward IdeS. Therefore, peptides and peptide analogues were also screened for activity toward the streptococcal cysteine protease SpeB and the family protease papain (Figure 5). Interestingly, very different inhibitory profiles were obtained that apparently distinguish the proteases from the profile obtained for IdeS and also from each other (Figures 4 and 5). In general, the inhibitory effect of all the tested compounds was much less pronounced on SpeB compared to inhibition of IdeS. Hardly any of the pip236G-fragment containing analogues had any inhibitory effect on SpeB activity (Figure 5A). Notable is however that an inhibitory capacity was observed for the LLGG peptide. In contrast to the effect observed on IdeS activity (Figure 4), the degree of inhibition was not improved when testing LLpipG (10) peptide analogues in which Gly236 was replaced with a piperidine moiety. However, a clearly improved degree of inhibition could be observed when testing the ethylated analogues (R)- and (S)-LLpipEtG (11). In addition, (R)- and (S)-LGpipP (23) containing a similar tertiary amine fragment as the ethylated analogues also inhibited SpeB activity, which is in contrast to the effect observed for IdeS. Thus, the absence of the possibility of hydrogen bond donation and the rigid structure of neighboring piperidine and proline rings appear to increase the interaction between the LGpipP (23) analogues and SpeB. Consequently, LpipGP (18) analogues do not affect SpeB activity. The specificity study was extended further to include the cysteine protease papain. Like SpeB also, papain exhibits a broad proteolytic activity and degrades a wide variety of substrates, including IgG. The analysis of putative inhibitory effects of peptides and piperidine-containing analogues on papain revealed that the enzyme was clearly very sensitive toward most compounds tested (Figure 5B). In analogy to SpeB, the positioning of the piperidine in the peptide sequence favors a replacement of the glycine corresponding to G237, resulting in the most efficient papain inhibitors in the series, with (R)-LGpipP ((R)-23) being the most active analogue. In summary, the most active inhibitors of IdeS, (R)-pipGP ((R)-16) and (S)-LpipGP ((S)-18), are even more active toward papain but show no inhibitory activity toward SpeB. measured predicted compd (R)-pipG (S)-pipG (R)-LpipGP (S)-LpipGP pKa1 4.41 4.35 5.13 5.21 pKa2 10.11 10.06 8.46 8.53 pKa1 5.40 7.35 pKa2 9.84 9.13 aDetermination of pKa values performed by a screening method based on pressure-assisted capillary electrophoresis and mass spectrometry.27 Prediction of pKa values was performed using the ACD/Laboratories software (Advanced Chemistry Development, Inc.).28 2554 dx.doi.org/10.1021/jm201517a | J. Med. Chem. 2012, 55, 2549−2560