Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C

Abstract

Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-013-1654-2) contains supplementary material, which is available to authorized users.

Differential effects of D-amino acids on the fusion forms of a cysteine proteinase/cystatin pair

“…2). This result was apparently not in agreement with the previously reported experimental results concerning the inhibitory effects of D amino acids or their unnat ural derivatives on cysteine, serine and aspartic types of proteases [13][14][15][16][17]. It might have 2 explanations as follows: 1-D amino acids do not interact with the fusion form of CP/CPI pair, or 2-D amino acids interact with the fusion form of CP/CPI pair in a man ner that does not affect the functionality of R1 prod uct.…”

“…To date, the develop ment and the use of efficient synthetic proteinase inhibitors including various non natural D amino acid derivatives are the most challenging task and tool to prevent their pathogenicities, in vivo [13,14]. In a recent study, the activity and the specificity of syn thetic peptides containing unnatural amino acids have been reported to be increased for human and malarial cysteine proteinases [17].…”

The influence of α-aminophosphonic acids on the activity of aminopeptidase from barley seeds—an approach to determine the enzyme specificity

“…In this manner, the sets of data that may be useful for design and synthesis of selective inhibitors are obtained (Drąg et al 2010;Kasperkiewicz et al 2012;Poras et al 2011Poras et al , 2013Węglarz-Tomczak et al 2013;Poręba et al 2014).…”

Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies differences in amino acid preferences between peptide‐based substrates and covalent inhibitors

“…The heat map shown in Fig. B compares the specificities of DPAP3 with those previously obtained for DPAP1 and CatC at the same substrate concentration. Note that D ‐Phg is the only D‐ AA in P2 that is cleaved by DPAP3, albeit poorly.…”

“… For DPAP1, k cat = 0.22 ± 0.004 s −1 , K m = 0.67 ± 0.14 μ m and k cat / K m = 320 000 ± 50 000 m −1 ·s −1 ; for CatC, k cat = 10.8 ± 0.2 s −1 , K m = 91 ± 5 μ m and k cat / K m = 116 000 ± 11 000 m −1 ·s −1 . …”