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

Marcin Poreba,Marko Mihelic,Boris Turk,Rafal Latajka,Artur Krezel,Marcin Drag,Dusan Turk,Michael Klemba,Priscilla Krai,Jelena Rajkovic,Malgorzata Pawelczak

doi:10.1007/s00726-013-1654-2

Marcin Poreba, Marko Mihelic + Show 9 more

Open Access

https://doi.org/10.1007/s00726-013-1654-2

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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.

Highlights

The specificity ratio between enzyme orthologs or homologs is one of the most important factors in terms of drug design or specific enzyme activity monitoring using chemical probes
NH2-L-hPhe-ACC was split, and the parallel coupling of Fmoc-protected amino acids using a semiautomatic FlexChem synthesizer was performed, yielding individual substrates after cleavage. In this library, which consisted of 57 individual substrates, we used all natural amino acids, several L-amino acid enantiomers (D-amino acids) and a broad range of unnatural amino acids, of which the structures were chosen to cover a spectrum of the possible interactions in the S2 pocket of human cathepsin C
It is especially intriguing that during parasite infection, both enzymes are found in the human (DPAP1 is found in human red blood cells infected with P. falciparum parasite, while human cathepsin C is a lysosomal protease), and molecules designed to reach the dipeptidyl aminopeptidase 1 (DPAP1) active site must significantly differentiate between human and parasite orthologs

Summary

Introduction

The specificity ratio between enzyme orthologs or homologs is one of the most important factors in terms of drug design or specific enzyme activity monitoring using chemical probes. There are several families of proteases, such as caspases, cathepsins or aminopeptidases, which are able to cleave the same peptide sequences, which significantly complicate their targeting with specific chemical tools, such as substrates or activity-based probes. This goal is even more complicated when trying to design specific molecules for monitoring enzyme orthologs, for example, in the case of parasite infection in humans. Cathepsin C (DPPI, EC 3.4.14.1, dipeptidyl peptidase I) is a lysosomal cysteine protease expressed in the majority of mammalian tissues (Tallan et al 1952). Cathepsin C is a classic exopeptidase, which trims dipeptides from the N-terminus of peptide substrates

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