Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).

Agustín Correa,Sabino Pacheco,Pedro M Alzari,Pablo Oppezzo,Ariel E Mechaly,Barbara Mouratou,Ghislaine Béhar,Frédéric Pecorari,Gonzalo Obal

doi:10.1371/journal.pone.0097438

Abstract

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

Highlights

Glycosidases are involved in a variety of metabolic disorders and human diseases such as type II diabetes, Gaucher disease, cancers and asthma [1,2,3,4]
Library Designs Endo-glycosidases have cleft-shaped active sites and it is well known that loops can penetrate clefts
Selection of Anti-glycosidase Affitins Two pools of libraries were constituted including the presence of the short (L1+L2) or long (L3+L4) loop 2, and selections were performed in parallel by ribosome display using immobilized CelD as a target protein

Summary

Introduction

Glycosidases are involved in a variety of metabolic disorders and human diseases such as type II diabetes, Gaucher disease, cancers and asthma [1,2,3,4] They are actively studied to probe their functions, and as targets for inhibitor drugs to treat human diseases. The use of small-molecular weight compounds is a powerful approach to modulate the activity of individual glycosidases [6], and a number of small-molecule inhibitors have been described for these enzymes This class of inhibitors is attractive for the development of drugs, they can interact with non-target proteins and few high-quality inhibitors useful for therapy have been reported The Ecotin scaffold has been used to generate a highly specific inhibitor of the protease kallikrein with a Ki of 11 pM [14] while binders with inhibition properties for hen egg white lysozyme (HEWL) have been derived from various proteins (VHH, shark IgNAR and an anticodon recognition domain of the aspartyl tRNA synthetase), and have been structurally described to mimic the oligosaccharide substrate of this glycosidase [11,15,16,17,18]

Methods

Results

Conclusion