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

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 me...

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Autores principales:	Agustín Correa, Sabino Pacheco, Ariel E Mechaly, Gonzalo Obal, Ghislaine Béhar, Barbara Mouratou, Pablo Oppezzo, Pedro M Alzari, Frédéric Pecorari
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b257c58438d04cc4a4c882f220efb2f0
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spelling	oai:doaj.org-article:b257c58438d04cc4a4c882f220efb2f02021-11-18T08:19:37ZPotent and specific inhibition of glycosidases by small artificial binding proteins (affitins).1932-620310.1371/journal.pone.0097438https://doaj.org/article/b257c58438d04cc4a4c882f220efb2f02014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24823716/?tool=EBIhttps://doaj.org/toc/1932-6203Glycosidases 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.Agustín CorreaSabino PachecoAriel E MechalyGonzalo ObalGhislaine BéharBarbara MouratouPablo OppezzoPedro M AlzariFrédéric PecorariPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 5, p e97438 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Agustín Correa Sabino Pacheco Ariel E Mechaly Gonzalo Obal Ghislaine Béhar Barbara Mouratou Pablo Oppezzo Pedro M Alzari Frédéric Pecorari Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).
description	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.
format	article
author	Agustín Correa Sabino Pacheco Ariel E Mechaly Gonzalo Obal Ghislaine Béhar Barbara Mouratou Pablo Oppezzo Pedro M Alzari Frédéric Pecorari
author_facet	Agustín Correa Sabino Pacheco Ariel E Mechaly Gonzalo Obal Ghislaine Béhar Barbara Mouratou Pablo Oppezzo Pedro M Alzari Frédéric Pecorari
author_sort	Agustín Correa
title	Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).
title_short	Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).
title_full	Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).
title_fullStr	Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).
title_full_unstemmed	Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).
title_sort	potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/b257c58438d04cc4a4c882f220efb2f0
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Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).

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