title: The Chaperoning Activity of Amino-oxyacetic Acid on Folding-Defective Variants of Human Alanine:Glyoxylate Aminotransferase Causing Primary Hyperoxaluria Type I.
abstract: The rare disease Primary Hyperoxaluria Type I (PH1) results from the deficit of liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene frequently leading to protein misfolding. Pharmacological chaperone (PC) therapy is a newly developed approach for misfolding diseases based on the use of small molecule ligands able to promote the correct folding of a mutant enzyme. In this report, we describe the interaction of amino-oxyacetic acid (AOA) with the recombinant purified form of two polymorphic species of AGT, AGT-Ma and AGT-Mi, and with three pathogenic variants bearing previously identified folding defects: G41R-Ma, G170R-Mi, and I244T-Mi. We found that for all these enzyme AOA (i) forms an oxime at the active site, (ii) behaves as a slow, tight-binding inhibitor with KI values in the nanomolar range, and (iii) increases the thermal stability. Furthermore, experiments performed in mammalian cells revealed that AOA acts as a PC by partly preventing the intracellular aggregation of G41R-Ma and by promoting the correct peroxisomal import of G170R-Mi and I244T-Mi. Based on these data, we carried out a small-scale screening campaign. We identified four AOA analogues acting as AGT inhibitors, even if only one was found to act as a PC. The possible relationship between the structure and the PC activity of these compounds is discussed. Altogether, these results provide the proof-of-principle for the feasibility of a therapy with PCs for PH1-causing variants bearing folding defects and provide the scaffold for the identification of more specific ligands.
authors.affiliation: Department of Life Sciences and Reproduction, Section of Biological Chemistry, University of Verona , Strada Le Grazie 8 37134 Verona, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Oppici
authors.affiliation: Department of Life Sciences and Reproduction, Section of Biological Chemistry, University of Verona , Strada Le Grazie 8 37134 Verona, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Montioli
authors.affiliation: Department of Life Sciences and Reproduction, Section of Biological Chemistry, University of Verona , Strada Le Grazie 8 37134 Verona, Italy.
authors.forename: Mirco
authors.initials: M
authors.lastname: Dindo
authors.affiliation: Siena Biotech S.p.A. , Strada del Petriccio e Belriguardo, 35 53100 Siena, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Maccari
authors.affiliation: Siena Biotech S.p.A. , Strada del Petriccio e Belriguardo, 35 53100 Siena, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Porcari
authors.affiliation: Department of Life Sciences and Reproduction, Section of Biological Chemistry, University of Verona , Strada Le Grazie 8 37134 Verona, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Lorenzetto
authors.affiliation: Siena Biotech S.p.A. , Strada del Petriccio e Belriguardo, 35 53100 Siena, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Chellini
authors.affiliation: Department of Life Sciences and Reproduction, Section of Biological Chemistry, University of Verona , Strada Le Grazie 8 37134 Verona, Italy.
authors.forename: Carla Borri
authors.initials: CB
authors.lastname: Voltattorni
authors.affiliation: Department of Life Sciences and Reproduction, Section of Biological Chemistry, University of Verona , Strada Le Grazie 8 37134 Verona, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Cellini
mesh_terms: D000409:Alanine / Q000235:genetics*; D000625:Aminooxyacetic Acid / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D005455:Fluorescent Antibody Technique; D014644:Genetic Variation; D006801:Humans; D006960:Hyperoxaluria, Primary / Q000201:enzymology* / Q000235:genetics*; D018832:Molecular Chaperones / Q000378:metabolism; D017510:Protein Folding / Q000187:drug effects; D055550:Protein Stability; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*
chemical_list: D018832:Molecular Chaperones; D000625:Aminooxyacetic Acid; D000637:Transaminases; C023067:glyoxylate aminotransferase; D000409:Alanine