title: Discovery of compounds that protect tyrosine hydroxylase activity through different mechanisms.
abstract: Pharmacological chaperones are small compounds that correct the folding of mutant proteins, and represent a promising therapeutic strategy for misfolding diseases. We have performed a screening of 10,000 compounds searching for pharmacological chaperones of tyrosine hydroxylase (TH), the tetrahydrobiopterin (BH4)-dependent enzyme that catalyzes the rate-limiting step in the synthesis of catecholamines. A large number of compounds bound to human TH, isoform 1 (hTH1), but only twelve significantly protected wild-type (hTH1-wt) and mutant TH-R233H (hTH1-p.R202H), associated to the rare neurological disorder TH deficiency (THD), from time-dependent loss of activity. Three of them (named compounds 2, 4 and 5) were subjected to detailed characterization of their functional and molecular effects. Whereas compounds 2 and 4 had a characteristic pharmacological chaperone (stabilizing) effect, compound 5 protected the activity in a higher extent than expected from the low conformational stabilization exerted on hTH1. Compounds 4 and 5 were weak competitive inhibitors with respect to the cofactor BH4 and, as seen by electron paramagnetic resonance, they induced small changes to the first coordination sphere of the catalytic iron. Molecular docking also indicated active-site location with coordination to the iron through a pyrimidine nitrogen atom. Interestingly, compound 5 increased TH activity in cells transiently transfected with either hTH1-wt or the THD associated mutants p.L205P, p.R202H and p.Q381K without affecting the steady-state TH protein levels. This work revealed different mechanisms for the action of pharmacological chaperones and identifies a subtype of compounds that preserve TH activity by weak binding to the catalytic iron. This article is part of a Special Issue entitled: Cofactor-dependent proteins: Evolution, chemical diversity and bio-applications.
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway.
authors.forename: Magnus
authors.initials: M
authors.lastname: Hole
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway.
authors.forename: Jarl
authors.initials: J
authors.lastname: Underhaug
authors.affiliation: Department of Neurology, Hospital Sant Joan de Déu (HSJD), Barcelona, Spain; Laboratory of Synaptic Metabolic Diseases, Fundació Sant Joan de Déu (HSJD), Barcelona, Spain.
authors.forename: Hector
authors.initials: H
authors.lastname: Diez
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway.
authors.forename: Ming
authors.initials: M
authors.lastname: Ying
authors.affiliation: Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Åsmund Kjendseth
authors.initials: ÅK
authors.lastname: Røhr
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway.
authors.forename: Ana
authors.initials: A
authors.lastname: Jorge-Finnigan
authors.affiliation: Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Spain.
authors.forename: Noèlia
authors.initials: N
authors.lastname: Fernàndez-Castillo
authors.affiliation: Department of Neurology, Hospital Sant Joan de Déu (HSJD), Barcelona, Spain; Laboratory of Synaptic Metabolic Diseases, Fundació Sant Joan de Déu (HSJD), Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Spain.
authors.forename: Angels
authors.initials: A
authors.lastname: García-Cazorla
authors.affiliation: Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: K Kristoffer
authors.initials: KK
authors.lastname: Andersson
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway.
authors.forename: Knut
authors.initials: K
authors.lastname: Teigen
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway. Electronic address: aurora.martinez@biomed.uib.no.
authors.forename: Aurora
authors.initials: A
authors.lastname: Martinez
mesh_terms: D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D062105:Molecular Docking Simulation; D011487:Protein Conformation; D017510:Protein Folding; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D014446:Tyrosine 3-Monooxygenase
keywords: Dopamine synthesis; Enzyme stabilization; Iron coordination; Pharmacological chaperone; Tyrosine hydroxylase deficiency