Sak 03807 O Vedlegg Fagleg Rapportering 2006 - Helse Vest

406Forskningsprosjekt 911298:Therapeutic approaches for the correction of misfoldingmutationsProsjektansvarlig: Aurora Martinez (aurora.martinez@biomed.uib.no), Universitetet i BergenMany inherited diseases are caused by the enhanced tendency of mutant proteins tomisfold and to either undergo intracellular degradation or deleterious aggregation withplaque formation.For several of these diseases evidence has been accumulated that chemical or pharmacologicchaperones (pharmacoperones), which usually resemble natural ligands and rescue the misfoldedproteins by stimulating their renaturation in vitro and in vivo, result in successful therapeuticintervention. Some of the mutations are associated, however, with a total absence of translatedprotein. In these instances alternative therapeutic approaches as enzyme replacement therapyappears to be promising. Enzyme forms to be considered for this purpose should have prolongedsurvival in vivo and reduced immunogeneicity. The main objective of this project is to further developthe pharmacoperone concept for the rescue of misfolding mutants of the aromatic amino acidhydroxylases and the optimization of enzyme forms for direct replacement therapy. This goal will beachieved through a translational methodological approach integrating biochemical, biophysical andcomputational techniques, as well as animal models. The results are expected to lead to a betterunderstanding of the structure-function-stability relationships in selected enzymes, and to result innovel therapeutic approaches.Since the start of the project and in collaboration with other member of the group we have obtainedrelevant results and competence in three areas:1) By using a high-throughput (HTP) experimental approach with a Thermofluor® installed at thelaboratory of Prof. Javier Sancho (Univ. Zaragoza, Spain), we have screened 1500 compounds from aHitfinder compound library. Four compounds have been selected due to their binding and stabilizationand rescue of phenylalanine hydroxylase mutants which are associated to phenylketonuria (PKU). Weare also characterizing the effect of these compounds on the activity and expression of the enzyme ineukaryote cells (A293). Very positive results measured as 2-3-fold increase of specific activity andprotein levels. Our collaborator at the University of Zurich (Prof. Beat Thöny, Univ. Zürich) is settingup the experiments with mice (enu1 and enu2) to test the in vivo viability of these compounds.2) With respect to the virtual screening and computational structural biology the group, and notablyDr. Knut Teigen, has further developed competence for molecular dynamics simulations and molecularinteraction fields, contributing to ligand-binding analysis and drug design in important biomedicalsystems (see following publications):• Pey AL, Martinez A, Charubala R, Maitland DJ, Teigen K, Calvo A, Pfleiderer W, Wood JM & Schallreuter KU(2006)Specific interaction of the diastereomers 7(R) and 7(S)-tetrahydrobiopterin with phenylalanine hydroxylase -Implications for understanding primapterinuria and vitiligo. FASEB J. 20, 2130-2132• Dao KK, Teigen K, Kopperud R, Hodneland E, Schwede F, Christensen AE, Martinez A & Døskeland SO (2006), Epac1and PKA holoenzyme have similar cAMP affinity, but their cAMP domains have distinct structural features and cyclicnucleotide recognition. J. Biol. Chem. 281, 21500-21511.• Teigen K, McKinney JA, Haavik J & Martínez, A. (2007), Selectivity and affinity determinants for ligand binding to thearomatic amino acid hydroxylases, Curr. Med. Chem. 14, 1-23.3) In the subproject aimed to strategies directed towards enzyme replacement therapy we haveinitiated the studies on the functional, conformational and stability effect of the adhesion of tyrosinehydroxylase into gold-nanoparticles.Translasjonsforskning - Pediatrics