title: Looking for protein stabilizing drugs with thermal shift assay.
abstract: Thermal shift assay can be used for the high-throughput screening of pharmacological chaperones. These drugs are small molecules that bind a mutant protein and stabilize it. We demonstrated the robustness, reproducibility and versatility of the method using two molecules that are in clinical trial for Fabry or Pompe disease, Deoxygalactonojirimycin and N-Butyldeoxynojirimycin, and their target enzymes, lysosomal alpha-galactosidaseA and alpha-glucosidase, as test cases. We assessed the influence of solvents and of scanning rate on the measures. We showed that a value that is equivalent to the melting temperature can be obtained by the first derivatives of raw data. We discuss the advantages of the method and the precaution to be taken in running the experiments.
authors.affiliation: Istituto di Chimica Biomolecolare -CNR, Pozzuoli, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Andreotti
authors.affiliation: Istituto di Chimica Biomolecolare -CNR, Pozzuoli, Italy.|Dipartimento di Biologia, Università Federico II, Napoli, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Monticelli
authors.affiliation: Dipartimento di Biologia, Università Federico II, Napoli, Italy.
authors.forename: Maria Vittoria
authors.initials: MV
authors.lastname: Cubellis
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004795:Enzyme Stability / Q000187:drug effects*; D000795:Fabry Disease / Q000188:drug therapy / Q000201:enzymology; D006009:Glycogen Storage Disease Type II / Q000188:drug therapy / Q000201:enzymology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D013696:Temperature; D000519:alpha-Galactosidase / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D017485:1-Deoxynojirimycin; C059896:miglustat; C090092:migalastat; D000519:alpha-Galactosidase
keywords: pharmacological chaperones; rare disease; thermal shift assay
references.citation: Biochemistry. 2005 Apr 5;44(13):5258-66
references.pmid: 15794662
references.citation: Drug Test Anal. 2015 Sep;7(9):831-4
references.pmid: 25845367
references.citation: Mol Genet Metab. 2009 Apr;96(4):233-8
references.pmid: 19181556
references.citation: Anal Biochem. 2006 Oct 15;357(2):289-98
references.pmid: 16962548
references.citation: J Mol Biol. 2005 Oct 21;353(2):459-73
references.pmid: 16169011
references.citation: Mol Ther. 2012 Apr;20(4):717-26
references.pmid: 22215019
references.citation: EMBO Mol Med. 2009 Aug;1(5):268-79
references.pmid: 20049730
references.citation: Proc Natl Acad Sci U S A. 2006 Oct 24;103(43):15835-40
references.pmid: 17035505
references.citation: Mol Genet Genomic Med. 2013 May;1(1):32-44
references.pmid: 24498599
references.citation: Anal Biochem. 2004 Sep 1;332(1):153-9
references.pmid: 15301960
references.citation: Chem Biol. 2011 Dec 23;18(12):1521-6
references.pmid: 22195554
references.citation: Biochim Biophys Acta. 2014 Mar;1840(3):1214-24
references.pmid: 24361605
references.citation: J Biol Chem. 2014 Dec 12;289(50):34900-10
references.pmid: 25324542
references.citation: J Med Chem. 2012 Nov 26;55(22):9817-30
references.pmid: 23106316
references.citation: Curr Protoc Protein Sci. 2015 Feb 02;79:28.9.1-28.9.14
references.pmid: 25640896
references.citation: Mol Ther. 2014 Nov;22(11):2004-12
references.pmid: 25052852
references.citation: Orphanet J Rare Dis. 2013 Jul 24;8:111
references.pmid: 23883437
references.citation: J Biomol Screen. 2001 Dec;6(6):429-40
references.pmid: 11788061
references.citation: Hum Mutat. 2009 Dec;30(12):1683-92
references.pmid: 19862843
references.citation: Biochemistry. 2009 Jun 9;48(22):4816-27
references.pmid: 19374450