title: UHPLC-MS-based HDAC Assay Applied to Bio-guided Microfractionation of Fungal Extracts.
abstract: BACKGROUND
  Histone deacetylases (HDAC) are considered as promising targets for cancer treatment. Today, four HDAC inhibitors, vorinostat, romidepsin, belinostat, and panobinostat, have been approved by the Food and Drug Administration (FDA) for cancer treatment, while others are in clinical trials. Among them, several are naturally occurring fungal metabolites.
  
  
  OBJECTIVE
  To develop and optimise an enzyme assay for bio-guided identification of HDAC inhibitors in fungal strains.
  
  
  METHODS
  Fluorescence and MS-based HDAC enzymatic assays were compared during the bio-guided fractionation of Penicillium griseofulvum. The MS-based approach was then optimised to evaluate HDAC selectivity using the human recombinant class I isoform HDAC1 and the class II isoform HDAC6.
  
  
  RESULTS
  Fluorescence-based assays have several drawbacks when used for bio-guided fractionation because of the native fluorescence and the trypsin inhibitory ability of compounds present in many extracts. The MS-based method led to the isolation of gliocladride C, which is selective for HDAC1 and salirepol, which showed an HDAC6 selectivity. Their activity and presence in P. griseofulvum is described here for the first time.
  
  
  CONCLUSIONS
  The UHPLC-ESI-MS/MS-based method using specific HDAC isoforms is suitable to isolate selective HDAC inhibitors by bio-guided fractionation of fungal strains. Also, it decreases potential interferences with natural products compared to the fluorescence-based assay.
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211, Geneva, 11, Switzerland.
authors.forename: Vincent
authors.initials: V
authors.lastname: Zwick
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211, Geneva, 11, Switzerland.
authors.forename: Pierre-Marie
authors.initials: PM
authors.lastname: Allard
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211, Geneva, 11, Switzerland.
authors.forename: Lucie
authors.initials: L
authors.lastname: Ory
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211, Geneva, 11, Switzerland.
authors.forename: Claudia A
authors.initials: CA
authors.lastname: Simões-Pires
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211, Geneva, 11, Switzerland.
authors.forename: Laurence
authors.initials: L
authors.lastname: Marcourt
authors.affiliation: Mycology and Biotechnology group, Institute for Plant Production Sciences IPS, Agroscope, Route de Duillier 50, P.O. Box 1260, Nyon, Switzerland.
authors.forename: Katia
authors.initials: K
authors.lastname: Gindro
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211, Geneva, 11, Switzerland.
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Wolfender
authors.affiliation: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211, Geneva, 11, Switzerland.
authors.forename: Muriel
authors.initials: M
authors.lastname: Cuendet
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005658:Fungi / Q000378:metabolism*; D006655:Histone Deacetylases / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006655:Histone Deacetylases
keywords: UHPLC-ESI-MS/MS; bio-guided fractionation; fungi; histone deacetylase; isoform selectivity