1. A direct label-free MALDI-TOF mass spectrometry based assay for the characterization of inhibitors of protein lysine methyltransferases
- Author
-
Gérard Bolbach, Robert H. Dodd, Emmanuelle Sachon, Olivier Pamlard, Sandra Beaupierre, Dominique Guianvarc'h, Thierry Drujon, Sandrine Sagan, Fabienne Burlina, Catherine Guillou, Karine Guitot, Laboratoire des biomolécules (LBM UMR 7203), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Analyse, Interactions Moléculaires et Cellulaires (LBM-E2), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Chimie Moléculaire de Paris Centre (FR 2769), Spectrométrie de Masse et Protéomique [IBPS] (IBPS-SPM), Institut de Biologie Paris Seine (IBPS), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,Methyltransferase ,Adenosine ,Pyrrolidines ,Histone lysine methylation ,[SDV]Life Sciences [q-bio] ,Lysine ,MESH: Sulfonamides ,Drug Evaluation, Preclinical ,H3K4 methylation ,Biochemistry ,Methylation ,MESH: Tetrahydroisoquinolines ,Analytical Chemistry ,Chemical library ,MESH: Methylation ,Small Molecule Libraries ,03 medical and health sciences ,Sinefungin ,chemistry.chemical_compound ,MESH: Small Molecule Libraries ,Tetrahydroisoquinolines ,Protein lysine methyltransferase ,MALDI-TOF MS ,Humans ,Enzyme Inhibitors ,chemistry.chemical_classification ,Sulfonamides ,MESH: Humans ,MESH: Pyrrolidines ,030102 biochemistry & molecular biology ,MESH: Histone-Lysine N-Methyltransferase ,Enzymatic assay ,Inhibitor characterization ,Histone-Lysine N-Methyltransferase ,MESH: Adenosine ,Matrix-assisted laser desorption/ionization ,030104 developmental biology ,Enzyme ,MESH: Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,chemistry ,MESH: Enzyme Inhibitors ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,(R)-PFI-2 ,MESH: Drug Evaluation, Preclinical - Abstract
International audience; Histone lysine methylation is associated with essential biological functions like transcription activation or repression, depending on the position and the degree of methylation. This post-translational modification is introduced by protein lysine methyltransferases (KMTs) which catalyze the transfer of one to three methyl groups from the methyl donor S-adenosyl-l-methionine (AdoMet) to the amino group on the side chain of lysines. The regulation of protein lysine methylation plays a primary role not only in the basic functioning of normal cells but also in various pathologies and KMT deregulation is associated with diseases including cancer. These enzymes are therefore attractive targets for the development of new antitumor agents, and there is still a need for direct methodology to screen, identify, and characterize KMT inhibitors. We report here a simple and robust in vitro assay to quantify the enzymatic methylation of KMT by MALDI-TOF mass spectrometry. Following this protocol, we can monitor the methylation events over time on a peptide substrate. We detect in the same spectrum the modified and unmodified substrates, and the ratios of both signals are used to quantify the amount of methylated substrate. We first demonstrated the validity of the assay by determining inhibition parameters of two known inhibitors of the KMT SET7/9 ((R)-PFI-2 and sinefungin). Next, based on structural comparison with these inhibitors, we selected 42 compounds from a chemical library. We applied the MALDI-TOF assay to screen their activity as inhibitors of the KMT SET7/9. This study allowed us to determine inhibition constants as well as kinetic parameters of a series of SET7/9 inhibitors and to initiate a structure activity discussion with this family of compounds. This assay is versatile and can be easily adapted to other KMT substrates and enzymes as well as automatized.
- Published
- 2016
- Full Text
- View/download PDF