title: A direct label-free MALDI-TOF mass spectrometry based assay for the characterization of inhibitors of protein lysine methyltransferases.
abstract: 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.
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005, Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), 75005, Paris, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Guitot
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005, Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), 75005, Paris, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Drujon
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005, Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), 75005, Paris, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Burlina
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005, Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), 75005, Paris, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Sagan
authors.affiliation: Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS UPR 2301, 1 Avenue de la Terrasse, 91198, Gif-sur-Yvette, France.
authors.forename: Sandra
authors.initials: S
authors.lastname: Beaupierre
authors.affiliation: Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS UPR 2301, 1 Avenue de la Terrasse, 91198, Gif-sur-Yvette, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Pamlard
authors.affiliation: Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS UPR 2301, 1 Avenue de la Terrasse, 91198, Gif-sur-Yvette, France.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Dodd
authors.affiliation: Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS UPR 2301, 1 Avenue de la Terrasse, 91198, Gif-sur-Yvette, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Guillou
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005, Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), 75005, Paris, France.|UPMC Univ Paris 06, IBPS/FR3631, Plateforme de Spectrométrie de Masse et Protéomique, 7-9 Quai Saint Bernard, 75005, Paris, France.
authors.forename: Gérard
authors.initials: G
authors.lastname: Bolbach
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005, Paris, France.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), 75005, Paris, France.|UPMC Univ Paris 06, IBPS/FR3631, Plateforme de Spectrométrie de Masse et Protéomique, 7-9 Quai Saint Bernard, 75005, Paris, France.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Sachon
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005, Paris, France. dominique.guianvarch@upmc.fr.|Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), 75005, Paris, France. dominique.guianvarch@upmc.fr.
authors.forename: Dominique
authors.initials: D
authors.lastname: Guianvarc'h
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D011495:Histone-Lysine N-Methyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D008745:Methylation / Q000187:drug effects; D011759:Pyrrolidines / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013449:Sulfonamides / Q000494:pharmacology; D044005:Tetrahydroisoquinolines / Q000494:pharmacology
chemical_list: C000592938:(R)-PFI-2; D004791:Enzyme Inhibitors; D011759:Pyrrolidines; D054852:Small Molecule Libraries; D013449:Sulfonamides; D044005:Tetrahydroisoquinolines; D011495:Histone-Lysine N-Methyltransferase; C540312:SETD7 protein, human; D000241:Adenosine; C006235:sinefungin
keywords: (R)-PFI-2; Enzymatic assay; H3K4 methylation; Inhibitor characterization; MALDI-TOF MS; Protein lysine methyltransferase