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A direct label-free MALDI-TOF mass spectrometry based assay for the characterization of inhibitors of protein lysine methyltransferases

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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.

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Abbreviations

AdoHcy:

S-Adenosyl-l-homocysteine

AdoMet:

S-Adenosyl-l-methionine

CHCA:

α-Cyano-4-hydroxycinnamic acid

DMSO:

Dimethylsulfoxide

ELISA:

Enzyme-linked immunosorbent assays

HTS:

High-throughput screening

KMT:

Protein lysine methyltransferases

MALDI-TOF MS:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

PTM:

Post-translational modifications

TFA:

Trifluoroacetic acid

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Acknowledgements

We are grateful to Prof. Albert Jeltsch (University Stuttgart, Germany) for the gift of plasmid pET28a(+)H6-setd7.

Authors’ contributions

All authors contributed to data analysis and interpretation. All authors read and approved the final manuscript.

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Authors and Affiliations

  1. Sorbonne Universités, UPMC Univ Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules (LBM), 4 place Jussieu, 75005, Paris, France

    Karine Guitot, Thierry Drujon, Fabienne Burlina, Sandrine Sagan, Gérard Bolbach, Emmanuelle Sachon & Dominique Guianvarc’h

  2. Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Univ Paris 06, CNRS, Laboratoire des Biomolécules (LBM), 75005, Paris, France

    Karine Guitot, Thierry Drujon, Fabienne Burlina, Sandrine Sagan, Gérard Bolbach, Emmanuelle Sachon & Dominique Guianvarc’h

  3. Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS UPR 2301, 1 Avenue de la Terrasse, 91198, Gif-sur-Yvette, France

    Sandra Beaupierre, Olivier Pamlard, Robert H. Dodd & Catherine Guillou

  4. UPMC Univ Paris 06, IBPS/FR3631, Plateforme de Spectrométrie de Masse et Protéomique, 7-9 Quai Saint Bernard, 75005, Paris, France

    Gérard Bolbach & Emmanuelle Sachon

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  1. Karine Guitot
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  2. Thierry Drujon
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  3. Fabienne Burlina
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  4. Sandrine Sagan
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  9. Gérard Bolbach
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  10. Emmanuelle Sachon
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  11. Dominique Guianvarc’h
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Correspondence to Dominique Guianvarc’h.

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Guitot, K., Drujon, T., Burlina, F. et al. A direct label-free MALDI-TOF mass spectrometry based assay for the characterization of inhibitors of protein lysine methyltransferases. Anal Bioanal Chem 409, 3767–3777 (2017). https://doi.org/10.1007/s00216-017-0319-5

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