Optimization of High-Throughput Methyltransferase Assays for the Discovery of Small Molecule Inhibitors.

ACS Combinatorial Science
Guangping DongRong Huang

Abstract

Methyltransferases (MTases) play diverse roles in cellular processes. Aberrant methylation levels have been implicated in many diseases, indicating the need for the identification and development of small molecule inhibitors for each MTase. Specific inhibitors can serve as probes to investigate the function and validate therapeutic potential for the respective MTase. High-throughput screening (HTS) is a powerful method to identify initial hits for further optimization. Here, we report the development of a fluorescence-based MTase assay and compare this format with the recently developed MTase-Glo luminescence assay for application in HTS. Using protein N-terminal methyltransferase 1 (NTMT1) as a model system, we miniaturized to 1536-well quantitative HTS format. Through a pilot screen of 1428 pharmacologically active compounds and subsequent validation, we discovered that MTase-Glo produced lower false positive rates than the fluorescence-based MTase assay. Nevertheless, both assays displayed robust performance along with low reagent requirements and can potentially be employed as general HTS formats for the discovery of inhibitors for any MTase.

References

Feb 17, 1999·Bioorganic & Medicinal Chemistry Letters·P ImbachJ Zimmermann
Jun 6, 2000·Journal of Biomolecular Screening·J H ZhangK R Oldenburg
Jan 9, 2004·Journal of Biomolecular Screening·Ge WuC Nicholas Hodge
Jun 17, 2005·Analytical Biochemistry·Evys CollazoRaymond C Trievel
Jul 26, 2006·Proceedings of the National Academy of Sciences of the United States of America·James IngleseChristopher P Austin
Mar 28, 2008·Journal of Medicinal Chemistry·Anton SimeonovJames Inglese
May 23, 2008·JALA : Journal of the Association for Laboratory Automation·Adam YasgarAnton Simeonov
Apr 21, 2010·Proceedings of the National Academy of Sciences of the United States of America·David KastDavid D Thomas
Jul 30, 2010·Nature·Christine E Schaner TooleyIan G Macara
Feb 19, 2011·Current Chemical Genomics·Yuhong WangDac-Trung Nguyen
Oct 4, 2011·Current Chemical Genomics·Amy M Quinn, Anton Simeonov
Dec 24, 2013·Journal of Structural Biology·Kenneth A TaylorKathleen M Trybus
Jun 14, 2014·Assay and Drug Development Technologies·Katherine M DrakeAndrew D Napper
Feb 26, 2015·Organic & Biomolecular Chemistry·Gang ZhangRong Huang
Mar 17, 2015·The Journal of Biological Chemistry·Stacie L RichardsonRong Huang
Apr 7, 2015·Mechanisms of Ageing and Development·Lindsay A BonsignoreChristine E Schaner Tooley
Aug 30, 2016·Cell Chemical Biology·Matthieu Schapira
Mar 25, 2017·Chemical Reviews·H Ümit KaniskanJian Jin
Apr 26, 2017·Clinical Epigenetics·Sheraz Gul
Apr 14, 2018·Nature Reviews. Drug Discovery·Christopher J Gerry, Stuart L Schreiber
Jul 31, 2018·Nature Reviews. Drug Discovery·Asher Mullard
Nov 28, 2018·Chembiochem : a European Journal of Chemical Biology·Rong Huang
Mar 19, 2019·Journal of Medicinal Chemistry·Dongxing ChenRong Huang
Nov 17, 2019·Cancer Letters·Alyncia D RobinsonSooryanarayana Varambally

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Citations

Jan 14, 2021·International Journal of Molecular Sciences·Yi-Hsun HoRong Huang
Aug 31, 2021·RSC Medicinal Chemistry·Iredia D Iyamu, Rong Huang

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