Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

Summary

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time. 

Abstract

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.

This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors — anthraquinones or anthraquinone-like molecules with one to three substituents — at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

Introduction

DNA methylation is an important epigenetic mark that regulates gene expression and chromatin structure. Methylation occurs predominately in CpG dinucleotides — cytosine followed by guanosine; the methyl group is added to the 5-position of cytosine. Correct DNA methylation patterns, and thus proper gene expression, are needed for appropriate cellular development and function. Many disease states have been associated with changes to the normal methylation pattern^1,2,3. For example, there is a link between cancer initiation and progression and alterations to the DNA methyl....

Protocol

1. Prepare assay solutions for the screen

NOTE: The concentrations of substrates used in this assay can be adapted. For RFTS-lacking DNMT1, the experimentally determined K_m values for the hairpin DNA substrate and SAM are 1–2 nM and 2 µM, respectively^26,40.

1. Prepare 600 µL of each assay condition being tested in microcentrifuge tubes on ice.
   NOTE: The total volume of assay solution needed depends on the number of replicates being conducted. Each assay condition was run in triplicate for this protocol.
   1. Add ddH<....

Results

Active DNMT1 is a prerequisite for this analysis. RFTS-lacking DNMT1 was expressed in E.coli and purified to homogeneity following previously published procedures⁴¹. To ensure the purified enzyme was active, a discontinuous endonuclease-coupled assay was used to examine DNA methylation activity³⁶. This assay utilizes a 32 base pair duplex DNA with a single hemimethylated CpG positioned in a Sau3A1 cleavage site. Sau3A1 can cleave the hemimethylated substrate DNA; h.......

Discussion

To identify and characterize inhibitors of DNA methyltransferases, the activity of the enzyme must be measured. Several methods for examining DNA methyltransferase activity exist. Activity is commonly monitored using radioactivity; transfer of the labeled methyl group of SAM can be quantified^{29,30,31,32,33,34}. Gel-based assay.......

Materials

Name	Company	Catalog Number	Comments
96-well Half Area Black Flat Bottom Polystyrene Not Treated Microplate	Corning	3694
96-Well Polystyrene Conical Bottom Plates	ThermoFisher	249570
Bovine Serum Albumin	NEB	B9000S
compound 1	ChemBridge	5812086	screening compound; resuspended in DMSO to 10 mM
compound 2	ChemBridge	6722175	screening compound; resuspended in DMSO to 10 mM
compound 3	ChemBridge	5249376	screening compound; resuspended in DMSO to 10 mM
Dithiothreitol	Sigma	D0632
Gla I	SibEnzyme	E494	methyl-sensitive endonuclease
Glycerol	RPI	G22025
Magnesium Chloride	Sigma	M0250
Oligonucleotide (5'-FAM-CCTATGCGmCATCAGTTTTCTGATGmCGmCATAGG-3'-Iowa Black Quencher)	IDT	custom synthesized	internally quenched hairpin DNA (substrate)
Potassium Glutamate	Sigma	G1501
S-adenosylmethionine	Sigma	A4377	methyl-donating co-factor (substrate)
Tris Base	RPI	T60040