C5‐DNA Methyltransferase Inhibitors: From Screening to Effects on Zebrafish Embryo Development

Abstract

DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.

Laccaic Acid A Is a Direct, DNA-competitive Inhibitor of DNA Methyltransferase 1

“…The K m,DNA values reported for full-length Dnmt3a are 2.5 M for 30-mer duplex non-methylated and hemi-methylated DNAs (34), 2.7 M for poly(dIdC), and 3.5 M for poly(dGdC) (35). All of these reported values are more than 10-fold larger than the DNA concentration used in the IC 50 experiment. Yet, as can been seen in Fig.…”

“…5B). The higher IC 50 for LCA inhibition of Dnmt1 (351-1600) than for Dnmt1 (621-1600) can be explained in two ways. First, the RFTS domain-containing enzyme must be assayed at a 10-fold higher concentration of DNA substrate.…”

“…In the presence of 100 M LCA, the observed melting temperature is shifted more than 2°C to the right from 43.0 Ϯ 0.1°C in the absence of LCA to 45.3 Ϯ 0.1°C, indicating a direct interaction between LCA and Dnmt3a. To further investigate specificity, IC 50 values were determined for LCA inhibition of 200 nM DNA substrate was used. This is ϳ13 times the reported K m,DNA for Dnmt1 (351-1600) (24).…”

“…Recently, 3-nitroflavanones were identified by screening against Dnmt3a and suggested by molecular docking to compete for DNA binding (50). We suggest that specifically substituted flavones, anthraquinones, and related compounds may produce multiple hits against Dnmts.…”

DNA methylation in cancer

“…For example, SGI-1027 belongs to a family of minor groove DNA binders developed by Denny and collaborators (Datta et al, 2009) and has been shown to inhibit DNA methylation in enzymatic tests and in cancer cells. We developed two families of DNMT inhibitors: procainamide conjugates that are selective of DNA methyltransferases versus histone methyltransferases and flavanoid derivatives that also inhibit DNA methylation in an in vivo model of zebrafish development (Ceccaldi et al, 2011). Since the small molecules identified so far showed less anti-cancer activities than nucleoside analogs and present limited therapeutic interest, we and others have developed high-throughput screening and drug design strategies to discover new inhibitors (Ceccaldi et al, 2013;Kilgore et al, 2013;Weng et al, 2014).…”