Cytosine-Based TET Enzyme Inhibitors

Abstract

DNA methylation is known as the prima donna epigenetic mark for its critical role in regulating local gene transcription. Changes in the landscape of DNA methylation across the genome occur during cellular transition, such as differentiation and altered neuronal plasticity, and become dysregulated in disease states such as cancer. The TET family of enzymes is known to be responsible for catalyzing the reverse process that is DNA demethylation by recognizing 5-methylcytosine and oxidizing the methyl group via an Fe(II)/alpha-ketoglutarate-dependent mechanism. Here, we describe the design, synthesis, and evaluation of novel cytosine-based TET enzyme inhibitors, a class of small molecule probes previously underdeveloped but broadly desired in the field of epigenetics. We identify a promising cytosine-based lead compound, Bobcat339, that has mid-μM inhibitor activity against TET1 and TET2, but does not inhibit the DNA methyltransferase, DNMT3a. In silico modeling of the TET enzyme active site is used to rationalize the activity of Bobcat339 and other cytosine-based inhibitors. These new molecular tools will be useful to the field of epigenetics and serve as a starting point for new therapeutics that target DNA methylation and gene transcription.

Scheme 1. Active DNA Methylation and Demethylation

DNA methylation is dynamically regulated by methyl writing enzymes (DNMTs) and methyl erasing enzymes (TETs), allowing the mechanism to govern local gene expression, a process important for cell function and identity.

Figure 1

Crystal structure of TET2-DNA complex. (a) TET2 binds dsDNA, breaks the double helix, and inserts 5mC into its active site. (b) View of the TET2 active site binding 5mC by forming hydrogen bonds with Asn1387, His1904, and Arg1261, all of which are critical residues for TET2 catalytic activity and methylated DNA binding. The oxidative iron center is shown in proximity to the methyl group on 5mC. (c) A 2D rendering of the 5mC-bound TET2 active site and critical residue interactions; blue = basic residue, red = acidic residue, and black = neutral residue.

Scheme 2. Cytosine-Based TET Enzyme Inhibitor Synthesis

The 5-position of cytosine was chlorinated using NCS in acetic acid and then coupled to phenylboronic acid using copper-mediated Ullman conditions. All other N1 and 5-substituted cytosine derivatives were synthesized using similar conditions.

Figure 2

Examining possible methyl bioisosteres at the R₁ position. Chloro, bromo, and trifluoromethyl-substituted derivatives were tested for inhibition of TET1- or TET2-mediated oxidation of methylated dsDNA. Each compound was tested at 100 μM in an ELISA. All data presented are N = 3, error bars indicate ± SEM. Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Inhibitor optimization. (a) Several aryl groups were tested at the R₂ position for inhibition of TET1 and TET2 at 100 μM. 3-Biphenyl substitution significantly increased TET1 inhibition (P = 0.002) over simple phenyl substitution, while 2-biphenyl (P = 0.0001) and 4-biphenyl (P < 0.0001) substitution significantly reduced TET1 inhibition as compared to a phenyl substitution. (b) 5-Chloro substitution at the R₁ position is necessary to maintain the activity of 3-biphenyl substitution at the R₂ position for both TET1 (P < 0.0001) and TET2 (P = 0.0003). (c) Bobcat339 inhibits TET1 (IC₅₀ = 33 μM) and TET2 (IC₅₀ = 73 μM), but not DNMT3a. (d) Bobcat339 docked into a homology model of TET1. (e) Predicted binding conformations of Bobcat339 (teal), its 2-biphenyl isomer (blue), and its 4-biphenyl isomer (gold). Molecular surface; white = hydrophobic, blue = electropositive, and red = electronegative. All data presented are N = 3, error bars indicate ± SEM.

Figure 4

Bobcat339 reduces DNA 5hmC levels in hippocampal neurons. Lead compounds Bobcat212 and Bobcat339 were dosed at 10 μM in 1% DMSO onto HT-22 mouse hippocampal neurons for 24 h. Cells were then lysed, DNA extracted, and TET enzyme inhibition determined by 5hmC-specfic antibody binding. Data presented are N = 6 biological replicates per group, error bars indicate ± SEM. One-way ANOVA, ns = not significant, *P < 0.05, **P < 0.01.