Kinase experiment:

To determine the inhibition of Moz activity by the test compounds, assay reactions are conducted in a volume of 8 μL in 384-well low volume assay plates. The reactions are performed in assay buffer (100 mM Tris-HCl, pH 7.8, 15 mM NaCl, 1 mM EDTA, 0.01% Tween-20, 1 mM Dithiothreitol, and 0.02% m/v chicken egg white albumin). Reactions are set up with 0.4 μM Acetyl coenzyme A (AcCoA), 50 nM N-terminal histone H4 peptide (sequence SGRGKGGKGLGKGGAKRHRKV-GGK-biotin), 10 nM MOZ enzyme, and an acetyl-lysine specific antibody (final dilution 1: 10000). 11-point dilution series of the compounds (WM-8014) of the invention are prepared in DMSO; a volume of 100 nL is transferred using a pin tool into assay plates containing substrates, before adding enzyme to start the reaction. Positive (no compound) and negative (AcCoA omitted) control reactions are included on the same plates and receive the same amount of DMSO as the compound treated wells. After adding all reagents, the plates are sealed with adhesive seals and incubated for 90 minutes at room temperature[1].

Target: lysine acetyltransferases, KAT6A(MOZ), KAT6B(MORF/QKF)

WM-8014 induces cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma.

Data:

Treatment of MEFs with WM-8014 leads to cellular senescence.

Because KAT6A suppresses senescence, the ability of WM-8014 to induce cell cycle arrest is tested in embryonic day (E)14.5 mouse embryonic fibroblasts (MEFs). Cells treated with WM-8014 failed to proliferate after 10 days of treatment (IC₅₀2.4 μM).

(Left: effects of WM-8014 compared with the inactive compound WM-2474 or DMSO vehicle control on cell growth of MEFs grown in 3% O2. Right: effects of the dose of WM-8014 and the duration of treatment.)

Western blot of MEFs treated with increasing doses of WM-8014 and controls as indicated.

K_D(KAT6A)=0.005μM K_D(KAT5) = 0.9μM K_D(KAT7) = 0.09 μM

References

1. Baell JB, et al. Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth. Nature. 2018 Aug;560(7717):253-257.