Identification of catalytic and non-catalytic activity inhibitors against PRC2-EZH2 complex through multiple high-throughput screening campaigns.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) along with embryonic ectoderm development (EED) and suppressor of zeste 12 (SUZ12), which implements transcriptional repression mainly by depositing trimethylation marks at lysine 27 of histone H3 (H3K27me3). Its catalytic activity is closely correlated with the stability of PRC2, and somatic activating mutation of EZH2 Y641F within the catalytic SET domain drives tumor aggressiveness, drug resistance, and poor prognosis. Here, we report two high-throughput screening (HTS) campaigns targeting EZH2 Y641F and EZH2-EED interaction, respectively. For the EZH2 Y641F mutant, the HTS campaign involved a library of 250,000 compounds using a homogenous time-resolved fluorescence (HTRF) assay and identified 162 hits, while 60,160 compounds were screened against EZH2-EED interaction with a fluorescence polarization (FP) assay resulting in 97 hits. Among the 162 EZH2 Y641F inhibitors, 38 also suppressed EZH2-EED interaction and 80 showed inhibitory effects on the wide-type (WT) EZH2. Meanwhile, 10 of the 97 EZH2-EED interaction inhibitors were active against WT EZH2. These hit compounds provide useful tools for the development of novel PRC2-EZH2 inhibitors targeting its catalytic and non-catalytic activities.

Valproic acid exposure leads to upregulation and increased promoter histone acetylation of sepiapterin reductase in a serotonergic cell line.

Valproic acid (VPA) is a widely used antiepileptic drug and first-line treatment in bipolar disorder, although the mechanisms underlying its therapeutic effects are largely unknown. Recently, the recognition of VPA as an epigenetic drug offers new opportunities for understanding its therapeutic actions. In a rat serotonergic cell line (RN46A) we observed that VPA exposure has a strong upregulatory effect on the gene for sepiapterin reductase (SPR), a key enzyme involved in the tetrahydrobiopterin (BH4) synthetic pathway. BH4 is an essential cofactor in the biosynthesis of neurotransmitters like serotonin, dopamine and noradrenalin, and the BH4 pathway may thus be important in mood biology. Using real-time quantitative PCR we show that VPA, at therapeutically relevant doses, increases the expression of the Spr gene by about 8-fold in RN46A cells. In addition, Spr protein levels in VPA-exposed cells were elevated, as were the intracellular BH4 levels. HDAC inhibitors (HDACI) trichostatin A and sodium butyrate also upregulated Spr, but this was not observed using the VPA-analogue valpromide, which lacks HDAC inhibitory activity. Further examination of this effect revealed that exposure to VPA increased the acetylated histone mark H3K9/K14ac at the Spr promoter. The DNMT inhibitor 5'aza-dC also upregulated Spr by over 8-fold. However, DNA methylation status across the Spr promoter did not change in response to VPA. The BH4 pathway is fundamental to the regulation of neurotransmitters relevant to mood disorders, and this epigenetic effect of VPA at the Spr promoter may represent a novel mechanism through which VPA achieves its therapeutic action.