GSK343: A Selective EZH2 Inhibitor for Precision Epigenetic Research

Executive Summary: GSK343 is a highly selective, cell-permeable inhibitor targeting the histone methyltransferase EZH2, the catalytic subunit of PRC2, with an in vitro IC50 of 4 nM for EZH2 enzymatic activity (product source). It blocks H3K27 trimethylation, a repressive histone mark central to cancer and stem cell gene regulation (Kotian et al., 2024). GSK343’s SAM-competitive inhibition is highly selective for EZH2 over other methyltransferases, but it also inhibits EZH1 at higher concentrations. The compound is insoluble in water/ethanol, soluble in DMF, and is primarily used in vitro due to high in vivo clearance. GSK343 advances mechanistic and translational studies in epigenetic modulation, enabling researchers to dissect PRC2 function and downstream gene repression with high precision (see prior review).

Biological Rationale

Polycomb repressive complex 2 (PRC2) orchestrates epigenetic silencing by catalyzing trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2, the core catalytic subunit, is frequently upregulated in cancers and stem cell compartments, contributing to silencing of tumor suppressor genes such as RUNX3, FOXC1, and BRCA1 (Kotian et al., 2024). H3K27me3 is a stable, repressive chromatin mark, and its deposition is tightly coupled to cell fate decisions, proliferation, and oncogenesis. Inhibiting EZH2 activity with GSK343 enables precise investigation of the dynamics and functional consequences of H3K27me3 in cancer and pluripotent stem cell models (GSK343: A Selective EZH2 Inhibitor—this article provides extended mechanistic benchmarking and workflow guidance beyond prior reviews).

Mechanism of Action of GSK343

GSK343 is a potent, competitive inhibitor of EZH2’s methyltransferase activity. It targets the S-adenosylmethionine (SAM) binding site of EZH2, preventing transfer of methyl groups to H3K27. The compound exhibits an enzymatic IC50 of 4 nM for recombinant EZH2 under standard in vitro assay conditions (25°C, 50 mM Tris-HCl, pH 8.5) (ApexBio product spec). GSK343 is over 60-fold selective for EZH2 versus its homolog EZH1 (IC50 for EZH1: 240 nM), and shows minimal inhibition of other SAM-dependent methyltransferases including DNMT, MLL, PRMT, and SETMAR at concentrations up to 10 μM. By blocking H3K27 trimethylation, GSK343 derepresses PRC2 target genes and disrupts cancer cell proliferation and survival. The compound is cell-permeable, enabling robust modulation of chromatin methylation in cultured cells (GSK343: Selective EZH2 Inhibitor—contrasts this mechanism section by including additional selectivity and workflow constraints).

Evidence & Benchmarks

• GSK343 inhibits recombinant EZH2 with an IC50 of 4 nM in biochemical assays (ApexBio: product page).
• GSK343 reduces H3K27 trimethylation in HCC1806 breast cancer cells (IC50: 174 nM, 72 h treatment, DMEM, 10% FBS, 37°C) (ApexBio).
• Inhibits proliferation of LNCaP prostate cancer cells with an IC50 of 2.9 μM (96 h, RPMI 1640, 10% FBS, 37°C) (ApexBio).
• Cellular selectivity confirmed: minimal off-target inhibition of DNMT, MLL, PRMT, and SETMAR at ≤10 μM (ApexBio).
• Induces apoptosis and autophagy in cancer cells (breast/prostate lines) (ApexBio; see also Unlocking Precision Epigenetic Modulation—this article expands on translational use cases).
• Potentiates sorafenib response in HepG2 liver cancer cells (combination index studies, 48 h, DMEM, 10% FBS, 37°C) (ApexBio).
• High in vivo clearance in rodents; limited utility as a systemic drug (ApexBio).
• PRC2 inhibition by GSK343 can rescue loss of TERT transcription caused by MEK inhibition in human pluripotent stem cells (Kotian et al., 2024, Fig. 4).

Applications, Limits & Misconceptions

GSK343’s primary application is as an in vitro tool compound for dissecting PRC2 function and chromatin regulation in cancer and stem cell models. It is widely used to study gene silencing, epigenetic therapy, and the molecular basis of oncogenic transformation. The compound’s high selectivity makes it ideal for mechanistic studies, including rescue experiments in pluripotent stem cells and combinatorial screening with other epigenetic or kinase inhibitors (Illuminating EZH2 Inhibition—this article adds unique benchmarks for telomerase/TERT axis modulation).

Common Pitfalls or Misconceptions

• GSK343 is not suitable for in vivo or clinical studies due to rapid clearance in animal models.
• It is not water- or ethanol-soluble; must be solubilized in DMF (≥7.58 mg/mL with gentle warming).
• Not a pan-PRC2 inhibitor: efficacy is much lower against EZH1 versus EZH2 (IC50: 240 nM vs 4 nM).
• Does not inhibit DNA methyltransferase (DNMT), PRMT, or non-SET domain methyltransferases at relevant concentrations.
• Functional rescue of gene expression depends on cellular context; derepression requires transcriptionally competent loci.

Workflow Integration & Parameters

Preparation: Dissolve GSK343 in DMF at ≥7.58 mg/mL with gentle warming. Store solid at -20°C, protected from light (GSK343 protocol).

Typical in vitro use: Treat cultured cells at 100 nM–5 μM for 24–96 h. Monitor H3K27me3 by ChIP, western blot, or mass spectrometry. Validate target gene activation by qPCR or RNA-seq. For combinatorial studies, co-treat with kinase inhibitors (e.g., MEK/ERK) or established chemotherapeutics (Kotian et al., 2024).

Controls: Use vehicle (DMF) controls and, if possible, orthogonal EZH2 inhibitors to confirm specificity. Assess cell viability and off-target toxicity at each concentration.

For detailed protocol steps and troubleshooting, users may consult the A3449 kit documentation and compare with Precision Epigenetic Modulation—this article updates workflow integration with new guidance on pluripotent stem cell systems.

Conclusion & Outlook

GSK343 is a highly validated, cell-permeable, and selective EZH2 inhibitor that enables precise, reproducible interrogation of PRC2-driven epigenetic silencing. Its robust biochemical and cellular activity, combined with high selectivity and in vitro compatibility, make it a gold-standard probe for cancer and stem cell epigenetics. Future work will focus on leveraging GSK343 in multi-omic platforms and combinatorial screening to unravel context-specific dependencies on PRC2 and H3K27me3. For more details or to order, refer to the GSK343 product page.