# Biological Evaluation of a Novel Compound with Predicted EZH2 and EED Binding Against Human Malignant Melanoma Cells

## TL;DR

This paper introduces a new compound, SG-8, which may effectively target melanoma cells by interacting with EZH2 and EED proteins, potentially offering improved brain penetration compared to existing drugs.

## Contribution

The novel compound SG-8 is designed to overcome limitations of current EZH2 inhibitors, particularly brain penetration and cytotoxicity in melanoma cells.

## Key findings

- SG-8 showed passive blood–brain barrier permeability in in vitro PAMPA assays.
- SG-8 and PF-06726304 reduced EED and EZH2 protein levels in melanoma cells without affecting H3K27me3 levels.
- SG-8 treatment reduced phosphorylation of EZH2 and Akt, suggesting modulation of the Akt–EZH2 signaling axis.

## Abstract

Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), mediates histone H3 lysine 27 trimethylation (H3K27me3), an epigenetic modification associated with transcriptional repression. EZH2 inhibitors (EZH2is) gained attention after the first-in-class drug Tazemetostat received FDA approval for treating epithelioid sarcoma. Preclinical studies suggest that EZH2is could be effective against melanoma, but their general inability to cross the blood–brain barrier (BBB), among others, limits the treatment of secondary brain metastases. Based on these limitations, we designed SG-8, a novel compound derived from TDI-6118 (a known brain-penetrant EZH2i). In silico docking predicted that SG-8 may exhibit high affinity for EZH2 as well as for another PRC2 subunit, Embryonic Ectoderm Development (EED). In addition, in vitro PAMPA assays suggested passive BBB permeability of SG-8. In cell-based assays, SG-8 and the structurally related EZH2i PF-06726304 displayed lower cytotoxicity than Tazemetostat in both primary (A375) and metastatic (Colo-679) human melanoma cells. Western blot analysis showed that SG-8 and PF-06726304 markedly reduced EED protein levels and, to a lesser extent, EZH2 levels, without affecting total H3K27me3, consistent with preserved canonical PRC2 activity. Instead, treatment with both compounds—most prominently SG-8—was associated with reduced phosphorylation levels of EZH2 (Ser21) and its upstream regulator Akt (Ser473), suggesting that modulation of the Akt–EZH2 signaling axis may at least partially contribute to their anti-melanoma activity.