Discovery of a potent PROTAC targeting G9a suppresses proliferation of triple-negative breast cancer cells and psoriasis-like keratinocytes.

G9a is a SET domain-containing histone methyltransferase that catalyzes H3K9 methylation to regulate gene transcription. Recent studies have revealed that G9a exerts both catalytic and non-catalytic functions in tumor progression and inflammatory diseases, establishing it as a promising therapeutic target. Herein, we developed L4 by using proteolysis targeting chimera (PROTAC) technology. L4 induces G9a degradation through the ubiquitin-proteasome system (UPS) in both a concentration- and time-dependent manner (DC50 = 1.29 μM), while substantially reducing H3K9me2 expression levels. Through molecular dynamics (MD) simulations, we elucidated the binding mode and key interactions of L4, as well as the stable conformation of the G9aSET-L4-CRBNTBD ternary complex. Quantitative proteomics results demonstrated that L4 selectively targets G9a. In tumor models, L4 not only inhibits triple-negative breast cancer (TNBC) cell proliferation in vitro but also promotes apoptosis and suppresses cell migration. Furthermore, we investigated the therapeutic potential of L4 in inflammatory disorders, particularly psoriasis. L4 inhibited HaCaT cell proliferation, promoted G9a degradation, and suppressed NF-κB signaling. In vivo results showed that L4 dose-dependently alleviated skin inflammation, reduced epidermal hyperplasia, and decreased Ki67 expression, showing superior efficacy to BIX01294. Mechanistically, these effects were associated with the downregulation of G9a and NF-κB, while L4 also exhibited favorable local safety. Taken together, the development of L4 presents an innovative approach for designing G9a-targeting PROTAC molecules and offers new therapeutic possibilities for cancer and inflammatory diseases driven by non-enzymatic functions of G9a.