GPX4-AUTAC is an autophagy-mediated degrader (AUTAC) targeting GPX4. It consists of the inhibitor ML162-yne, a degradation tag FBnG, and a glycol linker. GPX4-AUTAC facilitates the ubiquitination of GPX4 by the E3 ligase TRAF6 and enhances its interaction with GPX4 and p62, leading to selective autophagy-dependent degradation of GPX4. This compound significantly induces ferroptosis and demonstrates potent anticancer activity in breast cancer cells, patient-derived organoids (PDOs), and MDA-MB-231 tumor xenograft mouse models. It shows strong synergy when used in combination with Sulfasalazine (SAS) or chemotherapy drugs (Paclitaxel or Cisplatin).

GPX4-AUTAC reduces GPX4 protein levels in a dose- and time-dependent manner in MDA-MB-231 and MCF-7 cells (5-80 μM, 12-72 hours) while minimally affecting GPX4 mRNA levels. In ovarian, lung, melanoma, and glioma cells, GPX4-AUTAC (20-40 μM, 24 hours) effectively decreases GPX4 protein levels. The compound consistently lowers GPX4 protein levels for up to 96 hours and significantly increases the thermal stability of GPX4 in full cells and cell lysates from MDA-MB-231 and MCF-7 cells under denaturing conditions (10 μM, 24-96 hours, 37-57°C). GPX4-AUTAC (5-80 μM, 24 hours) selectively degrades GPX4 without affecting the expression of other selenoproteins like GPX1, TXNRD1 (a GPX4 homolog), or HK2 (a TRAF6/p62 substrate). It induces ferroptosis in tumor cells (MDA-MB-231) with little impact on normal cells (MCF-10A). In HEK293T cells, GPX4-AUTAC (10 μM, 24 hours) accelerates the autophagy-dependent degradation of GPX4, enhancing both endogenous and exogenous K63-linked ubiquitination. This downregulation effect in MDA-MB-231 cells by GPX4-AUTAC (10-40 μM, 12 hours) can be reversed by PYR-41, NH4Cl, and 3-MA. GPX4-AUTAC (24 hours) promotes colocalization of GPX4 and p62, enhancing GPX4 colocalization with LC3B or LAMP2 in PDOs. Targeting GPX4 selectively at 40 μM for 24 hours leads to substantial ferroptosis accumulation in MDA-MB-231 cells. With GPX4-AUTAC (5-40 μM, 72 hours), specific induction of ferroptosis increases lipid ROS and Fe²⁺ accumulation in MDA-MB-231 cells, only salvageable in MCF-7 cells by Ferrostatin-1 (Fer-1). GPX4-AUTAC (5-80 μM) induces ferroptosis, mitochondrial dysfunction, and significantly raises PTGS2 mRNA levels in MDA-MB-231 cells. It inhibits cell viability and proliferation in MDA-MB-231 and MCF-7 cells dose-and-time-dependently (5-40 μM, 24-96 hours), regardless of GPX4 knockdown sensitivity. A pronounced suppression of PDOs growth, reducing diameter and phase contrast, is observed with GPX4-AUTAC (5-40 μM, 4 days), with GPX4-overexpressing PDOs showing increased sensitivity. Expression of GPX4 is attenuated, and high levels of 4-HNE-induced ferroptosis suppress cancer cell proliferation in PDOs with low Ki67 (10-20 μM, 4 days). Combined with Sulfasalazine, GPX4-AUTAC exhibits significantly stronger anticancer activity and induces more pronounced ferroptosis in MDA-MB-231, MCF-7 cells, and PDOs than Sulfasalazine alone. It also sensitizes MDA-MB-231 and HCC1806 cells to chemotherapy, showing synergistic inhibition of cell viability, proliferation, and PDOs growth when combined with Paclitaxel or Cisplatin (10 μM, 72 hours or 4 days).

The compound GPX4-AUTAC (10-20 mg/kg, intraperitoneal injection, once daily for 10-12 days) demonstrates potent anti-cancer activity by accumulating preferentially and selectively degrading GPX4 in tumor tissues within the MDA-MB-231 xenograft mouse model, significantly reducing tumor weight, volume, GPX4 protein levels, and Ki67 percentage, while increasing 4-HNE levels with minimal impact on normal tissues and no noticeable toxicity in vital organs. Additionally, GPX4-AUTAC (10-20 mg/kg combined with Sulfasalazine 100 mg/kg, intraperitoneal injection, once daily for 10-12 days) acts synergistically with Sulfasalazine, exhibiting strong tumor suppression through ferroptosis induction in the MDA-MB-231 xenograft mouse model without detectable toxicity.