Abstract B108: NTS231 is a novel covalent allosteric molecular glue of the KEAP1-CUL3 E3-ligase complex that selectively degrades NRF2 and inhibits tumors with aberrant NRF2 activation

Abstract Loss-of-function mutations in the E3-ligase complex of Kelch-like ECH-associated protein 1 (KEAP1) and Cullin 3 (CUL3) or gain-of-function mutations in its primary substrate – nuclear factor erythroid 2-related factor 2 (NRF2) are frequently observed in non-small cell lung cancer (NSCLC) and various other cancers. Such mutations lead to aberrant activation of NRF2 transcriptional activity and cause therapeutic resistance. Due to the intrinsically disordered structure of NRF2 and lack of a well-defined binding pocket for drug molecules, direct inhibition of NRF2 has been historically challenging. However, activating the KEAP1-CUL3 E3-ligase complex offers a promising alternative by promoting NRF2 degradation through the ubiquitin-proteasome system. NTS231, developed by Nutshell's proprietary AlloStarTM, an AI-powered computational platform for allosteric drug design and optimization, is an oral small molecule that covalently binds to the Cysteine-151 (C151) residue of KEAP1 and allosterically enhances the protein-protein interaction between KEAP1 and CUL3 as demonstrated by the KEAP1-CUL3 homogeneous time-resolved fluorescence (HTRF) assay. NTS231 potently induces NRF2 degradation, suppresses NRF2-dependent gene expression and inhibits the viability of various NRF2/KEAP1/CUL3-mutated or NRF2-amplified cell lines cultured in 3D microtissue spheroids with the lowest of single-digit nanomolar activities. Mechanism of action (MOA) study using western blot confirmed that NTS231 selectively degraded NRF2 without affecting other reported substrates of KEAP1-CUL3 E3-ligase complex such as MCM3, p62, PGAM5, Bcl-2, and IKKβ; Additionally, NTS231 exhibits no obvious off-target effects in the Eurofins Safety Panel screening at 10 μM, indicating high target specificity and safety potential. The in vivo antitumor efficacy of NTS231 was validated across multiple CDX and PDX models harboring diverse NRF2/KEAP1/CUL3 mutations, with pronounced NRF2 degradation and significantly reduced expression of NRF2-dependent genes. Furthermore, NTS231 elicits synergy with existing treatment methods including chemotherapy and targeted therapies such as EGFR, KRAS G12C, pan-KRAS, and PI3Kα inhibitors, etc, showing robust synergistic effects both in vitro and in vivo. Overall, NTS231 exhibits exceptional potency, high selectivity and desirable ADME properties without CYP or hERG inhibition liability. It has also demonstrated good tolerability in preclinical toxicology studies in rats and dogs and is progressing toward IND filing in 2026. Citation Format: Qiang Li, Chengyi Zhang, Chengxiang Wang, Xiaowei Ma, Guoqiang Zhou. NTS231 is a novel covalent allosteric molecular glue of the KEAP1-CUL3 E3-ligase complex that selectively degrades NRF2 and inhibits tumors with aberrant NRF2 activation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B108.