Abstract A079: Structure-guided discovery of first-in-class TRIB2 small-molecule inhibitors for therapy-resistant neuroendocrine prostate cancer
Therapy-induced lineage plasticity is a major mechanism of resistance in advanced prostate cancer, enabling tumor progression despite androgen receptor (AR)-directed therapies and promoting emergence of lethal neuroendocrine prostate cancer (NEPC). Our prior studies identified the pseudokinase Tribbles homolog 2 (TRIB2) as a key regulator of therapy resistance, lineage plasticity, and neuroendocrine (NE) differentiation. Despite strong biological rationale, TRIB2 remains difficult to target because pseudokinases lack catalytic activity and often possess shallow or poorly defined ligand-binding pockets. However, structural analyses indicate that TRIB2 retains a conserved ATP-binding pocket involved in regulating protein stability and scaffolding functions, suggesting a tractable therapeutic vulnerability. We employed a structure-guided drug discovery workflow integrating computational docking, biochemical validation, thermal shift assay (TSA), cellular thermal shift assay (CETSA), and functional screening to identify compounds targeting the TRIB2 pseudokinase domain. Lead compounds were evaluated for target engagement, selectivity, and biologic activity in enzalutamide-resistant and NEPC models. Mechanistic studies assessed downstream signaling, lineage-plasticity markers, apoptosis, and viability in antiandrogen-resistant and engineered TRIB2-expressing systems. Structure-guided optimization identified TBI-001 as a bioactive TRIB2-directed small molecule with favorable pharmacologic properties. TSA and CETSA confirmed direct interaction between TBI-001 and TRIB2 in biochemical and cellular contexts. TBI-001 induced TRIB2 destabilization and suppressed downstream survival signaling, including AKT and BCL2 pathways. Treatment reduced expression of TRIB2 regulated lineage-plasticity and NE regulators, including MYCN, EZH2, ASCL1, BRN2 and SOX2. Mechanistically, TRIB2 actively suppresses AR signaling to facilitate resistance; accordingly, TRIB2 inhibition restored AR pathway activity and resensitized resistant cells to enzalutamide. Functionally, TBI-001 selectively reduced viability and induced apoptosis in enzalutamide-resistant and NEPC models while sparing nonmalignant prostate epithelial cells. In xenograft studies, TBI-001 inhibited tumor growth, reduced NE marker expression, and was well tolerated without overt toxicity. Human liver microsomal stability assays demonstrated low intrinsic clearance and prolonged microsomal half-life, supporting favorable drug-like pharmacokinetic properties. These findings establish TRIB2 as a druggable vulnerability in enzalutamide-resistant and NE prostate tumors despite the inherent challenges of targeting pseudokinases. Pharmacologic disruption of TRIB2 signaling using TBI-001 suppresses oncogenic and lineage-plasticity programs, restores sensitivity to AR-directed therapy, and selectively impairs survival of resistant tumor cells. This work provides a framework for development of first-in-class TRIB2-directed therapeutics for aggressive prostate cancer subtypes. JITENDER MONGA, Sang-Yoon Lee, Deepak Rohila, Sharad K. Suthar, Shirish Gadgeel, Craig Rogers. Structure-guided discovery of first-in-class TRIB2 small-molecule inhibitors for therapy-resistant neuroendocrine prostate cancer [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A079.