Design, synthesis and biological evaluation of CYP1B1 targeted degraders for sensitizing immune checkpoint inhibitor therapy.

Abstract

Despite its established role in tumor progression and therapy resistance, Cytochrome P450 1B1 (CYP1B1) has remained largely underexplored as a therapeutic target in immunotherapy, with no prior reports using CYP1B1-targeted agents to enhance immune checkpoint inhibitor efficacy. To address this, we designed a series of symmetric degraders in which an α-naphthoflavone (ANF) derivative 1 was conjugated to both ends of a polyethylene glycol (PEG) linker. This design enables each terminus to independently engage the CYP1B1 active site, while the unbound ANF moiety, owing to its hydrophobic nature, may protrude from the protein surface and serve as an intrinsic hydrophobic tag to trigger degradation. The symmetrical architecture also increases binding probability by allowing either end to occupy the pocket. Notably, compound 2 induced potent, dose-dependent degradation of CYP1B1 with selectivity over CYP1A1, and did not exhibit the hook effect typically observed in conventional bifunctional degraders. Mechanistic studies, supported by molecular docking and co-immunoprecipitation, revealed that the dual-binding configuration acts as a hydrophobic tag, promoting protein misfolding and recruiting chaperones HSP70 and HSP90 to facilitate proteasomal clearance. Proteomic profiling further demonstrated that compound 2 broadly modulates immune signaling, metabolic pathways, and ferroptosis, with a particular impact on pathways associated with tumor immune resistance. In syngeneic colon cancer models, compound 2 exhibited favorable pharmacokinetics and, at a subtherapeutic dose, synergized with anti-PD-L1 therapy to induce complete tumor regression in a subset of animals. Importantly, a Palbociclib-based Cyclin-dependent kinases (CDK) 4/6 degrader was designed as a proof-of-concept that the ANF derivative 1 serves as a hydrophobic tag, supporting its broader applicability. Collectively, this work provides a novel chemical tool for probing CYP1B1 biology and positions its targeted degradation as a promising avenue to sensitize tumors to immune checkpoint blockade.