A cell-based degrader assessment platform facilitates discovery of functional NUDT5 PROTACs

Abstract Targeted protein degradation (TPD) via PROTACs and molecular glues holds significant therapeutic promise but demands detailed mechanistic evaluation in live cells to fully understand compound behavior and optimize efficacy. Here, we present an integrated, cell-first platform that combines a modular degradation assay with E3 ligase target engagement readouts for comprehensive assessment of TPD molecules in cells and use it to evaluate PROTACs towards NUDT5. To mimic endogenous degradation conditions and TPD amenability, we established a fusion protein expression system consisting of a lysine-free FKBP12 F36V PROTAC handle (FKBPV K0 ) and used a HiBiT/akaLuc dual luciferase reporter to accurately measure degradation dynamics. This set-up identified a VHL-dependent NUDT5 PROTAC, DDD2, that induced robust NUDT5 degradation, despite impaired NUDT5 binding in vitro and in cellulo , but no CRBN-dependent degraders. NUDT5 lysine availability mapping with DDD2 and FKBP12 F36V-directed PROTACs suggested that the CRL4 CRBN complex is more sensitive to target lysine accessibility than CRL2 VHL , which may have implications for E3 ligase choice and therapeutic resistance. CeTEAM drug biosensors were also established towards CRBN and VHL to quantitatively monitor degrader engagement in living cells and confirmed that the tested CRBN-directed NUDT5 PROTACs poorly engaged the E3. All together, this platform provides a versatile and scalable framework for TPD molecule discovery in a cellular context.