VHL-recruiting PROTAC attenuates renal fibrosis and preserves renal function via simultaneous degradation of Smad3 and stabilization of HIF-2α

Background: Renal fibrosis is the pathological foundation of various chronic kidney diseases progressing to end stage renal failure. However, there are currently no nephroprotective drugs targeted to the fibrotic process in clinical practice. Proteolytic targeting chimeras (PROTACs), which reversibly degrade target proteins through the ubiquitin-proteasome pathway, is a novel therapeutic modality. SMAD3 is a key pathogenic factor in fibrogenesis while HIF-2α exhibits prominent renal protective effects, which is the natural substrate of von Hippel-Lindau (VHL) E3 Ligase. We hypothesied the construction of VHL-recruiting, Smad3-targeting PROTAC might combine the effects of SMAD3 degradation and HIF-2α stabilization, which not only improving the clinical efficacy of PROTAC but also avoiding its potential off-target effects, could greatly improve the possibility of its translation into clinical drugs.

Methods: By joining the Smad3-binding small molecule compound (SMC) to VHL-binding SMC with a linker, we designed and synthesized a Smad3-targeting, VHL-based PROTAC. The effects of this PROTAC on targeted proteins were verified both in vitro and in vivo. The toxicity and pharmacokinetic (PK) evaluations were conducted with both male and female mice. The renal protection effects and mechanism of PROTAC were evaluated in unilateral ureteral obstruction (UUO) and 5/6 subtotal nephrectomy (5/6Nx) mouse model.

Results: By optimizing the linker and the Smad3-binding SMC, we got a stable and high efficient PROTAC which simultaneously degraded SMAD3 and stabilized HIF-2α both in vivo and in vitro. The acute toxicity evaluation showed a pretty large therapeutic window of the PROTAC. The prominent renal protection effects and its underlying mechanism including anti-fibrosis and anti-inflammatory, improving renal anemia and promoting kidney repair, had all been verified in UUO and 5/6Nx mouse model.

Conclusion: By accurate combination of PROTAC targeted protein and E3 ligase, we got a Smad3-targeting, VHL-recruting PROTAC which caused SMAD3 degradation and HIF-2α stabilization effects simultaneously, and led to the strong renal function protection effects.

HIF-2α; PROTACs; Renal fibrosis; Smad3.