Rational design and discovery of potent PROTAC degraders of ASK1: a targeted therapy in MASH.

Abstract

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease marked by hepatic steatosis, inflammation, and fibrosis, with limited therapeutic options. This study introduces a novel PROTAC-based strategy for the selective degradation of apoptosis signal-regulating kinase 1 (ASK1), a key mediator of MASH pathology. We first developed <b>dASK1</b> (35), a cereblon (CRBN)-based PROTAC, which successfully formed a stable ternary complex with ASK1, facilitating its rapid and sustained degradation <i>via</i> the ubiquitin-proteasome pathway. <i>In vitro</i> evaluations demonstrated potent ASK1 degradation in the 10-100 nM range (70% degradation at 100 nM) in HepG2 and HEK293A cell lines, validating the efficacy of <b>dASK1</b> (35). To enhance the degradation mechanism and explore broader E3 ligase utility, we designed and synthesized <b>dASK1-VHL</b> (60), leveraging the von Hippel-Lindau (VHL) E3 ligase, known for its regulatory functions in hepatic physiology. We optimized the linker length through molecular docking and MMGBSA calculations, achieving efficient ASK1-VHL engagement and stable ternary complex formation. Detailed ADME and pharmacokinetic studies confirmed that <b>dASK1-VHL</b> (60) exhibited enhanced solubility, moderate clearance, and improved bioavailability, making it suitable for <i>in vivo</i> application. In an MCD diet-induced murine model of MASH, <b>dASK1-VHL</b> (60) effectively reduced ASK1 protein levels, suppressed p38 MAPK activation, and decreased hepatic lipid content, indicating significant therapeutic benefits. This work underscores the importance of rational PROTAC design, precise linker engineering, and innovative E3 ligase selection in optimizing target protein degradation. Our findings pave the way for developing VHL-based PROTACs, offering a novel therapeutic approach for metabolic and inflammatory liver diseases.