Compound B3(Hangzhou City University)

Under hypoxic stress, cardiomyocytes predominantly depend on 20S proteasome-mediated degradation to clear accumulated and misfolded proteins. The resultant proteotoxic stress from impaired protein homeostasis contributes significantly to the pathogenesis of cardiovascular disorders. Pharmacological enhancement of 20S proteasome activity thus represents a novel therapeutic paradigm for ischemic cardiomyopathy by restoring proteostasis in myocardial cells. In this study, the hit compound X-1 was identified through screening of proteasome activation profile. Subsequent structure-activity relationship optimization yielded a series of highly potent activators. Intracellular protein degradation assessment revealed that these compounds possessed abilities to alleviate endoplasmic reticulum stress, as demonstrated by the luciferase reporter system. Additionally, selected compound B3 exhibited superior cytoprotection, increasing viability of hypoxia-injured cardiomyocytes while downregulating ER stress markers CHOP and Grp78 at the protein level. AlphaFold3-predicted binding modes and 100 ns molecular dynamics simulations revealing its stabilization of the α region to induce proteasome gate opening, thereby establishing a structure-function rationale for its 20S activation mechanism.