A Pyrazole-Based Small Molecule, KB3409, Restores Mitochondrial Function via VCP Activation and RCN2-Dependent Ca2+ Regulation

Cellular senescence is characterized by the accumulation of reactive oxygen species (ROS), and the selective elimination of excessive ROS remains a key therapeutic challenge. Through antioxidant screening, we identified the pyrazole-based small molecule KB3409 as a potent regulator that reduces ROS levels in senescent fibroblasts. To elucidate its mechanism of action, we performed target identification and found that KB3409 directly binds to valosin-containing protein (VCP) and reticulocalbin-2 (RCN2). Functionally, KB3409 acts as an allosteric activator of VCP, enhancing its ATPase activity and promoting autophagic flux. This activation facilitates the selective clearance of dysfunctional mitochondria, thereby improving mitochondrial quality control and limiting ROS generation at its source. Concurrently, KB3409 modulates RCN2-dependent Ca2+ homeostasis, alleviating mitochondrial Ca2+ overload and suppressing the opening of the mitochondrial permeability transition pore (mPTP). This coordinated regulation preserves mitochondrial structural integrity and sustains oxidative phosphorylation efficiency. Collectively, these findings identified the novel mechanism in which KB3409 restores mitochondrial function, reduces ROS levels, and functionally reverses cellular senescence phenotypes.