Experimental and computational analysis of SERCA inhibition by naphthoquinone derivatives

Naphthoquinones are small aromatic compounds with diverse biological activities and established applications in medicine and industry. Previous studies have identified a limited number of naphthoquinones that inhibit the sarco/endoplasmic reticulum calcium ATPase (SERCA), an ion-translocating enzyme that plays a central role in cellular calcium homeostasis. To further explore naphthoquinone-mediated SERCA inhibition and its molecular determinants, we evaluated the inhibitory potencies of 13 commercially available naphthoquinones using enzyme activity assays with SERCA1a, enabling the establishment of structure-activity relationships (SARs). The most potent compounds inhibited SERCA at low micromolar concentrations and were characterized by the presence of a hydroxy or nitro substituent at the C5 position. In contrast, electronegative substituents at the C2 position produced mixed effects on potency whereas increased steric bulk reduced or eliminated inhibitory potency. Possible inhibition mechanisms were considered, including covalent modification of cysteine residues and reversible binding to defined sites on the enzyme, with the latter being more consistent with the observed SARs. Molecular simulations of naphthoquinone binding to SERCA further supported this model and provided insight into potential ligand-protein interactions within the ATP-binding pocket.