Rosmarinic Acid Is an hCtr1 Inhibitor that Interferes with Copper Transport Activity.

Human copper transporter 1 (hCtr1) is a transport protein essential for cellular copper uptake and homeostasis, yet excess copper influx is toxic to cells. Currently, no inhibitors of hCtr1 are available to modulate intracellular copper levels. Here, we discovered that the natural phenolic compound rosmarinic acid (RA), as a moderate-strength copper chelator, can directly interact with hCtr1 and inhibit its copper-transport function. RA binds to the N-terminal copper-binding domain of hCtr1, lowering the Cu(II)-binding affinity of hCtr1. More crucially, RA and hCtr1 form a ternary complex with Cu(II) ions (RA/Cu/hCtr1). Cellular assays indicated that RA inhibited the copper-mediated membrane interaction of the hCtr1 N-terminal copper-binding domain, a crucial step in triggering cellular copper internalization. Functional assays showed that RA significantly inhibits hCtr1-mediated copper uptake under high copper conditions, hence effectively alleviating cellular copper overload and attenuating copper-induced cytotoxicity. Notably, the moderate copper-binding affinity of RA minimizes adverse effects while modulating the cellular copper levels. This work identifies RA as a promising lead compound for modulating copper-related pathologies and provides a strategy for regulating intracellular copper homeostasis by targeting hCtr1.