A broad-spectrum inhibitor of copper-exporting P_1B-type ATPases.

Abstract

Copper (Cu) transporting ATPases represent a highly conserved subclass of P-type ATPases with critical roles in Cu export and metalloenzyme synthesis. Despite their important biological roles and association with a wide range of human diseases, no high-affinity small-molecule inhibitors have been described. Here, we identify MKV3 as a small molecule inhibitor of Cu-transporting P-type ATPases that targets a conserved Cu⁺ entry site to the translocation pathway. In silico docking against the <i><i>Xenopus</i></i> ATP7B structure revealed a highly conserved pocket suitable for pharmacological inhibition. MKV3 bound human ATP7A and ATP7B with nanomolar affinity, competed with N-terminal metal-binding domains for access to the Cu⁺ entry site, and selectively inhibited <i><i>Escherichia coli</i></i> CopA ATPase activity and Cu⁺ transport. Mechanistically, MKV3 blocked chaperone-mediated Cu⁺ delivery to the intramembranous CPC site of CopA that is essential for its transport function. We further identified a single charged P-domain residue that governed MKV3 affinity and potency across species. Functionally, MKV3 phenocopied the genetic loss of Cu⁺-ATPases in bacteria, fungi, plants, zebrafish, and mammals, impairing copper-dependent enzymes, transporter trafficking, and copper tolerance. These findings establish a conserved, druggable vulnerability in Cu⁺-ATPases and introduce MKV3 as a broadly active chemical tool to modulate copper homeostasis across biological kingdoms.