A Dual-Function Guanidinium Scaffold that Couples Copper Sequestration with Redox Protection in Wilson disease

Abstract

Wilson disease (WD) is caused due to mutations in the copper ATPase gene ATP7B, that result in accumulation of labile copper pools and the consequent disruption of cellular redox homeostasis through uncontrolled copper-mediated reactive oxygen species generation. Current therapies mainly depend on high-affinity copper chelation to lower metal burden which sometimes also strip copper from cuproproteins and may disturb physiological copper-dependent processes. It also does not directly suppress pathological copper reactivity i.e., free radical generation, a major driver of WD progression. To overcome these limitations, we have rationally designed Gua-Cu-3, a C-symmetric guanidinium-based molecule that can chelate labile copper without metal stripping from cuproproteins due to moderate binding affinity and it has intrinsic antioxidant activity within a single nanosheet-forming supramolecular self-assembly. Spectroscopic, calorimetric, and computational analyses revealed controlled multivalent copper coordination (Kd = 95.4 M) while radical-scavenging and hydroxyl-radical inhibition assays further revealed potent redox-regulatory activity. In cellular models of copper overload, Gua-Cu-3 treatment reduces ATP7B trafficking from trans-Golgi network, confirming effective intracellular copper sequestration. This was accompanied by a marked reduction in oxidative stress, reduction of translocation of Nrf2 in nucleus and of HO-1 expression, thereby limiting lipid peroxidation which leads to restoration of cellular homeostasis, including improved lysosomal integrity, reduced endoplasmic reticulum stress, decreased mitochondrial superoxide levels, and diminished apoptosis. Importantly, the protective effects extended, beyond cultured cells, Gua-Cu-3 attenuates oxidative stress in ATP7B-homolog-deficient C. elegans and rescues copper-induced developmental defects in zebrafish, outperforming D-penicillamine, which is currently in use for Wilson disease management. These findings establish Gua-Cu-3 as a molecularly designed supramolecular copper-reactivity buffer that couples-controlled copper sequestration with redox regulation which is distinct from conventional copper depletion and provides a framework for treating Wilson disease and other disorders associated with metal dyshomeostasis and oxidative stress.