Developing and Characterizing Small Molecules for the Therapy of Wilson Disease

Pandey, Raviranjan (2026) Developing and Characterizing Small Molecules for the Therapy of Wilson Disease. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Raviranjan Pandey (20RS126)) 20RS126.pdf - Submitted Version Restricted to Repository staff only Download (10MB)

Abstract

Wilson Disease (WD) is a genetic disorder caused by mutations in the ATP7B gene, leading to copper accumulation in the liver and brain. This excess copper induces oxidative stress, resulting in severe hepatic and neurological symptoms. Current treatments, such as copper chelators (Penicillamine and Trientine), often lack antioxidant properties, cause significant side effects, and fail to effectively mitigate the downstream consequences of copper accumulation, including oxidative stress and excessive autophagy. In this study, we explore the therapeutic potential of melatonin, an FDA-approved antioxidant supplement, and Gua-Cu-3, a guanidium-based compound with both potent antioxidant and potential metal-chelating properties for Wilson Disease therapeutics. Using wild-type and ATP7B knockout HepG2 cells as a cellular model of WD, along with zebrafish embryos and C. elegans, we investigated their ability to mitigate copper-induced cytotoxicity and oxidative stress. Our findings demonstrate that melatonin and Gua-Cu-3 effectively reduce oxidative stress, apoptosis, and Nrf2 pathway activation while chelating excess copper with moderate affinity, as validated through molecular dynamics simulations, fluorescence spectroscopy, mass spectrometry, and NMR analyses. Additionally, In-Cellulo studies revealed that melatonin and Gua-Cu-3 promote ATP7B trafficking back to the trans-Golgi network under high copper conditions, suggesting that melatonin and Gua-Cu-3 can reduce labile copper pool within the cellular environment. We further found that both molecules exert their antioxidant effects by upregulating key antioxidant enzymes, including GPX4, NRF2, and HO-1, while modulating the glutathione cycle by increasing the expression of GSR to protect cells from oxidative damage. To enhance therapeutic efficacy and minimize off-target effects, we developed glutathione-sensitive polymeric nanocapsules (MNCs) for targeted, stimuli-responsive, and sustained drug release. These MNCs leverage the elevated glutathione levels in hepatic cells, enabling disease-specific controlled drug delivery. Notably, MNCs demonstrated threefold greater efficiency than free melatonin in alleviating oxidative stress. These results highlight melatonin and Gua-Cu-3 as promising therapeutic candidates for WD and other copper metabolism disorders. Furthermore, our nanotechnology-based drug delivery approach represents a significant advancement in WD therapy, offering greater treatment precision while minimizing off-target effects.

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