Modulation of the alpha3-Na, K-ATPase isoform by digoxin derivatives in SHSY-5Y cells with the D923Y mutation in the ATP1A3 gene associated with alternating hemiplegia of childhood (AHC)

• Grant number: 26/08779-7
• Support Opportunities: Regular Research Grants
• Start date: August 01, 2026
• End date: July 31, 2029
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: Cristoforo Scavone
• Grantee: Cristoforo Scavone
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• City of the host institution: São Paulo
• Associated researchers: Jacqueline Alves Leite ; Leandro Augusto de Oliveira Barbosa ; Luciana Biagini Lopes ; Maria Rita dos Santos e Passos Bueno

Abstract

Cardiotonic steroids (CTSs) are compounds that interact with the Na/KATPase (NKA) enzyme, which is fundamental for maintaining ionic balance andintracellular conditions in the brain. Beyond their classic role in ion transport, thisinteraction can also activate specific signaling pathways. CTSs exhibit a high capacity forstructural modification, resulting in distinct affinities for NKA isoforms. Compoundsderived from digoxin (¿-benzylidene-digoxin - BD), such as BD-14 and BD-15, havedemonstrated selectivity for the ¿3 isoform and the ability to stimulate NKA activity.Studies from our group show that others, such as BD-15 and BD-21, exhibit significanteffects at low concentrations, including anti-inflammatory and neuroprotective actions.Cytotoxicity assays in Wistar rats indicate that BD-15 has a possible cardioprotectiveeffect, without causing liver or kidney damage, exhibiting low toxic potential and greatersafety than digoxin. Alternating hemiplegia of childhood (AHC) results from mutationsin the ATP1A3 gene, affecting the ¿3 isoform of NKA. The disease manifests before 18months of age with hemiplegia, epileptic seizures, ataxia, and cognitive deficit, with nocure and only symptomatic treatments available. Therefore, it is essential to investigatenew cardiotonic steroids (CTS) as therapeutic alternatives. Compounds derived fromBD-15 (BD15-7, BD15-8, BD15-9, ISO-BD15, and BOUA15) have been developed withstructural modifications that confer distinct affinities for NKA isoforms. Their efficientsynthesis, low cost, and ease of implementation make them promising for AHC studies.This study proposes using SHSY-5Y cells, with CRISPR gene editing, to generate aknock-in cell model of the D923Y variant associated with AHC. The project also intendsto use an in vivo model that carries the same ATP1A3 mutation associated with AHC toassess the toxicity and effects observed in vitro. This model will enable optimizedevaluation of modulation of the ¿3-NKA isoform by the compounds and characterizationof oxidative and mitochondrial parameters related to the pathophysiology of the disease.The development of these drugs, in conjunction with BD nanocomposites, could expandaccess to innovative therapies, integrate nanomedicine and pharmacology, andstrengthen translational medicine and international scientific cooperation. (AU)