Sylvia Stockler: “Development of a Creatine Uptake Assay to Evaluate Repurposable Drugs in SLC6A8-Deficient Fibroblasts”
ABSTRACT
Introduction: Therapeutic strategies for cerebral creatine transporter deficiency (CTD) include gene therapy, creatine analogues, and small molecules to restore transporter function.
Previous Work: We focused on small-molecule approaches. Using an AI-based screening tool, we identified eight repurposable drug candidates that enhanced SLC6A8 activity in ΔF408-transfected HEK cells, measured by single-cell patch-clamp analysis (reported previously).
Aim: To assess translational relevance, we developed a sensitive LC–MS/MS assay to quantify creatine uptake in human control and SLC6A8-deficient fibroblasts, including ΔF408.
Methods & Results: Fibroblasts were incubated with D3-labelled creatine (50 µM) for 6 hours, and intracellular levels were quantified in lysates by LC–MS/MS. Experiments used ~9.6 cm² confluent monolayers (~400–600 µg protein per well), performed in triplicate and replicated across independent experiments, yielding robust reproducibility. Drug incubation ranged from 3–96 hours. Uptake variability correlated with passage number and growth rate but did not affect outcomes. Toxicity was minimal, with 7/8 compounds tolerated at 10–100 µM; one compound was excluded due to solubility issues. None of the drugs increased creatine uptake above baseline under any condition. Phenylbutyrate, currently in clinical evaluation, is currently under investigation.
Relevance: Findings highlight a potential translational gap between overexpression systems and patient-derived cells. Although no compounds enhanced creatine uptake, this work establishes a valuable platform for preclinical evaluation of additional therapeutic candidates in SLC6A8 deficiency. The assay also supports functional validation of newly identified SLC6A8 variants.
Supported by: Association for Creatine Deficiencies; BC Children’s Hospital Research Institute.