Alkaptonuria (AKU) is a rare genetic disorder caused by mutations in the homogentisate 1,2-dioxygenase (HGD) gene. The HGD enzyme forms a complex hexameric structure (dimer of trimers), which is highly susceptible to destabilization by missense mutations, accounting for 64.2 % of AKU-causing variants. Current treatment with nitisinone (NTBC) in combination with a low-protein diet, is associated with severe side effects, underscoring the unmet need for alternative therapies. To explore pharmacological chaperones that stabilize mutant HGD and restore enzymatic activity, we developed and optimized a high-throughput screening (HTS) assay in Escherichia coli expressing human HGD variants. The assay quantifies time-dependent formation of maleylacetoacetate, the product of homogentisic acid conversion, and was validated for robustness (Z'-value > 0.4, Signal window > 2). We screened a library of 2320 FDA-approved drugs and identified 30 compounds that increased the catalytic activity of the prevalent HGDG161R variant by at least 3-fold. Compound 21 showed a dose-dependent effect, doubling activity at 100 and 250 μM compared to the untreated variant. Molecular docking using CB-Dock indicated that compound 21 binds at multiple sites on the unbound form of HGD, particularly at the active site loop and C-terminal β-sheet, suggesting stabilization prior to substrate and cofactor binding. Our newly developed HTS assay provides a powerful tool for ranking HGD missense variants based on their residual activity, offering insights into genotype-phenotype correlations and enabling compound screening for their stabilization. These findings support the development of targeted therapies and a promising personalized alternative to NTBC for AKU patients.
Keywords: Alkaptonuria; High-throughput screening system; Human homogentisate 1,2-dioxygenase; Missense variants; Personalized medicine; Pharmacological chaperone therapy.
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