A robust bacterial high-throughput screening assay to identify pharmacological chaperones targeting human homogentisate 1,2-dioxygenase missense variants in alkaptonuria

Pros, cons & Future potential

• - Enables high-throughput, quantitative screening of HGD activity across multiple variants and compound simultaneously.
• - Uses E. coli as an efficient, low-cost expression system, avoiding the need for complex eukaryotic expression.
• - Simplified workflow without enzyme purification maintains throughput while retaining sufficient assay sensitivity.
• - Demonstrates robust performance with Z' values > 0.4 and signal window > 2, confirming assay reliability.
• - Supports drug repurposing efforts by identifying existing compounds that enhance HGD activity.
• - Facilitates variant-specific functional assessment, contributing to personalized medicine approaches for AKU.
• - Provides a scalable and adaptable platform that can be expanded to other protein-misfolding disorders.

• - Cannot model compound heterozygosity or hetero-oligomer formation of HGD variants.
• - The use of E. coli limits physiological relevance, as bacterial cells lack the endoplasmic reticulum where chaperone action occurs in humans.
• - The non-purififed enzyme preparation leads to higher apparent Km values and may reduce sensitivity to weak binders.
• - Stabilizing effects observed from bacterial lysates require confirmation in human cell-based systems and further validation in in vivo models.
• - Hit reproducibility may vary between primary and dose-response screens, requiring confirmation and optimization.

The method could be further optimized in several ways. For example, adaptation to mammalian or human cell-based systems could provide a more physiologically relevant environment for assessing HGD stabilization. The use of partially purified enzyme preparations might enhance kinetic accuracy and enable clearere interpretation of compound effects. Additionally, further miniaturization to higher-density plate formats and increased automation could further improve throughput and reproducibility.

This method could serve as a primary screening platform for identifying stabilizing compounds in other inborn errors of metabolism (IEMs) caused by enzyme misfolding or instability. The overall workflow provides a useful basis for adaptation to other target enzymes, but would require further optimization and validation to account for differences in enzyme structure, cofactors, and assay conditions. it could also be used for variant functional studies to assess residual activity and support genotype-phenotype correlation analyses. In addition, this approach may contribute to drug repurposing and precision medicine research by enabling rapid identification of candidate compounds for follow-up in more physiologically relevant systems.