Development of Specific Fluorogenic Substrates for Human β-N-Acetyl-D-hexosaminidase A for Cell-Based Assays.

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• Human beta-N-acetyl-D-hexosaminidase A (hHEXA) and O-GlcNAcase (hOGA) are implicated in diseases like Sandhoff, Tay-Sachs, Alzheimer's, and progressive supranuclear palsy.
• Pharmacological chaperone (PC) therapy using hHEXA inhibitors shows promise for Sandhoff disease (SD) and Tay-Sachs disease (TSD).
• Existing diagnostic substrates for hHEXA are insufficient for screening inhibitors from compound libraries, especially in cell-based assays.

Purpose of the Study:

• To address the lack of suitable hHEXA substrates for cell-based inhibitor screening.
• To develop novel, highly specific, and sensitive fluorogenic substrates for hHEXA.
• To enable efficient screening of potential pharmacological chaperone therapies for lysosomal storage diseases.

Main Methods:

• Comparative analysis of tertiary structures of hHEXA and hOGA to identify active site differences.
• Application of a quinone methide cleavage substrate design platform for molecular design.
• Synthesis and evaluation of novel three-color fluorogenic substrates in three human cell lines.

Main Results:

• Novel three-color fluorogenic substrates for hHEXA were successfully synthesized.
• The developed substrates demonstrated excellent specificity and sensitivity in human cell lines.
• These substrates are the first of their kind for screening hHEXA inhibitors in adherent cultured cells.

Conclusions:

• The new fluorogenic substrates are ideal tools for screening hHEXA inhibitors.
• This advancement facilitates the development of pharmacological chaperone therapies for hHEXA-related diseases.
• The findings pave the way for improved diagnostics and therapeutic strategies for lysosomal storage diseases.