Select azo compounds post-translationally modulate HTRA1 abundance and activity potentially through interactions at...

Area of Science:

• Molecular Biology
• Neuroscience
• Ophthalmology

Background:

• High-temperature requirement protein A1 (HTRA1) is a serine protease implicated in neurodegenerative diseases, including age-related macular degeneration (AMD).
• Genetic studies link the 10q26 locus containing HTRA1 to AMD susceptibility, with risk alleles potentially causing age-related defects in retinal HTRA1 synthesis.

Purpose of the Study:

• To identify small molecules that enhance HTRA1 transcription or protein levels using a high-throughput screening approach.
• To explore potential therapeutic strategies for AMD by targeting HTRA1.

Main Methods:

• CRISPR/Sp.Cas9 gene editing was used to tag HTRA1 with a HiBiT peptide in ARPE-19 cells.
• A library of 1920 compounds was screened for their effect on HTRA1 secretion and intracellular levels.
• Counter-screening, HiBiT blotting, serine hydrolase activity-based protein profiling (SH-ABPP), and computational modeling were employed.

Main Results:

• Chicago Sky Blue 6B (CSB), an azo compound, significantly enhanced HTRA1 secretion (2.0-fold) and intracellular levels (1.7-fold) without altering transcription or enzymatic activity.
• Congo Red, another azo compound, increased intracellular HTRA1 (3.6-fold) but impaired its enzymatic activity.
• Computational modeling suggested CSB and Congo Red bind to HTRA1's trimer interface.

Conclusions:

• Select azo dyes, including CSB, can serve as chemical probes to modulate HTRA1 levels.
• These findings identify potential starting points for developing novel small molecule therapeutics targeting HTRA1 for AMD and other related conditions.