Rational engineering of isoform-specific hSULT1E1 fluorogenic substrates for functional analysis and inhibitor...

Area of Science:

• Biochemistry and Molecular Biology
• Enzyme Engineering
• Drug Discovery

Background:

• Human estrogen sulfotransferase (hSULT1E1) is vital for estrogen homeostasis and metabolizing foreign compounds.
• Developing specific tools to study hSULT1E1 activity is crucial for understanding its role in health and disease.

Purpose of the Study:

• To rationally design and synthesize highly specific fluorogenic substrates for hSULT1E1.
• To establish a sensitive assay for measuring hSULT1E1 activity and screening for inhibitors.

Main Methods:

• Structure-based virtual screening identified 4-hydroxyl-1,8-naphthalimide as a scaffold.
• Iterative structural modifications led to the development of HN-375, a selective and reactive substrate.
• Fluorescence-based assays were employed for activity detection and inhibitor screening.

Main Results:

• HN-375 was synthesized and demonstrated high specificity and sensitivity for hSULT1E1.
• The substrate produced a bright green fluorescent signal (510 nm) upon sulfation, forming HN-375 4-O-sulfate.
• HN-375 enabled sensing of hSULT1E1 activity in hepatocellular carcinoma specimens and live organs, and facilitated the identification of two potent inhibitors.

Conclusions:

• A groundbreaking strategy for engineering specific fluorogenic substrates for conjugative enzymes was developed.
• HN-375 is a practical tool for sensing hSULT1E1 activity in biological contexts.
• The developed assay supports high-throughput screening of hSULT1E1 inhibitors for potential therapeutic applications.