A fluorescence-based assay for measuring aminopropyltransferase activity.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Polyamines (PAs) are vital for cellular processes and implicated in diseases like cancer and neurodegenerative disorders.
• Aminopropyl transferases (APTs) are key enzymes in polyamine biosynthesis, making them therapeutic targets.
• Existing methods for inhibiting APTs lack selectivity, and high-throughput screening assays are needed.

Purpose of the Study:

• To develop a novel, high-throughput fluorescence-based assay for measuring aminopropyl transferase (APT) activity.
• To facilitate the discovery of novel inhibitors for APT enzymes involved in polyamine biosynthesis.

Main Methods:

• Development of the 1,2-diacetyl benzene (DAB)-APT assay, a fluorescence-based method.
• Utilizing 1,2-diacetyl benzene (DAB) to react with polyamines (putrescine, spermidine, spermine) forming fluorescent conjugates.
• Validation of the DAB-APT assay using APT enzymes from Saccharomyces cerevisiae and Plasmodium falciparum.

Main Results:

• The DAB-APT assay provides a reliable measure of APT activity, with fluorescence intensity correlating to polyamine carbon chain length.
• The assay demonstrated suitability for high-throughput screening of large chemical libraries.
• Successful validation using enzymes from different organisms indicates broad applicability.

Conclusions:

• The novel DAB-APT assay is a significant advancement for APT enzyme research.
• This assay accelerates the identification of potential inhibitors for APT enzymes.
• It offers a valuable tool for drug discovery in oncology, neurobiology, and infectious diseases.