A fluorescence-based assay for measuring aminopropyltransferase activity.

Abstract

Polyamines (PAs) are small polycationic alkylamines that are essential for numerous cellular processes and found in all living cells. The three principal polyamines, putrescine (PUT), spermidine (SPD), and spermine (SPM), have been shown to play crucial roles in cellular function and implicated in several diseases including infectious diseases, cancer and neurodegenerative disorders. As such, the enzymes involved in polyamine biosynthesis are promising targets for developing antimicrobial, antineoplastic and neuroprotective therapies. Aminopropyl transferases (APTs) are key enzymes in this pathway, catalyzing the formation of spermidine from putrescine and spermine from spermidine. While in most eukaryotes and prokaryotes, the spermidine synthase and spermine synthase activities are catalyzed by distinct enzymes, some organisms such as Plasmodium falciparum have a single enzyme, which catalyzes both reactions with varying efficiency. To date, efforts to inhibit APTs have focused primarily on substrate analogs, often with limited selectivity. A major challenge in discovering novel inhibitors has been the lack of an assay suitable for high-throughput chemical screening. We have recently developed DAB-APT, the first fluorescence-based assay for measuring APT activity, using 1,2-diacetyl benzene (DAB) which reacts with putrescine, spermidine, and spermine to form fluorescent conjugates, with fluorescence intensity correlating to carbon chain length. The DAB-APT assay has been validated using APT enzymes from Saccharomyces cerevisiae, and P. falciparum, and has been found to be suitable for high-throughput screening of large chemical libraries. This assay represents a significant advancement, offering a valuable tool for identifying potential inhibitors of APT enzymes and accelerating drug discovery efforts in cancer, neurobiology, and infectious diseases.