A multipurpose probe for identification and mechanistic analysis of prenylation inhibitors.

Abstract

Protein prenylation is a critical post-translational modification that controls many cancer-related signalling pathways and represents an important therapeutic target currently lacking effective pharmacological agents. Here, we establish the fluorescent prenyl diphosphate analogue MANT-O-GPP as a multipurpose probe for simultaneous analysis of ligand binding and catalysis in geranylgeranyltransferase I (GGTaseI) and farnesyltransferase (FTase). Using a tryptophan-to-MANT-O-GPP FRET assay, we found that MANT-O-GPP bound to GGTaseI and FTase with high affinity and reported occupancy of the isoprenoid donor site, as confirmed by displacement with the native substrates. In parallel, a FRET-based activity assay employing CFP-tagged protein substrates enabled direct monitoring of prenyl transfer and product formation. Notably, the known inhibitor L-778123 blocked catalysis without displacing MANT-O-GPP, demonstrating that the combined platform distinguishes prenyl-site competitors from inhibitors acting through the adjacent protein-substrate region. This fluorescence-based system provides a practical, mechanistically informative, and high-throughput-compatible platform for prenyltransferase inhibitor discovery.