Design and synthesis of a fluorescent probe to develop a fluorescence polarization assay for the E3 ligase FEM1C

Abstract

A proteolysis targeting chimera (PROTAC) is a bivalent molecule consisting of an E3 ligase ligand and a protein of interest ligand, which promotes the degradation of specific proteins by recruiting the ubiquitin-proteasome system. Although VHL and CRBN ligands have been extensively used in PROTAC development, the availability of small molecule E3 ligase ligands remains limited. Therefore, identifying novel E3 ligase ligands would expand the repertoire for PROTAC development. FEM1C, an E3 ligase that recognizes proteins with an R/K-X-R or R/K-X-X-R motif at the C-terminus, is a promising candidate for this purpose. In this study, we present the design and synthesis of a fluorescent probe ES148, exhibiting a Ki value of 1.6 ± 0.1 µM for FEM1C. Utilizing this fluorescent probe, we have established a robust fluorescence polarization (FP) based competition assay to characterize FEM1C ligands, with a Z’ factor of 0.80 and a S/N ratio > 20 in a high-throughput format. Furthermore, we have validated binding affinities of FEM1C ligands using isothermal titration calorimetry, consistently corroborating the results from our FP assay. Thus, we anticipate that our FP competition assay will expedite the discovery of FEM1C ligands, offering new tools for PROTAC development.