Specific C or N-terminal sequences of proteins can act as degradation signals for the ubiquitin-proteasome system, a pathway vital for the degradation of damaged or excess proteins. E3 ligases are responsible for the recruitment of a protein substrate, allowing ubiquitin-conjugating enzymes to mark the target protein for degradation by adding a ubiquitin chain. FEM1C is an E3 ligase that recognizes C-degrons with the sequence K/R-X-R or K/R-X-X-R, where X can be any amino acid. FEM1C has been observed to have roles in apoptosis, while downregulation of FEM1C increases proliferation and metastasis in colorectal cancer (CRC) cells, illustrating its importance in healthy physiological function. Further, there is a correlation of FEM1C downregulation in CRC tissues, and low FEM1C expression is correlated to poor patient outcomes. This preliminary data implies that FEM1C and its downstream degradation substrates may be targets of interest for the development of CRC therapeutics. However, there are currently no reported binders of FEM1C to further investigate its function and disease relevance. Therefore, we aimed to develop an assay to rapidly screen potential FEM1C binders. Here we report the development of a HTS-amendable fluorescent polarization assay for FEM1C. We designed a fluorescent probe that binds to FEM1C with a K<sub>i</sub> of 1.6 ± 0.4 µM utilizing the sequence RERQR bound to a fluorescent dye. The assay demonstrates an excellent performance for HTS with a Z’ factor of 0.80, signal to noise ratio of 5.81, and DMSO tolerance up to 10%. Preliminary screens of several in-house chemical libraries have yielded 2 potential lead compounds with K<sub>i</sub> values &lt; 20 µM. Our assay will enable the discovery of the first selective binders of FEM1C that will promote future studies to understand the function and cancer relevance of FEM1C.

Development of a high throughput screening-amenable assay for the E3 ligase FEM1C

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