Nuclear magnetic resonance鈥恇ased fragment screen yields novel E3 ligases for use in PROTAC therapy
By Shelby A. Harris
PROteolysis TArgeting Chimeras, known as PROTACs, are a recently developed group of therapeutics that utilize the ubiquitin-proteasome system to target and degrade disease associated proteins by recruiting E3 ligase. Of the many different E3 ligases found in human cells, over 600 are known to be expressed and only a few are used with PROTACs.
Pre-clinical studies have shown that PROTACs can develop resistance when mutations occur in the E3 ligase being used, only to be rescued when a different E3 ligase-based PROTACs is used. The resistance compounds with the fact that the commonly used E3 ligases, cereblon (CRBN) and von Hippel Lindau (VHL) can have increased off-target effects and restricted chemical design, respectively. Therefore, expanding the availability of E3 ligases for PROTAC therapy is necessary for the development of future PROTACs not only to increase diversity of the therapy but also minimize off-target effect and allow for more flexibility in the design.
This led 91黑料网 postdoc Dr. Jade Katinas, of the , and colleagues to the protein fem-1 homolog B (FEM1B). FEM1B recognizes substrate in the Cullin-RING E3 ligase, which is found in most cells and is crucial in maintaining the balance of proteins with roles in redox sensing and management. To achieve this, it is thought that FEM1B has multiple recognition sites for binding to substrates with interesting structural aspects in the binding pocket that induces strong interactions.
Previous library screening with FEM1B had shown that it has good potential as a degrader, thus the researchers aimed to run an NMR-based fragment screen to try and identify small molecules that bind to FEM1B. 
Due to protein instability the team had to develop multiple mutants of the protein, however, once they overcame this, 1H鈥13C HMQC spectra using selective 13C鈥恗ethyl labeling of Leu, Ile, Val, and Met sites of FEM1B was performed and several hits were identified.
These hits were then characterized by X-ray co-crystal structures. These FEM1B co-crystals structures gave valuable insight into the binding potential of these fragments. One molecule (VU0416476) was found to mimic previously known ligands by binding covalently to a cysteine residue, while the other identified hits bound non-covalently, the first reported of its kind.
This research is exciting, as it offers a starting template for discovering new forms of PROTACs, especially for proteins such as FEM1B.
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