Doug Auld, Ph.D. Novartis Institutes for BioMedical Research

The Boc3Arg moiety may provide a general strategy to construct inhibitors that induce targeted protein degradation.

Ligand-based stabilization of proteins is a well-known phenomenon in protein chemistry. Stabilization of proteins through ligand binding can extend a protein’s half-life in cells and result in increased protein thermostability and protection from proteolysis. However certain protein ligands can destabilize protein structure resulting in increased susceptibility to proteolysis and a shorter intracellular half-life.

Changes in protein stability upon ligand binding have been used to identify protein targets for small molecules through drug affinity responsive target stability (DARTS; Nishiya et al., Anal Biochem 2009;385:314–320). Inducing protein degradation has been achieved by linking a protein to a ligand that binds an E3 ubiquitin ligase (proteolysis targeting chimeric molecules [PROTAC]; Riana and Crews, JBC 2010;285:11057–11060) or attaching hydrophobic tags to a dehalogenase fusion protein (HaloTag™, Promega; Neklesa et al., Nat Chem Biol 2011;7:538–543). However, techniques using fusion proteins are unable to modulate the endogenous levels of a specific protein. In this article* it is shown that attaching tert-butyl carbamate-protected arginine (Boc3Arg) to a ligand results in degradation of proteins that bind the Boc3Arg-ligand.

The arginine tag targets proteins for degradation based on the N-end rule, which states that the N-terminal sequence of amino acids determines the half-life of the protein with those proteins starting with amino acids such as arginine having short (<1 h) half-lives, while those starting with hydrophobic amino acids show much longer half-lives. To initially test this tag, ethacrynic acid (EA), which could be modified with Boc3Arg, was used. The drug EA is used as a diuretic and works through covalent modification of gluthanione-S-transferases through modification of cysteine residues. The presence of a Michael acceptor in EA provides the site of covalent attachment, and the compound is of interest because it is an example of a drug that works through a covalent mechanism. Boc3Arg-EA induced degradation of GST-α1 in cell lysates and in whole cells. The authors also tagged trimethoprim (TMP), a specific inhibitor of Escherichia coli dihydrofoloate reductase (eDHFR). Whole cell assays were enabled using fusions of GST or eDHFR with GFP, which were co-expressed with a red fluorescent protein from the same mRNA (using a bicistronic construct). In this way, changes in the amount of GFP fusion protein present could be measured using the GFP/RFP ratio by FACS analysis (see Figure).

No change in eDHFR-GFP/RFP ratio was observed when TMP alone was used to treat cells, but this ratio was reduced when Boc3Arg-TMP was used (see Figure). Cycloheximide-treated cells were then monitored to measure the effect on protein synthesis on the degradation kinetics, which showed rapid degradation (levels reduced to ∼10% in 3 h) for either GST or eDHFR using Boc3Arg-tagged EA or TMP, respectively. Using specific inhibitors for the ubiquitin-proteasome pathway, the degradation was shown to be dependent on the proteasome but not ubiquitylation or ATP, suggesting that the 20S subunit of the proteasome may be involved. GFP is a very stable protein (half-life ∼24 h) and the fast degradation of the GFP fusions suggests that the Boc3Arg degron should work with a wide range of protein stabilities.

Drugs that induce degradation of their target proteins can have improved pharmacodynamics compared to drugs that only inhibit the protein function. In the case of protein degradation both functional and scaffolding effects of the target protein are removed. Investigating the structural requirements for linking the Boc3Arg degron to compounds could lead to chemical tools as well as new drug designs.

Figure. Degradation of eDHFR fusion proteins in whole cells. (A, B) Global protein stability assay. HeLa cells coexpress RFP and eDHFR-HA-GFP from a bicistronic construct. Red and green fluorescence was measured by flow cytometry. (A) TMP (80 mM). (B) TMP-Boc3Arg (80 mM). (C) Quantitation of four independent GPS experiments. Bar graphs show the average and the standard deviation. (D) Degradation of eDHFR-GFP was confirmed by anti-HA and anti-GFP immunoblotting.

*Abstract from Chemistry & Biology

The discovery of drugs that cause the degradation of their target proteins has been largely serendipitous. Here we report that the tert-butyl carbamate-protected arginine (Boc3Arg) moiety provides a general strategy for the design of degradation-inducing inhibitors. The covalent inactivators ethacrynic acid and thiobenzofurazan cause the specific degradation of glutathione-S-transferase when linked to Boc3Arg. Similarly, the degradation of dihydrofolate reductase is induced when cells are treated with the noncovalent inhibitor trimethoprim linked to Boc3Arg. Degradation is rapid and robust, with 30%–80% of these abundant target proteins consumed within 1.3–5 h. The proteasome is required for Boc3Arg-mediated degradation, but ATP is not necessary and the ubiquitin pathways do not appear to be involved. These results suggest that the Boc3Arg moiety may provide a general strategy to construct inhibitors that induce targeted protein degradation.

Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Chemistry & Biology titled “Inhibitor mediated protein degradation.” Authors of the paper are Long MJC, Gollapalli DR, and Hedstrom L.

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