Technologies Enabling Protein Degradation Studies

Targeted protein degradation has been touted as a new paradigm for therapeutic intervention that utilizes the cell’s natural degradation machinery to eliminate targets implicated in disease. Biocompare recently hosted a Bench Tips webinar where senior postdoctoral fellows actively involved in studying protein degradation discussed how they were using various technologies to structurally and mechanistically understand what cellular pathways are involved and how they can be targeted. They shared their experiences and best practices, and here are some key learnings from the discussion.

Protein-protein interactions initiate many cellular processes that play a key role in disease. Compounds that orchestrate some of these interactions are referred to as molecular glues. Molecular glues act as protein degraders by binding to the protein of interest and then recruiting a ubiquitin E3 ligase to trigger ubiquitination and subsequent degradation of the protein. Molecular glues are small, monovalent, drug-like molecules that trigger conformational changes in the protein upon binding, making them more vulnerable to degradation. Large, multivalent degraders like proteolysis-targeting chimeras (PROTACs) can also trigger degradation. These molecules include a linker attached to two different warheads. One warhead binds the protein of interest, while the other binds the E3 ligase, which triggers the degradation. This chemically induced degradation using molecular glues, PROTACs, and other modalities has given rise to a new paradigm in drug discovery called targeted protein degradation (TPD).

Matching the right tools with the strategy pursued

“The shift in traditional drug discovery from inhibition to binding has led to the targeting of many proteins that were previously considered to be undruggable,” explained Luis Nieto-Barrado, a doctoral student in the Targeted Protein Degradation & Drug Discovery lab led by Cristina Mayor-Ruiz at the Institute for Research in Biomedicine. He is working on unraveling the nuances of E3 ligase regulation and using innovative tools to develop new monovalent degraders. In a paper co-authored by Nieto-Barrado, the group cited work done by various groups to outline the serendipitous and intentional strategies that led to the development of molecular glues.

“We clustered the development strategies into four groups depending on whether the discovery was driven by a target or a specific E3 ligase, or if it was target or E3 ligase agnostic,” said Nieto-Barrado. A target and E3 ligase driven strategy pursued in Stuart Schreiber’s lab at Harvard involved using combinatorial chemistry to synthesize more than a million compounds. Enrichment and sequencing studies were then done to identify hit compounds that could form a stable ternary complex with the protein and the ligase. “A crystal structure of one of the hit compounds was a proof-of-concept that this approach could be used,” added Nieto-Barrado.

Phenotypic screening with cell-based assay readouts has been used to find molecular glues in a target and E3 ligase agnostic manner when trying to identify disease vulnerabilities or looking for regulators that govern sensitivity to degraders. Work done in Georg Winter’s lab at CeMM in Vienna used expression proteomics and western blotting techniques to find and validate the target protein, and genome-wide CRISPR screening to identify the E3 ligase involved in the degradation. While they performed interaction studies using time resolved fluorescence energy transfer (TR-FRET) to identify the two proteins glued together, other types of technologies such as pull-down, direct interaction, drug-induced proximity assays can be used as well. “These studies showed that molecular glues are more frequent than we thought, and they can be found in a rational way,” Nieto-Barrado concluded.

Tools to design and validate degraders

Only recently, it was shown that thalidomide and its derivatives lenalidomide and pomalidomide (referred to as iMiDs) bind to cereblon, an E3 ligase substrate receptor, and act as molecular glue degraders to neosubstrates that are typically not recognized by cereblon. Justin Cruite, Ph.D., a structural biologist and project lead in the Center for Protein Degradation at Dana-Farber Cancer Institute, uses different techniques to study the structure of lenalidomide bound to cereblon to look for covalent cereblon binders, which in turn will help find new degradable targets. He started by designing and synthesizing a number of iMiD derivatives with different covalent warheads.

“The first thing we wanted to do was to confirm that these covalent probes bind to the imide-binding pocket in cereblon in live cells,” explained Cruite. So, they developed a cereblon engagement assay based on the NanoLuc^® luciferase bioluminescence resonance energy transfer (NanoBRET) system. Using intact protein mass spectrometric analysis, they showed that the covalent probes specifically labeled cereblon. Protein digestion followed by peptide mapping was done to confirm that the labeling sites were indeed on cereblon, and proteomic assays were performed to show that these covalent derivatives induced degradation of new targets.

“While most of the compounds that we designed did not show any degradation, the compounds that did show degradation of a new target were then further analyzed,” Cruite said. “We showed the specific interaction of the ligand binding site on the cereblon with the target of interest, suggesting that it is the covalent modification of cereblon that is responsible for the interaction between the two proteins.”

Tools for understanding degradation pathways

Unlike mammalian cells that use ubiquitin, bacteria utilize several prokaryotic protein complexes for degradation. Francesca Ester Morreale, Ph.D., a postdoctoral researcher in Tim Clausen’s group at the Research Institute of Molecular Pathology in Vienna, focuses her work on understanding and modulating targeted protein degradation in bacteria. Her ultimate goal is to enable degradation of essential bacterial proteins leading to the development of better antibiotics.

Morreale works on the bacterial ClpC protease-induced degradation pathways using bacterial proteolysis-targeting chimeras (BacPROTACs) as degraders. BacPROTACs use a linker to bind the ClpC domain on one end and the target protein on the other. “We started our proof-of-concept studies by degrading simple proteins such as streptavidin and bromodomain 1, which have known ligands and binding domains,” noted Morreale. “For our affinity binding studies we always started out using isolated domains, and not the entire protein.”

Size exclusion chromatography was used to determine whether binding to BacPROTAC enabled a ternary complex formation. Once the binding and formation of the ternary complex was confirmed, they developed an assay to determine whether the protein was indeed degraded. “In eukaryotic cells, we can use western blotting to confirm protein degradation; however, in bacteria, finding the right antibody for the protein and the cell permeability can be a challenge,” Morreale said. Hence, they set up an in vitro degradation assay using recombinantly purified protein and ran an electrophoresis gel to show that BacPROTAC does indeed degrade the protein.

Using Alphafold models, Morreale was able to further analyze which structural aspects of the bacterial protein played an important role in affecting the extent of degradation. Using cryogenic electron microscopy, they were able to look at the protein in different activation states. The results showed that there were multiple sites on the ClpC protein that could be targeted for BacPROTAC binding, that some proteins were more degradable than others, and that specific protein characteristics determined the degradation efficiency. The next few years will likely showcase many more technologies and assays that can be used to uncover new pathways and modalities for targeted degradation.