Protein Degradation Methods

Pharmacology typically works either through addition or subtraction: by supplying a molecule the body lacks (for example thyroid hormone or growth factors) or by inhibiting the activity of bad molecular players through direct inhibition, genetic knockout, etc.

Targeted protein degradation (TPD), a relatively new idea belonging to the latter category, eliminates disease-causing proteins by marking them for destruction by the cell's native protein degradation machinery.

There are many ways to skin this particular cat.

Proteolysis-targeting chimeras (PROTACs^®: a trademark of TPD development firm Arvinas) consist of two chemically linked ligands. One binds to protein targeted for destruction, the other attracts E3 ubiquitin ligase, an enzyme that marks proteins for degradation by the proteosome.

Other approaches work similarly. For example, molecular glues also induce ubiquitination, leading to degradation and clearance of the troublesome protein. And lysosome-targeting chimeras (LYTACs) recruit lysosomes instead of the proteasome—an advantage when targeting extracellular or membrane-bound target proteins normally inaccessible to PROTACs.

Other variations on this theme include degradation tags (dTAGs), autophagy-targeting chimeras (AUTACs), and emerging approaches like trim-away, SNIPERs (Specific and Non-genetic IAP-dependent Protein ERasers), and chaperone-mediated autophagy.

By operating through subtraction vs. addition, TPD-based strategies greatly expand the universe of “druggable” proteins beyond those accessible to competitive or irreversible inhibition.

Since its effectiveness is non-stoichiometric but based on catalysis, TPD treatments may be administered in small doses to minimize dose-related side effects. As a related side benefit, tight binding to targets is strictly required. Other advantages include wide biodistribution, oral or parenteral delivery routes, and either systemic or tissue-targeted biodistribution.

Off-target effects are still possible, however, and the downside is these proceed catalytically as well.

Practice meets theory

The catalytic aspects of “therapy by subtraction” make TPD-based strategies unique among modern pharmaceuticals. Since our metabolism takes care of ubiquitylated proteins on its own one might imagine that the only discovery/development issue is attracting the target, but that is an over-simplification.

One end of a PROTAC, for example, binds to the drug target and the other binds to an E3 ligase, but simply reducing the targets’ physical proximity is insufficient.

“Bringing the two together in the correct orientation enables ubiquitin tagging and subsequent degradation of the target protein,” says Angela M. Cacace, Ph.D., Chief Scientific Officer at Arvinas. “However, this is not as simple as with single-target binding-site drugs.”

The PROTAC must be properly oriented so its complex with the target is accessible to the E3 ligase. Once tagged, the ubiquitylated protein becomes a substrate for the proteasome and is subsequently degraded while the PROTAC molecule is recycled.

“It’s also important to balance the affinities for both the target protein and the E3 ligase to optimize degradation without off-target effects,” Cacace says. “And since PROTAC degraders are larger small molecules that typically do not obey Lipinski’s rules for pharmacokinetics and bioavailability, addressing their size and complexity while optimizing their oral bioavailability and blood-brain barrier penetration can be challenging.”

Arvinas has developed proprietary PROTAC optimization rules intrinsic to the molecule under study, which help optimize pharmacokinetic parameters like hydrogen bond donor count and polar surface area, among others, to drive oral bioavailability and blood-brain barrier penetration.

“While tight binding isn’t strictly necessary, balancing target and ligase affinity is critical to ensure effective ternary complex recruitment and iterative cycling of the PROTAC,” Cacace says. “Other factors in play include binding affinity of the PROTAC in the ternary complex with target protein and ubiquitin ligase, the cooperativity of the ternary complex, and the kinetics of the degradation process.”

Cooperativity refers to the ratio of a PROTAC’s ternary complex to its binary complex. A cooperativity value greater than 1 means ternary complex binding is higher, while a value less than 1 suggests unfavorable protein-protein interactions.

PROTAC discovery

PROTAC discovery is similar to that for small molecule drugs in that it involves an iterative, optimization cycle. Researchers experiment with various linkers, ligands, and structural modifications to find the right combination that effectively induces protein degradation.

To accelerate discovery, Arvinas uses its PROTAC^® Discovery Engine, which incorporates understanding the PROTAC-target zone of ubiquitination, Arvinas’ Next Generation Linker Evolution (ANGLE) privileged linkers, computational modeling, and proteomics.

Once a PROTAC enters the pipeline, key challenges include determining formulation solubility, formulated stability, scale-up of GxP supplies, and ensuring a robust path for clinical development activities. “We engage relatively early with pharmaceutics and process chemistry because of the larger size of PROTAC degraders compared to typical small molecules,” Cacace explains.

Potential liabilities can be assessed early in optimization and are evaluated via proteomics and by knowledge of a particular PROTAC’s interactions with known E3 ligase substrates or to the target protein itself. PROTAC molecules follow the same regulatory toxicology studies required for typical small molecules.

“Optimizing PROTAC degraders can be confirmed in target and known off-target specific assays and by employing our researchers' unbiased proteomic studies. This can enable teams to incorporate additional assays for unanticipated off-target effects into the optimization cycles to inform medicinal chemistry and minimize these potential liabilities,” Cacace tells Biocompare.

Quantifying progress

Loss of target protein is a key measurement in PDT agent development but since the analysis involves a decrease in protein concentration (and therefore lower signals), methods must be both sensitive and specific to ensure the right protein is being measured.

“Many groups measure protein degradation in peripherally available samples such as blood or peripheral blood mononuclear cells, which enables mapping of in vivo degradation kinetics,” says Nick Dupuis, Senior Director, Scientific Business Development at CellCarta. In some cases, a sensitive immunoassay may be the best approach for measuring this. However, samples often contain several closely related isoforms of the target protein. In those cases liquid chromatography-mass spectrometry is more routinely deployed to get an accurate measure of degradation.”

How much degradation of a target protein is required for clinical effect, and is it possible to over- or under-do protein degradation?

“That depends mainly on the target’s mode of action,” Dupuis says. “Some targets require 70% to 80% degradation to achieve a clinical endpoint while others require complete degradation. The target protein and biology dictate the need for the depth of degradation.”