Advances in Protein Degradation

Proteolysis targeting chimeras (PROTACs) are heterobifunctional compounds that recruit the E3 ubiquitin ligase machinery to specific proteins of interest, resulting in their ubiquitination and subsequent proteasomal degradation.^1,2 This new approach has the potential to deliver transformative new therapies across a wide variety of diseases, in part due to the ability to target previously undrugged or poorly drugged proteins. As a result, this emerging modality is becoming an attractive pharmacologic strategy to overcome limitations of classical enzyme inhibitor-based/target occupancy approaches.

Over the past 5 years, the field of targeted protein degradation has grown exponentially, and numerous milestone achievements have been realized. A first wave of PROTAC drugs is now undergoing clinical development in cancer, and a pipeline of clinical candidates is emerging that promises to address both additional oncology indications and applications in inflammatory diseases. ³ In addition to PROTAC approaches, several modalities for targeted protein degradation are now well established and include monofunctional degraders or molecular glues, as exemplified by the IMiD class of drugs, and compounds that bind and disrupt protein structure to induce degradation, such as the selective estrogen receptor degraders (SERDs).^4,5 Recently, hybrid molecules that encode both a PROTAC function and molecular glue activity have been identified, thereby demonstrating that these distinct mechanisms can be merged to create molecules with unique and specific polypharmacology. ⁶ Further, while most of the efforts to date have focused on a select set of E3 ligases, including both cereblon and von Hippel–Lindau (VHL), the field is seeking to extend the chemistries that allow the hijacking of novel E3 ligases and expand the scope of targeted protein degradation. Finally, recent reports have demonstrated proof of concept for a range of applications that complement these “traditional” approaches. These approaches direct target protein degradation through additional, orthogonal cellular degradation processes, such as lysosomal trafficking and autophagy,^7,8 thereby significantly expanding the potential and diversity of the central targeted protein degradation concept.

This special issue of SLAS Discovery, “Targeted Protein Degradation,” includes contributions that range from perspectives to original scientific reports, with content that spans all aspects of this exciting field. As such, this issue will appeal to experts with its important new advances and detailed reviews of recent progress in specific themes unique to targeted protein degradation, but it also includes technical guides on assay development and degrader characterization that can serve as manuals for best practice to those new to the field.

Among these contributions, Nowak and Jones ⁹ provide an overarching strategic perspective on the targeted protein degradation approach through the application of a “four pillars” framework. This framework provides context in optimizing PROTAC development by informing pharmacokinetic–pharmacodynamic (PK/PD) understanding and helping elucidate structure–activity relationships. A number of assays are discussed to monitor PROTAC biodistribution and exposure at the site of action (pillar 1), engagement of its targets and ternary complex formation (pillar 2), functional pharmacological effects (pillar 3), and the resulting modulation of a relevant phenotype (pillar 4).

For those interested in the state of progress in E3 ligase ligand identification, Ishida and Ciulli ¹⁰ have assembled a comprehensive review of this area. This review covers the initial ligand identification for both cereblon and VHL that now serves as the basis for many of the first-wave PROTAC drugs, but also provides the definitive resource for ligand identification efforts across the E3 ligome to date. The authors also provide an overview of the various methods for E3 ligase binder discovery, ranging from DNA-encoded libraries to display techniques to covalent warhead approaches.

The perspective by Kastl et al. ¹¹ includes an overview of the various approaches to targeted protein degradation, including PROTACS, autophagy directed chimeras (AUTACs), and lysosomal trafficking chimeras (LYTACs); it also provides an informed discussion on the screening approaches to identify monofunctional degraders. This discussion is based on the authors’ experience upon completion of a small-molecule compound collection high-throughput screen using a cellular degradation assay. The authors describe the methods for hit triage and characterization to avoid the potential pitfalls that may be encountered in the discovery of these novel molecules.

The perspective by Simard et al. ¹² provides an overview of the range of cellular assays that can be used for quantification and a characterization of target protein degraders. A range of assay options for measuring cellular target levels are now commercially available, but they differ in key assay characteristics, including sensitivity, expense, and testing format. Simard et al. describe these techniques in detail, providing the relative pros and cons for each in turn. This report provides detailed guidance on best practice for characterizing degraders within and across drug discovery programs.

Zhang et al. ¹³ provide an overview of the use of mass spectrometry-based global proteomic approaches in characterizing targeted protein degraders. Mass spectrometry measurements of global protein levels provide the most robust assessment of degrader selectivity and represent the current gold standard for degrader specificity. The authors outline the approaches used to characterize protein degraders, which include both direct protein-level quantification and downstream cellular pathways. In addition, the authors outline the value of proteomic approaches in assessing E3 ligase levels, target levels, and intrinsic degradation and resynthesis rates across cell lines and tissues to inform on potential safety liabilities and PK/PD relationships.

Confirmation of target engagement, ternary complex formation, and subsequent ubiquitination events are essential steps for effective degradation. The original research by Chernobrovkin et al. ¹⁴ describes the application of the cellular thermal shift assay (CETSA) to simultaneously evaluate target engagement by observing changes in protein thermal stability as well as efficacy by simultaneous assessment of protein abundances. This assay enables correlation of target engagement between the target protein and E3 ligases with functional readout, such as degrader-induced protein degradation.

The original research by Vieux et al. ¹⁵ describes how in vitro effectiveness of the bifunctional degradation activating compounds (BiDACs) to form a ternary complex between the E3 ligase and the target protein and facilitate E3 ligase-catalyzed ubiquitination of the target protein is not determined by a single parameter such as half-maximal inhibition (IC50) or binding constant (K_i). Rather, these authors found that both thermodynamic and kinetic parameters govern the overall degradation process. This work utilized a cell-free ubiquitination assay to measure the kinetics of BiDAC-induced ubiquitination and compared these data to the active complex affinities. These insights provide a framework for optimizing BiDAC properties to improve not only the affinities of ternary complex formation but also the rate of ubiquitination.

Riching et al. ¹⁶ describe a novel finding for the field on selective PROTAC degraders of the cyclin-dependent kinase (CDK) family. This original scientific report highlights the unique feature of degraders in that they encode a kinetic signature, which can reveal time-based selectivity across a family of related protein targets. Using time-based measurements of protein levels, the authors report distinct differences in depth and potency across the CDK family as a function of time. Importantly, the authors further found that CDK2 degradation was also dependent on growth phase, suggesting that targeted protein degradation is highly context dependent and may be observed in specific cellular subpopulations.

Finally, Lee et al. ¹⁷ report on the development of a novel SNAP-epitope tag/near-infrared imaging assay that enables quantitative analysis of G-protein-coupled receptor (GPCR) degradation in human cells and investigated the utility of incorporating SNAP-tag technology into traditional cycloheximide (CHX)-chase assays to permit calculation of GPCR protein half-lives when expressed in cultured human cells. The assay could be readily adapted to a high-throughput format and is expected to quantify degradation for any membrane protein of interest.

In sum, this special issue provides a useful overview of this exciting and rapidly developing field. While we anticipate that the applications of targeted protein degradation will continue to expand and diversify, we believe that the articles within this issue will serve both as a definitive marker for the state of the current field and as valued resources for those pursuing targeted protein degradation drug discovery programs.