Targeted protein degradation with small molecules for cancer immunotherapy.

Advances and opportunities in targeted protein degradation

Targeted protein degradation using small chimeric molecules, such as proteolysis-targeting chimeras (PROTACs) and specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs), is a promising technology in drug discovery. We recently developed a novel class of chimeric compounds that recruit the aryl hydrocarbon receptor (AhR) E3 ligase complex and induce the AhR-dependent degradation of target proteins. However, these chimeras contain a hydrophobic AhR E3 ligand, and thus, degrade target proteins even in cells that do not express AhR. In this study, we synthesized new compounds in which the AhR ligands were replaced with a hydrophobic adamantane moiety to investigate the mechanisms of AhR-independent degradation. Our results showed that the compounds, 2, 3, and 16 induced significant degradation of some target proteins in cells that do not express AhR, similar to the chimeras containing AhR ligands. However, in cells expressing AhR, 2, 3, and 16 did not induce the degradation of other target proteins, in contrast with their response to chimeras containing AhR ligands. Overall, it was suggested that target proteins susceptible to the hydrophobic tagging system are degraded by chimeras containing hydrophobic AhR ligands even without AhR.

Targeted protein degradation (TPD) is a valuable strategy for investigating protein functionality in cell biology and drug discovery. Among the various emerging TPD technologies, antibody-guided TPD offers key advantages over other protein degradation methods in terms of compatibility with different proteins of interest (POIs) and cell types. However, increasing the efficiency of cellular antibody internalisation and protein degradation remains challenges. Inspired by viral infection, which often efficiently activates protein degradation pathways in host cells, we developed a strategy called virus infection-mimicking targeted protein degradation (ViTPD) as a universal platform for degrading intracellular proteins. By mimicking three features of viral infection, we produced ViTPD nanoparticles by biomineralising antibodies enveloped by viral membranes or mixed with IFN-α. The biomineralised shell enhanced the cellular uptake of ViTPD nanoparticles via clathrin-mediated endocytosis. Similar to viral neutralising antibodies entering cells, the Fab region of the antibody released from ViTPD nanoparticles binds the POI, while the Fc region can recruit TRIM21, a key enzyme that continuously consumes during protein degradation. Interestingly, viral membrane components or IFN-α in the ViTPD led to increased TRIM21 expression, which enhanced the efficiency of proteolysis. ViTPD can effectively degrade several POIs, including GFP, FAK, COPZ1 and TREX1. Collectively, our results demonstrate that ViTPD provides a novel design strategy and an efficient nanoplatform for targeting intracellular protein degradation. STATEMENT OF SIGNIFICANCE: Antibody-guided targeted protein degradation (TPD) exhibits superior versatility compared to conventional degradation methods, demonstrating broad compatibility with diverse proteins of interest (POIs) across various cell types. Despite these advantages, significant challenges persist in optimizing cellular antibody internalization efficiency and degradation kinetics. In this study, we developed ViTPD, a biomimetic TPD platform that mimicking three viral infection features: (1) virus-like cellular internalization pathways, (2) virus-neutralizing antibody behavior, and (3) host-mediated protein degradation responses during viral infection. The development of ViTPD provides not only a robust platform for degrading diverse intracellular POIs but also establishes new design principles for next-generation protein degradation systems. This platform establishes new design principles for next-generation TPD systems while expanding therapeutic potential for precision medicine.

Targeted protein degradation as an antiviral approach

A heterobifunctional molecule recruits cereblon to an RNA scaffold and activates its PROTAC function

Industry Perspective on the Pharmacokinetic and Absorption, Distribution, Metabolism, and Excretion Characterization of Heterobifunctional Protein Degraders.

Recent advances in small-molecule mediated protein degradation have fueled interest in applying such technology to drug development. Whereas conventional therapeutics require sustained target occupancy to ensure maximal clinical effect, induced protein degradation operates via an event driven model in which the drug, through a catalytic mechanism, mediates recruitment of the target protein to the cellular quality control machinery. Targeted protein degradation offers the potential of a new class of drug molecules, advantageous in terms of potency, efficacy, duration of action and target selection in comparison to traditional occupancy based therapeutics. Recent findings in the development of small molecule heterobifunctional degraders (which recruit target proteins to an E3 ubiquitin ligase) have positioned this technology front and center for taking on this therapeutic challenge. Such bifunctional degraders have demonstrated potent, selective and reversible protein depletion of a number of cellular protein targets. There are however a number of factors that play a role in what makes an optimal degrader and what defines an ideal target for this technology. This chapter discusses recent progress in the development of heterobifunctional degraders, assesses the scope and limitations of the technology, and touches on other emerging concepts in the area of small-molecule mediated targeted protein degradation.

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, ‘fit-for-purpose’ PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable. Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 981.

The therapeutic approach of targeted protein degradation(TPD)is gaining momentum due to its potentially superior effects comparedwith protein inhibition. Recent advancements in the biotech and pharmaceuticalsectors have led to the development of compounds that are currentlyin human trials, with some showing promising clinical results. However,the use of computational tools in TPD is still limited, as it hasdistinct characteristics compared with traditional computational drugdesign methods. TPD involves creating a ternary structure (protein–degrader–ligase)responsible for the biological function, such as ubiquitination andsubsequent proteasomal degradation, which depends on the spatial orientationof the protein of interest (POI) relative to E2-loaded ubiquitin.Modeling this structure necessitates a unique blend of tools initiallydeveloped for small molecules (e.g., docking) and biologics (e.g.,protein–protein interaction modeling). Additionally, degradermolecules, particularly heterobifunctional degraders, are generallylarger than conventional small molecule drugs, leading to challengesin determining drug-like properties like solubility and permeability.Furthermore, the catalytic nature of TPD makes occupancy-based modelinginsufficient. TPD consists of multiple interconnected yet distinctsteps, such as POI binding, E3 ligase binding, ternary structure interactions,ubiquitination, and degradation, along with traditional small moleculeproperties. A comprehensive set of tools is needed to address thedynamic nature of the induced proximity ternary complex and its implicationsfor ubiquitination. In this Perspective, we discuss the current stateof computational tools for TPD. We start by describing the seriesof steps involved in the degradation process and the experimentalmethods used to characterize them. Then, we delve into a detailedanalysis of the computational tools employed in TPD. We also presentan integrative approach that has proven successful for degrader designand its impact on project decisions. Finally, we examine the futureprospects of computational methods in TPD and the areas with the greatestpotential for impact.

Targeted protein degradation (TPD) has emerged as a revolutionary paradigm in drug discovery and development, offering a promising avenue to tackle challenging therapeutic targets. Unlike traditional drug discovery approaches that focus on inhibiting protein function, TPD aims to eliminate proteins of interest (POIs) using modular chimeric structures. This is achieved through the utilization of proteolysis-targeting chimeras (PROTACs), which redirect POIs to E3 ubiquitin ligases, rendering them for degradation by the cellular ubiquitin-proteasome system (UPS). Additionally, other TPD technologies such as lysosome-targeting chimeras (LYTACs) and autophagy-based protein degraders facilitate the transportation of proteins to endo-lysosomal or autophagy-lysosomal pathways for degradation, respectively. Despite significant growth in preclinical TPD research, many chimeras fail to progress beyond this stage in the drug development. Various factors contribute to the limited success of TPD agents, including a significant hurdle of inadequate delivery to the target site. Integrating TPD into delivery platforms could surmount the challenges of in vivo applications of TPD strategies by reshaping their pharmacokinetics and pharmacodynamic profiles. These proteolysis-targeting drug delivery systems (ProDDSs) exhibit superior delivery performance, enhanced targetability, and reduced off-tissue side effects. In this review, we will survey the latest progress in TPD-inspired drug delivery systems, highlight the importance of introducing delivery ideas or technologies to the development of protein degraders, outline design principles of protein degrader-inspired delivery systems, discuss the current challenges, and provide an outlook on future opportunities in this field.

Targeted protein degradation (TPD) strategies offer a significant advantage over traditional small molecule inhibitors by selectively degrading disease-causing proteins. While small molecules can lead to recurrence and resistance due to compensatory pathway activation, TPD addresses this limitation by promoting protein degradation, thereby reducing the likelihood of recurrence and resistance over the long-term. Despite these benefits, bifunctional TPD molecules face challenges such as low solubility, poor bioavailability, and limited tumor specificity. In this study, we developed polymer-based nanoparticles that combine TPD strategies with nanotechnology through a hydrophobic tagging method. Hydrophobic polymer-tagged nanoparticles facilitate targeted protein degradation by incorporating hydrophobic polymers that mimic hydrophobic residues in misfolded proteins. This system combines degradation and delivery capabilities within a polymer-based platform, inducing protein degradation while improving solubility, stability, and tumor targeting. These nanoparticles consist of a block copolymer composed of an androgen receptor ligand (ARL)-conjugated hydrophobic polylactic acid (PLA) and a hydrophilic polyethylene glycol (PEG), connected by a GSH-cleavable disulfide bond. In aqueous solutions, this block copolymer (ARL-PLA-SS-PEG) forms micelles that degrade in reducible cellular environments. The micelles demonstrated significant in vitro degradation of the target androgen receptor (AR). Furthermore, they achieved substantial tumor accumulation and significantly inhibited tumor growth in a tumor-bearing mouse model. A mechanistic study revealed that the micelle-mediated TPD follows a dual pathway involving both proteasome and autophagosome. This approach has the potential to serve as a universal platform for protein degradation, eliminating the need to develop disease-specific TPD molecules.

Targeted protein degradation (TPD) represents a revolutionary therapeutic strategy in disease management, providing a stark contrast to traditional therapeutic approaches like small molecule inhibitors that primarily focus on inhibiting protein function. This advanced technology capitalizes on the cell’s intrinsic proteolytic systems, including the proteasome and lysosomal pathways, to selectively eliminate disease-causing proteins. TPD not only enhances the efficacy of treatments but also expands the scope of protein degradation applications. Despite its considerable potential, TPD faces challenges related to the properties of the drugs and their rational design. This review thoroughly explores the mechanisms and clinical advancements of TPD, from its initial conceptualization to practical implementation, with a particular focus on proteolysis-targeting chimeras and molecular glues. In addition, the review delves into emerging technologies and methodologies aimed at addressing these challenges and enhancing therapeutic efficacy. We also discuss the significant clinical trials and highlight the promising therapeutic outcomes associated with TPD drugs, illustrating their potential to transform the treatment landscape. Furthermore, the review considers the benefits of combining TPD with other therapies to enhance overall treatment effectiveness and overcome drug resistance. The future directions of TPD applications are also explored, presenting an optimistic perspective on further innovations. By offering a comprehensive overview of the current innovations and the challenges faced, this review assesses the transformative potential of TPD in revolutionizing drug development and disease management, setting the stage for a new era in medical therapy.

Structure driven compound optimization in targeted protein degradation

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, ‘fit-for-purpose’ PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable. Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 981.

Although information has rapidly developed concerning the intracellular localization of plant proteins, relatively few reports concern the intracellular location of endo- and exo-proteolytic activities. Relatively few proteases have been purified, characterized, and associated with a specific cellular location. With the exception of the processing proteases involved in transport of proteins across membranes, little progress has yet been made concerning determination of in vivo products of specific proteases. Information on the turnover of individual proteins and the assessment of rate-limiting steps in pathways as proteins are turned over is steadily appearing. Since chloroplasts are the major site of both protein synthesis and, during senescence, degradation, it was important to show unambiguously that chloroplasts can degrade their own constituents. Another important contribution was to obtain evidence that the chloroplasts contain proteases capable of degrading their constituents. This work has been more tenuous because of the low activities found and the possibility of contamination by vacuolar enzymes during the isolation of organelles. The possible targeting of cytoplasmic proteins for degradation by facilitating their transport into vacuoles is a field which hopefully will develop more rapidly in the future. Information on targeting of proteins for degradation via the ubiquitin (Ub) degradation pathway is developing rapidly. Future research must determine how much unity exists across the different eukaryotic systems. At present, it has important implications for protein turnover in plants, since apparently Ub is involved in the degradation of phytochrome. Little information has been developed regarding what triggers increased proteolysis with the onset of senescence, although it appears to involve protein synthesis. Thus far, the evidence indicates that the complement of proteases prior to senescence is sufficient to carry out the observed protein degradation. This field of study has great practical implications, e.g. maintaining photosynthesis during seed-fill in order to obtain greater crop yields. The current use of stay green' variants in the populations of several crop plants to produce increased yields shows the potential for future development. The near future should see exciting discoveries in these areas of research that will have far reaching effects on the construction of transgenic plants for future research accomplishments and agricultural use.