Driving E3 Ligase Substrate Specificity for Targeted Protein Degradation: Lessons from Nature and the Laboratory.

Methods to direct the degradation of protein targets with proximity-inducing molecules that coopt the cellular degradation machinery are advancing in leaps and bounds, and diverse modalities are emerging. The most used and well-studied approach is to hijack E3 ligases of the ubiquitin-proteasome system. E3 ligases use specific molecular recognition to determine which proteins in the cell are ubiquitinated and degraded. This review focuses on the structural determinants of E3 ligase recruitment of natural substrates and neo-substrates obtained through monovalent molecular glues and bivalent proteolysis-targeting chimeras. We use structures to illustrate the different types of substrate recognition and assess the basis for neo-protein-protein interactions in ternary complex structures. The emerging structural and mechanistic complexity is reflective of the diverse physiological roles of protein ubiquitination. This molecular insight is also guiding the application of structure-based design approaches to the development of new and existing degraders as chemical tools and therapeutics.

Mapping the Degradable Kinome Provides a Resource for Expedited Degrader Development.

Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.

Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis.

Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed "multiplexed proteome dynamics profiling" (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems.

Cullin-RING ligases employ geometrically optimized catalytic partners for substrate targeting.

Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.

A synthetic KLHL20 ligand to validate CUL3KLHL20 as a potent E3 ligase for targeted protein degradation.

Targeted protein degradation (TPD) has risen as a promising therapeutic modality. Leveraging the catalytic nature of the ubiquitin-proteasome enzymatic machinery, TPD exhibits higher potency to eliminate disease-causing target proteins such as oncogenic transcription factors that may otherwise be difficult to abrogate by conventional inhibitors. However, there are challenges that remain. Currently, nearly all degraders engage CUL4CRBN or CUL2VHL as the E3 ligase for target ubiquitination. While their immediate efficacies are evident, the narrowed E3 ligase options make TPD vulnerable to potential drug resistance. In addition, E3 ligases show differential tissue expression and have intrinsic limitations in accessing varying types of disease-relevant targets. As the success of TPD is closely associated with the ability of E3 ligases to efficiently polyubiquitinate the target of interest, the long-term outlook of TPD drug development will depend on whether E3 ligases such as CUL4CRBN and CUL2VHL are accessible to the targets of interest. To overcome these potential caveats, a broad collection of actionable E3 ligases is required. Here, we designed a macrocyclic degrader engaging CUL3KLHL20 for targeting BET proteins and validated CUL3KLHL20 as an E3 ligase system suitable for TPD. This work thus contributes to the expansion of usable E3 ligases for potential drug development.

Definition of a small core transcriptional circuit regulated by AML1-ETO.

Transcription factors regulate gene networks controlling normal hematopoiesis and are frequently deregulated in acute myeloid leukemia (AML). Critical to our understanding of the mechanism of cellular transformation by oncogenic transcription factors is the ability to define their direct gene targets. However, gene network cascades can change within minutes to hours, making it difficult to distinguish direct from secondary or compensatory transcriptional changes by traditional methodologies. To overcome this limitation, we devised cell models in which the AML1-ETO protein could be quickly degraded upon addition of a small molecule. The rapid kinetics of AML1-ETO removal, when combined with analysis of transcriptional output by nascent transcript analysis and genome-wide AML1-ETO binding by CUT&RUN, enabled the identification of direct gene targets that constitute a core AML1-ETO regulatory network. Moreover, derepression of this gene network was associated with RUNX1 DNA binding and triggered a transcription cascade ultimately resulting in myeloid differentiation.

Acute perturbation strategies in interrogating RNA polymerase II elongation factor function in gene expression.

The regulation of gene expression catalyzed by RNA polymerase II (Pol II) requires a host of accessory factors to ensure cell growth, differentiation, and survival under environmental stress. Here, using the auxin-inducible degradation (AID) system to study transcriptional activities of the bromodomain and extraterminal domain (BET) and super elongation complex (SEC) families, we found that the CDK9-containing BRD4 complex is required for the release of Pol II from promoter-proximal pausing for most genes, while the CDK9-containing SEC is required for activated transcription in the heat shock response. By using both the proteolysis targeting chimera (PROTAC) dBET6 and the AID system, we found that dBET6 treatment results in two major effects: increased pausing due to BRD4 loss, and reduced enhancer activity attributable to BRD2 loss. In the heat shock response, while auxin-mediated depletion of the AFF4 subunit of the SEC has a more severe defect than AFF1 depletion, simultaneous depletion of AFF1 and AFF4 leads to a stronger attenuation of the heat shock response, similar to treatment with the SEC inhibitor KL-1, suggesting a possible redundancy among SEC family members. This study highlights the usefulness of orthogonal acute depletion/inhibition strategies to identify distinct and redundant biological functions among Pol II elongation factor paralogs.

Using Chemical Epigenetics to Target Cancer.

Transcription is epigenetically regulated by the orchestrated function of chromatin-binding proteins that tightly control the expression of master transcription factors, effectors, and supportive housekeeping genes required for establishing and propagating the normal and malignant cell state. Rapid advances in chemical biology and functional genomics have facilitated exploration of targeting epigenetic proteins, yielding effective strategies to target transcription while reducing toxicities to untransformed cells. Here, we review recent developments in conventional active site and allosteric inhibitors, peptidomimetics, and novel proteolysis-targeted chimera (PROTAC) technology that have deepened our understanding of transcriptional processes and led to promising preclinical compounds for therapeutic translation, particularly in cancer.

Chromatin Hyperacetylation Impacts Chromosome Folding by Forming a Nuclear Subcompartment.

Delineating how chromosomes fold at length scales beyond one megabase remains obscure relative to smaller-scale folding into TADs, loops, and nucleosomes. We find that rather than simply unfolding chromatin, histone hyperacetylation results in interactions between distant genomic loci separated by tens to hundreds of megabases, even in the absence of transcription. These hyperacetylated "megadomains" are formed by the BRD4-NUT fusion oncoprotein, interact both within and between chromosomes, and form a specific nuclear subcompartment that has elevated gene activity with respect to other subcompartments. Pharmacological degradation of BRD4-NUT results in collapse of megadomains and attenuation of the interactions between them. In contrast, these interactions persist and contacts between newly acetylated regions are formed after inhibiting RNA polymerase II initiation. Our structure-function approach thus reveals that broad chromatin domains of identical biochemical composition, independent of transcription, form nuclear subcompartments, and also indicates the potential of altering chromosome structure for treating human disease.

Development of an orally bioavailable mSWI/SNF ATPase degrader and acquired mechanisms of resistance in prostate cancer.

Mammalian switch/sucrose nonfermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, an orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 (ATP binding cassette subfamily B member 1) overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.

Applications of protein ubiquitylation and deubiquitylation in drug discovery.

The ubiquitin (Ub)-proteasome system (UPS) is the major machinery mediating specific protein turnover in eukaryotic cells. By ubiquitylating unwanted, damaged, or harmful proteins and driving their degradation, UPS is involved in many important cellular processes. Several new UPS-based technologies, including molecular glue degraders and PROTACs (proteolysis-targeting chimeras) to promote protein degradation, and DUBTACs (deubiquitinase-targeting chimeras) to increase protein stability, have been developed. By specifically inducing the interactions between different Ub ligases and targeted proteins that are not otherwise related, molecular glue degraders and PROTACs degrade targeted proteins via the UPS; in contrast, by inducing the proximity of targeted proteins to deubiquitinases, DUBTACs are created to clear degradable poly-Ub chains to stabilize targeted proteins. In this review, we summarize the recent research progress in molecular glue degraders, PROTACs, and DUBTACs and their applications. We discuss immunomodulatory drugs, sulfonamides, cyclin-dependent kinase-targeting molecular glue degraders, and new development of PROTACs. We also introduce the principle of DUBTAC and its applications. Finally, we propose a few future directions of these three technologies related to targeted protein homeostasis.

3D based generative PROTAC linker design with reinforcement learning.

Proteolysis targeting chimera (PROTAC), has emerged as an effective modality to selectively degrade disease-related proteins by harnessing the ubiquitin-proteasome system. Due to PROTACs' hetero-bifunctional characteristics, in which a linker joins a warhead binding to a protein of interest (POI), conferring specificity and a E3-ligand binding to an E3 ubiquitin ligase, this could trigger the ubiquitination and transportation of POI to the proteasome, followed by degradation. The rational PROTAC linker design is challenging due to its relatively large molecular weight and the complexity of maintaining the binding mode of warhead and E3-ligand in the binding pockets of counterpart. Conventional linker generation method can only generate linkers in either 1D SMILES or 2D graph, without taking into account the information of ternary structures. Here we propose a novel 3D linker generative model PROTAC-INVENT which can not only generate SMILES of PROTAC but also its 3D putative binding conformation coupled with the target protein and the E3 ligase. The model is trained jointly with the RL approach to bias the generation of PROTAC structures toward pre-defined 2D and 3D based properties. Examples were provided to demonstrate the utility of the model for generating reasonable 3D conformation of PROTACs. On the other hand, our results show that the associated workflow for 3D PROTAC conformation generation can also be used as an efficient docking protocol for PROTACs.

The emerging role of targeted protein degradation to treat and study cancer.

The evolution of cancer treatment has provided increasingly targeted strategies both in the upfront and relapsed disease settings. Small-molecule inhibitors and immunotherapy have risen to prominence with chimeric antigen receptor T-cells, checkpoint inhibitors, kinase inhibitors, and monoclonal antibody therapies being deployed across a range of solid organ and haematological malignancies. However, novel approaches are required to target transcription factors and oncogenic fusion proteins that are central to cancer biology and have generally eluded successful drug development. Thalidomide analogues causing protein degradation have been a cornerstone of treatment in multiple myeloma, but a lack of in-depth mechanistic understanding initially limited progress in the field. When the protein cereblon (CRBN) was found to mediate thalidomide analogues' action and CRBN's neo-targets were identified, existing and novel drug development accelerated, with applications outside multiple myeloma, including non-Hodgkin's lymphoma, myelodysplastic syndrome, and acute leukaemias. Critically, transcription factors were the first canonical targets described. In addition to broadening the application of protein-degrading drugs, resistance mechanisms are being overcome and targeted protein degradation is widening the scope of druggable proteins against which existing approaches have been ineffective. Examples of targeted protein degraders include molecular glues and proteolysis targeting chimeras (PROTACs): heterobifunctional molecules that bind to proteins of interest and cause proximity-induced ubiquitination and proteasomal degradation via a linked E3 ligase. Twenty years since their inception, PROTACs have begun progressing through clinical trials, with early success in targeting the oestrogen receptor and androgen receptor in breast and prostate cancer respectively. This review explores important developments in targeted protein degradation to both treat and study cancer. It also considers the potential advantages and challenges in the translational aspects of developing new treatments. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Ultrasound-Activated PROTAC Prodrugs Overcome Immunosuppression to Actuate Efficient Deep-Tissue Sono-Immunotherapy in Orthotopic Pancreatic Tumor Mouse Models.

The degradation of oncoproteins mediated by proteolysis-targeting chimera (PROTAC) has emerged as a potent strategy in cancer therapy. However, the clinical application of PROTACs is hampered by challenges such as poor water solubility and off-target adverse effects. Herein, we present an ultrasound (US)-activatable PROTAC prodrug termed NPCe6+PRO for actuating efficient sono-immunotherapy in a spatiotemporally controllable manner. Specifically, US irradiation, which exhibits deep-tissue penetration capability, results in Ce6-mediated generation of ROS, facilitating sonodynamic therapy (SDT) and inducing immunogenic cell death (ICD). Simultaneously, the generated ROS cleaves the thioketal (TK) linker through a ROS-responsive mechanism, realizing the on-demand activation of the PROTAC prodrug in deep tissues. This prodrug activation results in the degradation of the target protein BRD4, while simultaneously reversing the upregulation of PD-L1 expression associated with the SDT process. In the orthotopic mouse model of pancreatic tumors, NPCe6+PRO effectively suppressed tumor growth in conjunction with US stimulation.

Liposomal STAT3-Degrading PROTAC Prodrugs Promote Anti-Hepatocellular Carcinoma Immunity via Chemically Reprogramming Cancer Stem Cells.

Cancer stem cells (CSCs) with hyperactivated signal transducer and activator of transcription 3 (STAT3) are a major driver of hepatocellular carcinoma (HCC). Herein, we report a nanointegrative proteolysis-targeting chimera (PROTAC)-based STAT3 degradation strategy that enables efficient chemical reprogramming of HCC-associated CSCs, which potently inhibits CSC growth while evoking anti-HCC immune responses. The PROTAC prodrug was synthesized by conjugating the STAT3 binding domain (inS3) with a thioketal-caged E3 ligase ligand (VL-TK) via an oligo(ethylene glycol) linker (OEG) with tuned length and flexibility and encapsulating it in cRGD-modified cationic liposomes for CSC-targeted delivery while facilitating their lysosomal escape. The PROTAC prodrugs were activated by the upregulated ROS levels in CSCs and efficiently degraded STAT3 for chemical reprogramming, which would not only impair their stemness features but also remodel the immunosuppressive TME into an immunosupportive state to boost anti-HCC immunity. This strategy provides an approach for improving HCC treatment in clinics.

Progress in RAS-targeted therapeutic strategies: From small molecule inhibitors to proteolysis targeting chimeras.

As a widely considerable target in chemical biology and pharmacological research, rat sarcoma (RAS) gene mutations play a critical driving factor in several fatal cancers. Despite the great progress of RAS subtype-specific inhibitors, rapid acquired drug resistance could limit their further clinical applications. Proteolysis targeting chimera (PROTAC) has emerged as a powerful tool to handle "undruggable" targets and exhibited significant therapeutic benefit for the combat of drug resistance. Owing to unique molecular mechanism and binding kinetics, PROTAC is expected to become a feasible strategy to break the bottleneck of classical RAS inhibitors. This review aims to discuss the current advances of RAS inhibitors and especially focus on PROTAC strategy targeting RAS mutations and their downstream effectors for relevant cancer treatment.

The new direction of drug development: Degradation of undruggable targets through targeting chimera technology.

Imbalances in protein and noncoding RNA levels in vivo lead to the occurrence of many diseases. In addition to the use of small molecule inhibitors and agonists to restore these imbalances, recently emerged targeted degradation technologies provide a new direction for disease treatment. Targeted degradation technology directly degrades target proteins or RNA by utilizing the inherent degradation pathways, thereby eliminating the functions of pathogenic proteins (or RNA) to treat diseases. Compared with traditional therapies, targeted degradation technology which avoids the principle of traditional inhibitor occupation drive, has higher efficiency and selectivity, and widely expands the range of drug targets. It is one of the most promising and hottest areas for future drug development. Herein, we systematically introduced the in vivo degradation systems applied to degrader design: ubiquitin-proteasome system, lysosomal degradation system, and RNA degradation system. We summarized the development progress, structural characteristics, and limitations of novel chimeric design technologies based on different degradation systems. In addition, due to the lack of clear ligand-binding pockets, about 80% of disease-associated proteins cannot be effectively intervened with through traditional therapies. We deeply elucidated how to use targeted degradation technology to discover and design molecules for representative undruggable targets including transcription factors, small GTPases, and phosphatases. Overall, this review provides a comprehensive and systematic overview of targeted degradation technology-related research advances and a new guidance for the chimeric design of undruggable targets.

Development of the nonreceptor tyrosine kinase FER-targeting PROTACs as a potential strategy for antagonizing ovarian cancer cell motility and invasiveness.

Aberrant overexpression of nonreceptor tyrosine kinase FER (Fps/Fes Related) has been reported in various ovarian carcinoma-derived tumor cells and is a poor prognosis factor for patient survival. It plays an essential role in tumor cell migration and invasion, acting concurrently in both kinase-dependent and -independent manners, which is not easily suppressed by conventional enzymatic inhibitors. Nevertheless, the PROteolysis-TArgeting Chimera (PROTAC) technology offers superior efficacy over traditional activity-based inhibitors by simultaneously targeting enzymatic and scaffold functions. Hence in this study, we report the development of two PROTAC compounds that promote robust FER degradation in a cereblon-dependent manner. Both PROTAC degraders outperform a Food and Drug Administration-approved drug, brigatinib, in ovarian cancer cell motility suppression. Importantly, these PROTAC compounds also degrade multiple oncogenic FER fusion proteins identified in human tumor samples. These results lay an experimental foundation to apply the PROTAC strategy to antagonize cell motility and invasiveness in ovarian and other types of cancers with aberrant expression of FER kinase and highlight PROTACs as a superior strategy for targeting proteins with multiple tumor-promoting functions.

Single amino acid-based PROTACs trigger degradation of the oncogenic kinase BCR-ABL in chronic myeloid leukemia (CML).

Proteolysis-targeting chimera (PROTAC) that specifically targets harmful proteins for destruction by hijacking the ubiquitin-proteasome system is emerging as a potent anticancer strategy. How to efficiently modulate the target degradation remains a challenging issue. In this study, we employ a single amino acid-based PROTAC, which uses the shortest degradation signal sequence as the ligand of the N-end rule E3 ubiquitin ligases to degrade the fusion protein BCR (breakpoint cluster region)-ABL (Abelson proto-oncogene), an oncogenic kinase that drives the progression of chronic myeloid leukemia. We find that the reduction level of BCR-ABL can be easily adjusted by substituting different amino acids. Furthermore, a single PEG linker is found to achieve the best proteolytic effect. Our efforts have resulted in effective degradation of BCR-ABL protein by the N-end rule pathway and efficient growth inhibition of K562 cells expressing BCR-ABL in vitro and blunted tumor growth in a K562 xenograft tumor model in vivo. The PROTAC presented has unique advantages including lower effective concentration, smaller molecular size, and modular degradation rate. Demonstrating the efficacy of the N-end rule-based PROTACs in vitro and in vivo, our study further expands the limited degradation pathways currently available for PROTACs in vivo and is easily adapted for broader applications in targeted protein degradation.

Targeted protein posttranslational modifications by chemically induced proximity for cancer therapy.

Post-translational modifications (PTMs) regulate all aspects of protein function. Therefore, upstream regulators of PTMs, such as kinases, acetyltransferases, or methyltransferases, are potential therapeutic targets for human diseases, including cancer. To date, multiple inhibitors and/or agonists of these PTM upstream regulators are in clinical use, while others are still in development. However, these upstream regulators control not only the PTMs of disease-related target proteins but also other disease-irrelevant substrate proteins. Thus, nontargeted perturbing activities may introduce unwanted off-target toxicity issues that limit the use of these drugs in successful clinical applications. Therefore, alternative drugs that solely regulate a specific PTM of the disease-relevant protein target may provide a more precise effect in treating disease with relatively low side effects. To this end, chemically induced proximity has recently emerged as a powerful research tool, and several chemical inducers of proximity (CIPs) have been used to target and regulate protein ubiquitination, phosphorylation, acetylation, and glycosylation. These CIPs have a high potential to be translated into clinical drugs and several examples such as PROTACs and MGDs are now in clinical trials. Hence, more CIPs need to be developed to cover all types of PTMs, such as methylation and palmitoylation, thus providing a full spectrum of tools to regulate protein PTM in basic research and also in clinical application for effective cancer treatment.