Selective degradation of hyperphosphorylated tau by proteolysis-targeting chimeras ameliorates cognitive function in Alzheimer's disease model mice.

Alzheimer's disease (AD) is one of the most common chronic neurodegenerative diseases. Hyperphosphorylated tau plays an indispensable role in neuronal dysfunction and synaptic damage in AD. Proteolysis-targeting chimeras (PROTACs) are a novel type of chimeric molecule that can degrade target proteins by inducing their polyubiquitination. This approach has shown promise for reducing tau protein levels, which is a potential therapeutic target for AD. Compared with traditional drug therapies, the use of PROTACs to reduce tau levels may offer a more specific and efficient strategy for treating AD, with fewer side effects. In the present study, we designed and synthesized a series of small-molecule PROTACs to knock down tau protein. Of these, compound C8 was able to lower both total and phosphorylated tau levels in HEK293 cells with stable expression of wild-type full-length human tau (termed HEK293-htau) and htau-overexpressed mice. Western blot findings indicated that C8 degraded tau protein through the ubiquitin-proteasome system in a time-dependent manner. In htau-overexpressed mice, the results of both the novel object recognition and Morris water maze tests revealed that C8 markedly improved cognitive function. Together, our findings suggest that the use of the small-molecule PROTAC C8 to degrade phosphorylated tau may be a promising therapeutic strategy for AD.

Unveiling a new strategy for PDIA1 inhibition: Integration of activity-based probes profiling and targeted degradation.

The overexpression of PDIA1 in cancer has spurred the quest for effective inhibitors. However, existing inhibitors often bind to only one active site, limiting their efficacy. In our study, we developed a PROTAC-mimetic probe dPA by combining PACMA31 (PA) analogs with cereblon-directed pomalidomide. Through protein profiling and analysis, we confirmed dPA's specific interaction with PDIA1's active site cysteines. We further synthesized PROTAC variants with a thiophene ring and various linkers to enhance degradation efficiency. Notably, H4, featuring a PEG linker, induced significant PDIA1 degradation and inhibited cancer cell proliferation similarly to PA. The biosafety profile of H4 is comparable to that of PA, highlighting its potential for further development in cancer therapy. Our findings highlight a novel strategy for PDIA1 inhibition via targeted degradation, offering promising prospects in cancer therapeutics. This approach may overcome limitations of conventional inhibitors, presenting new avenues for advancing anti-cancer interventions.

C646 degrades Exportin-1 to modulate p300 chromatin occupancy and function.

Molecular glues can induce proximity between a target protein and ubiquitin ligases to induce target degradation, but strategies for their discovery remain limited. We screened 3,200 bioactive small molecules and identified that C646 requires neddylation-dependent protein degradation to induce cytotoxicity. Although the histone acetyltransferase p300 is the canonical target of C646, we provide extensive evidence that C646 directly targets and degrades Exportin-1 (XPO1). Multiple cellular phenotypes induced by C646 were abrogated in cells expressing the known XPO1C528S drug-resistance allele. While XPO1 catalyzes nuclear-to-cytoplasmic transport of many cargo proteins, it also directly binds chromatin. We demonstrate that p300 and XPO1 co-occupy hundreds of chromatin loci. Degrading XPO1 using C646 or the known XPO1 modulator S109 diminishes the chromatin occupancy of both XPO1 and p300, enabling direct targeting of XPO1 to phenocopy p300 inhibition. This work highlights the utility of drug-resistant alleles and further validates XPO1 as a targetable regulator of chromatin state.

Protease-Responsive Toolkit for Conditional Targeted Protein Degradation.

BioPROTACs are heterobifunctional proteins designed for targeted protein degradation. While they offer a potential therapeutic avenue for modulating disease-related proteins, the current strategies are static in nature and lack the ability to modulate protein degradation dynamically. Here, we introduce a synthetic framework for dynamic fine-tuning of target protein levels using protease control switches. The idea is to utilize proteases as an interfacing layer between exogenous inputs and protein degradation by modulating the recruitment of target proteins to E3 ligase by separating the two binding domains on bioPROTACs. By decoupling the external inputs from the primary protease layer, new conditional degradation phenotypes can be readily adapted with minimal modifications to the design. We demonstrate the adaptability of this approach using two highly efficient "bioPROTAC" systems: AdPROM and IpaH9.8-based Ubiquibodies. Using the TEV protease as the transducer, we can interface small-molecule and optogenetic inputs for conditional targeted protein degradation. Our findings highlight the potential of bioPROTACs with protease-responsive linkers as a versatile tool for conditional targeted protein degradation.

ProteoCure : an European network to fine-tune the proteome.

Proteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms to ensure that they fulfil cellular needs in proper and timely fashion. Proteostasis is a key process allowing cells or organisms to maintain an appropriate but dynamic equilibrium of their proteome (the ensemble of all their proteins). It relies on multiple mechanisms that together control the level, fate and function of individual proteins, and ensure elimination of abnormal ones. The proteostasis network is essential for development and adaptation to environmental changes or challenges. Its dysfunctions can lead to accumulation of deleterious proteins or, conversely, to excessive degradation of beneficial ones, and are implicated in many diseases such as cancers, neurodegeneration, or developmental and aging disorders. Manipulating this network to control abundance of selected target proteins is therefore a strategy with enormous therapeutic or biotechnological potential. The ProteoCure COST Action gathers more than 350 researchers and their teams (31 countries represented) from the academic, clinical, and industrial sectors, who share the conviction that our understanding of proteostasis is mature enough to develop novel and highly specific therapies based on selective tunning of protein levels. Towards this objective, the Action organizes community-building activities to foster synergies among its participants and reinforce training of the next generation of European researchers. Its ambition is to function as a knowledge-based network and a creative exchange hub on normal and pathologic proteostasis, focusing on developing innovative tools modulating the level of specific protein(s).

PROteolysis-Targeting Chimeras (PROTACs) in leukemia: overview and future perspectives.

Leukemia is a heterogeneous group of life-threatening malignant disorders of the hematopoietic system. Immunotherapy, radiotherapy, stem cell transplantation, targeted therapy, and chemotherapy are among the approved leukemia treatments. Unfortunately, therapeutic resistance, side effects, relapses, and long-term sequelae occur in a significant proportion of patients and severely compromise the treatment efficacy. The development of novel approaches to improve outcomes is therefore an unmet need. Recently, novel leukemia drug discovery strategies, including targeted protein degradation, have shown potential to advance the field of personalized medicine for leukemia patients. Specifically, PROteolysis-TArgeting Chimeras (PROTACs) are revolutionary compounds that allow the selective degradation of a protein by the ubiquitin-proteasome system. Developed against a wide range of cancer targets, they show promising potential in overcoming many of the drawbacks associated with conventional therapies. Following the exponential growth of antileukemic PROTACs, this article reviews PROTAC-mediated degradation of leukemia-associated targets. Chemical structures, in vitro and in vivo activities, pharmacokinetics, pharmacodynamics, and clinical trials of PROTACs are critically discussed. Furthermore, advantages, challenges, and future perspectives of PROTACs in leukemia are covered, in order to understand the potential that these novel compounds may have as future drugs for leukemia treatment.

A small-molecule degron with a phenylpropionic acid scaffold.

Targeted protein degradation (TPD), employing proteolysis-targeting chimeras (PROTACs) composed of ligands for both a target protein and ubiquitin ligase (E3) to redirect the ubiquitin-proteasome system (UPS) to the target protein, has emerged as a promising strategy in drug discovery. However, despite the vast number of E3 ligases, the repertoire of E3 ligands utilized in PROTACs remains limited. Here, we report the discovery of a small-molecule degron with a phenylpropionic acid skeleton, derived from a known ligand of S-phase kinase-interacting protein 2 (Skp2), an E3 ligase. We used this degron to design PROTACs inducing proteasomal degradation of HaloTag-fused proteins, and identified key structural relationships. Surprisingly, our mechanistic studies excluded the involvement of Skp2, suggesting that this degron recruits other protein(s) within the UPS.

Expanding the horizons of targeted protein degradation: A non-small molecule perspective.

Targeted protein degradation (TPD) represented by proteolysis targeting chimeras (PROTACs) marks a significant stride in drug discovery. A plethora of innovative technologies inspired by PROTAC have not only revolutionized the landscape of TPD but have the potential to unlock functionalities beyond degradation. Non-small-molecule-based approaches play an irreplaceable role in this field. A wide variety of agents spanning a broad chemical spectrum, including peptides, nucleic acids, antibodies, and even vaccines, which not only prove instrumental in overcoming the constraints of conventional small molecule entities but also provided rapidly renewing paradigms. Herein we summarize the burgeoning non-small molecule technological platforms inspired by PROTACs, including three major trajectories, to provide insights for the design strategies based on novel paradigms.

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.

Towards the Targeted Protein Degradation of PRMT1.

Targeting the protein arginine methyltransferase 1 (PRMT1) has emerged as a promising therapeutic strategy in cancer treatment. The phase 1 clinical trial for GSK3368715, the first PRMT1 inhibitor to enter the clinic, was terminated early due to a lack of clinical efficacy, extensive treatment-emergent effects, and dose-limiting toxicities. The incidence of the latter two events may be associated with inhibition-driven pharmacology as a high and sustained concentration of inhibitor is required for therapeutic effect. The degradation of PRMT1 using a proteolysis targeting chimera (PROTAC) may be superior to inhibition as proceeds via event-driven pharmacology where a PROTAC acts catalytically at a low dose. PROTACs containing the same pharmacophore as GSK3368715, combined with a motif that recruits the VHL or CRBN E3-ligase, were synthesised. Suitable cell permeability and target engagement were shown for selected candidates by the detection of downstream effects of PRMT1 inhibition and by a NanoBRET assay for E3-ligase binding, however the candidates did not induce PRMT1 degradation. This paper is the first reported investigation of PRMT1 for targeted protein degradation and provides hypotheses and insights to assist the design of PROTACs for PRMT1 and other novel target proteins.

PROTACs in Ovarian Cancer: Current Advancements and Future Perspectives.

Ovarian cancer is the deadliest gynecologic malignancy. The majority of patients diagnosed with advanced ovarian cancer will relapse, at which point additional therapies can be administered but, for the most part, these are not curative. As such, a need exists for the development of novel therapeutic options for ovarian cancer patients. Research in the field of targeted protein degradation (TPD) through the use of proteolysis-targeting chimeras (PROTACs) has significantly increased in recent years. The ability of PROTACs to target proteins of interest (POI) for degradation, overcoming limitations such as the incomplete inhibition of POI function and the development of resistance seen with other inhibitors, is of particular interest in cancer research, including ovarian cancer research. This review provides a synopsis of PROTACs tested in ovarian cancer models and highlights PROTACs characterized in other types of cancers with potential high utility in ovarian cancer. Finally, we discuss methods that will help to enable the selective delivery of PROTACs to ovarian cancer and improve the pharmacodynamic properties of these agents.

Discovery of a DCAF11-dependent cyanoacrylamide-containing covalent degrader of BET-proteins.

Targeted protein degradation is mediated by small molecules that induce or stabilize protein-protein interactions between targets and the ubiquitin-proteasome machinery. Currently, there remains a need to expand the repertoire of viable E3 ligases available for hijacking. Notably, covalent chemistry has been employed to engage a handful of E3 ligases, including DCAF11. Here, we disclose a covalent PROTAC that enables DCAF11-dependent degradation, featuring a cyanoacrylamide warhead. Our findings underscore DCAF11 as an interesting candidate with a capacity to accommodate diverse electrophilic chemistries compatible with targeted protein degradation.

Therapeutic antibody engineering for efficient targeted degradation of membrane proteins in lysosomes.

Targeted degradation of pathological proteins is a promising approach to enhance the effectiveness of therapeutic monoclonal antibodies (mAbs) in cancer therapy. In this study, we demonstrate that this objective can be efficiently achieved by the grafting of mannose 6-phosphate analogues called AMFAs2 onto the therapeutic antibodies trastuzumab and cetuximab, both directed against membrane antigens. The grafting of AMFAs confers to these antibodies the novel property of being internalized via the mannose 6-phosphate receptor (M6PR) pathway. AMFA conjugation to these mAbs significantly increases their cellular uptake and leads to enhanced degradation of the target antigens in cancer cells. This results in a drastic inhibition of cancer cell proliferation compared to unconjugated mAbs, as demonstrated in various cancer cell lines, and an increased therapeutic efficacy in mouse and zebrafish xenografted models. These findings highlight the potential of this technology to improve therapeutic outcomes in cancer treatment.

An overview of PROTACs targeting MDM2 as a novel approach for cancer therapy.

MDM2 genes amplification or altered expression is commonly observed in various cancers bearing wild-type TP53. Directly targeting the p53-binding pocket of MDM2 to activate the p53 pathway represents a promising therapeutic approach. Despite the development of numerous potent MDM2 inhibitors that have advanced into clinical trials, their utility is frequently hampered by drug resistance and hematologic toxicity such as neutropenia and thrombocytopenia. The emergence of PROTAC technology has revolutionized drug discovery and development, with applications in both preclinical and clinical research. Harnessing the power of PROTAC molecules to achieve MDM2 targeted degradation and p53 reactivation holds significant promise for cancer therapy. In this review, we summarize representative MDM2 PROTAC degraders and provide insights for researchers investigating MDM2 proteins and the p53 pathway.

Exosome-mediated PROTAC delivery for treatment of RNA viral infections and zoonosis.

The increase in diseases caused by RNA viruses, such as influenza, severe acute respiratory syndrome-coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS), and Ebola, presents a growing global health challenge as well as the threat of zoonosis. Traditional antiviral treatments are often undermined by fast-mutating viruses, drug resistance, and newly emerging pathogens. Here, we explore proteolysis-targeting chimeras (PROTACs), a novel protein degradation machinery that has the potential to reshape the way in which RNA viral infections can be managed. PROTACs excel at specifically degrading pathogenic proteins, offering a targeted and efficient antiviral strategy. We also investigate the potential of exosome-based diagnostic technologies, which harness cell-derived nanovesicles for non-invasive sampling and early viral infection detection. Addressing the challenge of PROTAC delivery, we introduce a groundbreaking strategy utilizing exosomes to deliver PROTACs with improved precision and as a targeted delivery vehicle. Integrating these innovative strategies provides a novel approach to combat RNA zoonotic viral diseases, paving the way for a new era in antiviral therapy.

Synthetic lethality: targeting the SMARCA2 bromodomain for degradation in SMARCA4-deficient tumors - a review of patent literature from 2019-June 2023.

INTRODUCTION: SMARCA2 and SMARCA4 are subunits of the SWI/SNF complex which is a chromatin remodeling complex and a key epigenetic regulator that facilitates gene expression. Tumors with loss of function mutations in SMARCA4 rely on SMARCA2 for cell survival and this synthetic lethality is a potential therapeutic strategy to treat cancer. AREAS COVERED: The current review focuses on patent applications that claim proteolysis-targeting chimeras (PROTAC) degraders that bind the bromodomain site of SMARCA2 and are published between January 2019-June 2023. A total of 29 applications from 9 different applicants were evaluated. EXPERT OPINION: SMARCA2/4 bromodomain inhibitors do not lead to desired effects on cancer proliferation; however, companies have converted bromodomain binders into PROTACs to degrade the protein, with a preference for SMARCA2 over SMARCA4. Selective degradation of SMARCA2 is most likely required to be efficacious in the SMARCA4-deficient setting, while allowing for sufficient safety margin in normal tissues. With several patent applications disclosed recently, interest in targeting SMARCA2 should continue, especially with a selective SMARCA2 PROTAC now in the clinic from Prelude Therapeutics. The outcome of the clinical trials will influence the evolution of selective SMARCA2 PROTACs development.

Accelerating PROTACs Discovery Through a Direct-to-Biology Platform Enabled by Modular Photoclick Chemistry.

Proteolysis targeting chimeras (PROTACs) have emerged as a promising strategy for drug discovery and exploring protein functions, offering a revolutionary therapeutic modality. Currently, the predominant approach to PROTACs discovery mainly relies on an empirical design-synthesis-evaluation process involving numerous cycles of labor-intensive synthesis-purification and bioassay data collection. Therefore, the development of innovative methods to expedite PROTAC synthesis and exploration of chemical space remains highly desired. Here, a direct-to-biology strategy is reported to streamline the synthesis of PROTAC libraries on plates, enabling the seamless transfer of reaction products to cell-based bioassays without the need for additional purification. By integrating amide coupling and light-induced primary amines and o-nitrobenzyl alcohols cyclization (PANAC) photoclick chemistry into a plate-based synthetic process, this strategy produces PROTAC libraries with high efficiency and structural diversity. Moreover, by employing this platform for PROTACs screening, we smoothly found potent PROTACs effectively inhibit triple-negative breast cancer (TNBC) cell growth and induce rapid, selective targeted degradation of cyclin-dependent kinase 9 (CDK9). The study introduces a versatile platform for assembling PROTACs on plates, followed by direct biological evaluation. This approach provides a promising opportunity for high-throughput synthesis of PROTAC libraries, thereby enhancing the efficiency of exploring chemical space and accelerating the discovery of PROTACs.

Targeting Moonlighting Enzymes in Cancer.

Moonlighting enzymes are multifunctional proteins that perform multiple functions beyond their primary role as catalytic enzymes. Extensive research and clinical practice have demonstrated their pivotal roles in the development and progression of cancer, making them promising targets for drug development. This article delves into multiple notable moonlighting enzymes, including GSK-3, GAPDH, and ENO1, and with a particular emphasis on an enigmatic phosphatase, PTP4A3. We scrutinize their distinct roles in cancer and the mechanisms that dictate their ability to switch roles. Lastly, we discuss the potential of an innovative approach to develop drugs targeting these moonlighting enzymes: target protein degradation. This strategy holds promise for effectively tackling moonlighting enzymes in the context of cancer therapy.

Targeting the undruggables-the power of protein degraders.

Undruggable targets typically refer to a class of therapeutic targets that are difficult to target through conventional methods or have not yet been targeted, but are of great clinical significance. According to statistics, over 80% of disease-related pathogenic proteins cannot be targeted by current conventional treatment methods. In recent years, with the advancement of basic research and new technologies, the development of various new technologies and mechanisms has brought new perspectives to overcome challenging drug targets. Among them, targeted protein degradation technology is a breakthrough drug development strategy for challenging drug targets. This technology can specifically identify target proteins and directly degrade pathogenic target proteins by utilizing the inherent protein degradation pathways within cells. This new form of drug development includes various types such as proteolysis targeting chimera (PROTAC), molecular glue, lysosome-targeting Chimaera (LYTAC), autophagosome-tethering compound (ATTEC), autophagy-targeting chimera (AUTAC), autophagy-targeting chimera (AUTOTAC), degrader-antibody conjugate (DAC). This article systematically summarizes the application of targeted protein degradation technology in the development of degraders for challenging drug targets. Finally, the article looks forward to the future development direction and application prospects of targeted protein degradation technology.

Ternary Complex-Templated Dynamic Combinatorial Chemistry for the Selection and Identification of Homo-PROTACs.

Dynamic combinatorial chemistry (DCC) leverages a reversible reaction to generate compound libraries from constituting building blocks under thermodynamic control. The position of this equilibrium can be biased by addition of a target macromolecule towards enrichment of bound ligands. While DCC has been applied to select ligands for a single target protein, its application to identifying chimeric molecules inducing proximity between two proteins is unprecedented. In this proof-of-concept study, we develop a DCC approach to select bifunctional proteolysis targeting chimeras (PROTACs) based on their ability to stabilize the ternary complex. We focus on VHL-targeting Homo-PROTACs as model system, and show that the formation of a VHL2 : Homo-PROTAC ternary complex reversibly assembled using thiol-disulfide exchange chemistry leads to amplification of potent VHL Homo-PROTACs with degradation activities which correlated well with their biophysical ability to dimerize VHL. Ternary complex templated dynamic combinatorial libraries allowed identification of novel Homo-PROTAC degraders. We anticipate future applications of ternary-complex directed DCC to early PROTAC screenings and expansion to other proximity-inducing modalities beyond PROTACs.