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INTRODUCTION: ClpP is a highly conserved serine protease that plays a crucial role in maintaining protein homeostasis in both bacterial cells and human mitochondria. Several studies have demonstrated the potential of ClpP as a drug target, with ClpP modulators, including both inhibitors and activators, showing promise in treating a range of conditions such as drug-resistant bacteria, malignant cancers, and fatty liver disease. AREA COVERED: This review provides an overview of patents related to ClpP modulators filed over the last five years, detailing their claims and therapeutic applications. The sources of patent information included databases of the European Patent Office, the China Patent Office and the U.S.A. patent Office, while relevant research articles were accessed through PubMed. EXPERT OPINION: The number of patents concerning ClpP modulators is on the rise, reflecting advancements in related research. By summarizing and outlining relevant patents, we aim to stimulate further interest among researchers, ultimately leading to the development of effective drugs based on ClpP modulators. The broad spectrum of diseases associated with ClpP dysfunction underscores the potential for ClpP modulators to address a wide range of therapeutic needs.

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Accumulating evidence reveals the oncogenic role of methyltransferase-like 3 (METTL3) in a variety of cancers, either dependent or independent of its m6A methyl transferase activity. We have explored PROTACs targeting METTL3 and identified KH12 as a potent METTL3 degrader. Treatment of KH12 on MOLM-13 cells causes degradation of METTL3 with a DC50 value of 220 nM in a dose-, time- and ubiquitin-dependent fashion. In addition, KH12 is capable of reversing differentiation and possesses anti-proliferative effects surpassing the small molecule inhibitors on MOLM-13 cells. Notably, we first present that METTL3 degrader significantly suppresses the growth of various gastric cancer (GC) cells, where the m6A-independent activity of METTL3 plays a crucial role in tumorigenesis. The anti-GC effects of KH12 were further confirmed in patient-derived organoids (PDOs). This study offers therapeutic potentials of targeted degradation of METTL3 against GC implicated with non-catalytic function of METTL3 as well as against AML.

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Protein aggregation causes a wide range of neurodegenerative diseases. Targeting and removing aggregates, but not the functional protein, is a considerable therapeutic challenge. Here, we describe a therapeutic strategy called "RING-Bait," which employs an aggregating protein sequence combined with an E3 ubiquitin ligase. RING-Bait is recruited into aggregates, whereupon clustering dimerizes the RING domain and activates its E3 function, resulting in the degradation of the aggregate complex. We exemplify this concept by demonstrating the specific degradation of tau aggregates while sparing soluble tau. Unlike immunotherapy, RING-Bait is effective against both seeded and cell-autonomous aggregation. RING-Bait removed tau aggregates seeded from Alzheimer's disease (AD) and progressive supranuclear palsy (PSP) brain extracts and was also effective in primary neurons. We used a brain-penetrant adeno-associated virus (AAV) to treat P301S tau transgenic mice, reducing tau pathology and improving motor function. A RING-Bait strategy could be applied to other neurodegenerative proteinopathies by replacing the Bait sequence to match the target aggregate.

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Targeted protein degradation (TPD) has revolutionized drug discovery by selectively eliminating specific proteins within and outside the cellular context. Over the past two decades, TPD has expanded its focus beyond well-established targets, exploring diverse proteins beyond cancer-related ones. This evolution extends the potential of TPD to various diseases. Notably, TPD can target proteins at demanding locations, such as the extracellular matrix (ECM) and cellular membranes, presenting both opportunities and challenges for future research. In this review, we comprehensively examine the exciting opportunities in the burgeoning field of TPD, highlighting different targets, their cellular environment, and innovative strategies for modern drug discovery.

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The emerging field of induced proximity therapeutics, which involves designing molecules to bring together an effector and target protein-typically to induce target degradation-is rapidly advancing. However, its progress is constrained by the lack of scalable and unbiased tools to explore effector-target protein interactions. We combine pooled endogenous gene tagging using a ligand-binding domain with generic small-molecule-based recruitment to screen for induction of protein proximity. We apply this methodology to identify effectors for degradation in two orthogonal screens: using fluorescence to monitor target levels and a cellular growth that depends on the degradation of an essential protein. Our screens revealed new effector proteins for degradation, including previously established examples, and converged on members of the C-terminal-to-LisH (CTLH) complex. We introduce a platform for pooled induction of endogenous protein-protein interactions to expand our toolset of effector proteins for protein degradation and other forms of induced proximity.

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Small molecule-responsive tags for targeted protein degradation are valuable tools for fundamental research and drug target validation. Here, we show that genetically incorporated unnatural amino acids bearing a strained alkene or alkyne functionality can act as a minimalist tag for targeted protein degradation. Specifically, we observed the degradation of strained alkene- or alkyne-containing kinases and E2 ubiquitin-conjugating enzymes upon treatment with hydrophobic tetrazine conjugates. The extent of the induced protein degradation depends on the identity of the target protein, unnatural amino acid, and tetrazine conjugate, as well as the site of the unnatural amino acid in the target protein. Mechanistic studies revealed proteins undergo proteasomal degradation after tetrazine tethering, and the identity of tetrazine conjugates influences the dependence of ubiquitination on protein degradation. This work provides an alternative approach for targeted protein degradation and mechanistic insight, facilitating the future development of more effective targeted protein degradation strategies.

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Targeted protein degradation technology has gained substantial momentum over the past two decades as a revolutionary strategy for eliminating pathogenic proteins that are otherwise refractory to treatment. Among the various approaches developed to harness the body's innate protein homeostasis mechanisms for this purpose, lysosome targeting chimeras (LYTACs) that exploit the lysosomal degradation pathway by coupling the target proteins with lysosome-trafficking receptors represent the latest innovation. These chimeras are uniquely tailored to degrade proteins that are membrane-bound and extracellular, encompassing approximately 40% of all proteome. Several novel LYTAC formulas have been developed recently, providing valuable insights for the design and development of therapeutic degraders. This review delineates the recent progresses of LYTAC technology, its practical applications, and the factors that dictate target degradation efficiency. The potential and emerging trends of this technology are discussed as well. LYTAC technology offers a promising avenue for targeted protein degradation, potentially revolutionizing the therapeutic landscape for numerous diseases.

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Rationale: Since oncogene expression products often exhibit upregulation or abnormally activated activity, developing a technique to regulate abnormal protein levels represent a viable approach for treating tumors and protein abnormality-related diseases. Methods: We first screened out eMIATAC components with high targeted degradation efficiency and explored the mechanism by which eMIATAC induced target protein degradation, and verified the degradation efficiency of the target protein by protein imprinting and flow cytometry. Next, we recombined eMIATAC with some controllable elements to verify the regulatable degradation performance of the target protein. Subsequently, we constructed eMIATAC that can express targeted degradation of AKT1 and verified its effect on GBM cell development in vitro and in vivo. Finally, we concatenated eMIATAC with CAR sequences to construct CAR-T cells with low BATF protein levels and verified the changes in their anti-tumor efficacy. Results: we developed a system based on the endosome-microautophagy-lysosome pathway for degrading endogenous proteins: endosome-MicroAutophagy TArgeting Chimera (eMIATAC), dependent on Vps4A instead of lysosomal-associated membrane protein 2A (LAMP2A) to bind to the chaperone Hsc70 and the protein of interest (POI). The complex was then transported to the lysosome by late endosomes, where degradation occurred similarly to microautophagy. The eMIATACs demonstrated accuracy, efficiency, reversibility, and controllability in degrading the target protein EGFP. Moreover, eMIATAC exhibited excellent performance in knocking down POI when targeting endogenous proteins in vivo and in vitro. Conclusions: The eMIATACs could not only directly knock down abnormal proteins for glioma treatment but also enhance the therapeutic effect of CAR-T cell therapy for tumors by knocking down T cell exhaustion-related proteins. The newly developed eMIATAC system holds promise as a novel tool for protein knockdown strategies. By enabling direct control over endogenous protein levels, eMIATAC has the potential to revolutionize treatment for cancer and genetic diseases.

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Degrons are minimal protein features that are sufficient to target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, all of the identified degrons only function within the cytosol. Using Saccharomyces cerevisiae, we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate proteasomal degradation through the ERAD system. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation in budding yeast and mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and builds toward therapeutic approaches that exploit the highly-conserved ERAD system.

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Targeted therapies, such as small molecule kinase inhibitors, have made significant progress in the treatment of hematologic malignancies by directly modulating protein activity. However, issues such as drug toxicity, drug resistance due to target mutations, and the absence of key active sites limit the therapeutic efficacy of these drugs. Targeted protein degradation (TPD) presents an emergent and rapidly evolving therapeutic approach that selectively targets proteins of interest (POI) based on endogenous degradation processes. With an event-driven pharmacology of action, TPD achieves efficacy with catalytic amounts, avoiding drug-related toxicity. Furthermore, TPD has the unique mode of degrading the entire POI, such that resistance derived from mutations in the targeted protein has less impact on its degradation function. Proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs) are the most maturely developed TPD techniques. In this review, we focus on both preclinical experiments and clinical trials to provide a comprehensive summary of the safety and clinical effectiveness of PROTACs and MGDs in hematologic malignancies over the past two decades. In addition, we also delineate the challenges and opportunities associated with these burgeoning degradation techniques. TPD, as an approach to the precise degradation of specific proteins, provides an important impetus for its future application in the treatment of patients with hematologic malignancies.

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Breast cancer (BC) accounts for 30% of cancer cases among women cancer patient globally, indicating the urgent need for the development of selective therapies targeting BCs. Recently, proteolysis-targeting chimera (PROTAC) has been emerged as promising strategy to target breast cancer. PROTAC is a chimeric molecule consisting with target protein ligand, E3 ligase ligand, and conjugating linkers, enabling it to facilitate the degradation of desired target proteins via recruiting E3 ligase in close proximity. Due to the catalytic behavior and direct degradation of BC-causing proteins, PROTAC could achieve high drug efficacy with low doses, resulting in a great attention for its potential as therapeutics. This review provides cases of the current developed PROTACs targeting BCs depending on the type of BCs, limitation, and perspective of PROTAC in targeting BCs.

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Targeted protein degradation (TPD) allows cells to maintain a functional proteome and to rapidly adapt to changing conditions. Methods that repurpose TPD for the deactivation of specific proteins have demonstrated significant potential in therapeutic and research applications. Most of these methods are based on proteolysis targeting chimaeras (PROTACs) which link the protein target to an E3 ubiquitin ligase, resulting in the ubiquitin-based degradation of the target protein. In this study, we introduce a method for ubiquitin-independent TPD based on nanobody-conjugated plant ubiquitin regulatory X domain-containing (PUX) adaptor proteins. We show that the PUX-based NAnobody Degraders (P-NADs) can unfold a target protein through the Arabidopsis and human orthologues of the CDC48 unfoldase without the need for ubiquitination or initiating motifs. We demonstrate that P-NAD plasmids can be transfected into a human cell line, where the produced P-NADs use the endogenous CDC48 machinery for ubiquitin-independent TPD of a 143 kDa multidomain protein. Thus, P-NADs pave the road for ubiquitin-independent therapeutic TPD approaches. In addition, the modular P-NAD design combined with in vitro and cellular assays provide a versatile platform for elucidating functional aspects of CDC48-based TPD in plants and animals.

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Eukaryotic elongation factor 2 kinase (eEF2K) is a member of the α-kinase family that is activated by calcium/calmodulin. Of note, eEF2K is crucial for regulating translation and is often highly overexpressed in malignant cells. Therefore in this review, we summarize the molecular structure of eEF2K and its oncogenic roles in cancer. Moreover, we further discuss the inhibition of eEF2K with small-molecule inhibitors and other new emerging therapeutic strategies in cancer therapy. Taken together, these inspiring findings provide new insights into a promising strategy for inhibiting eEF2K to greatly improve future cancer therapy.

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Targeted protein degradation is an emergent and rapidly evolving therapeutic strategy. In particular, biologics-based targeted degradation modalities (bioPROTACs) are relatively under explored compared to small molecules. Here, we investigate how target affinity, cellular localization, and valency of bioPROTACs impact efficacy of targeted degradation of the oncogenic phosphatase src-homology 2 containing protein tyrosine phosphatase-2 (SHP2). We identify bivalent recruitment of SHP2 by bioPROTACs as a broadly applicable strategy to improve potency. Moreover, we demonstrate that SHP2-targeted bioPROTACs can effectively counteract gain-of-function SHP2 mutants present in cancer, which are otherwise challenging to selectively target with small molecule constructs. Overall, this study demonstrates the utility of bioPROTACs for challenging targets, and further explicates design principles for therapeutic bioPROTACs.

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Resistance to cancer drugs is a complex phenomenon that poses a significant challenge in the treatment of various malignancies. This review comprehensively explores cancer resistance mechanisms and discusses emerging strategies and modalities to overcome this obstacle. Many factors contribute to cancer resistance, including genetic mutations, activation of alternative signaling pathways, and alterations in the tumor microenvironment. Innovative approaches, such as targeted protein degradation, immunotherapy combinations, precision medicine, and novel drug delivery systems, hold promise for improving treatment outcomes. Understanding the intricacies of cancer resistance and leveraging innovative modalities are essential for advancing cancer therapy.

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Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in preclinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four-residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit can disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are powerful tools for expanding the CAR T cell engineering toolbox.

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Vepdegestrant (ARV-471) is an oral PROTAC ER degrader that binds an E3 ubiquitin ligase and ER to directly trigger ubiquitination of ER and its subsequent proteasomal degradation. In a first-in-human Phase I/II study, vepdegestrant monotherapy was well tolerated with clinical activity in pretreated patients with ER+/HER2- advanced breast cancer. The global, randomized Phase III VERITAC-2 study compares efficacy and safety of vepdegestrant versus fulvestrant in adults with ER+/HER2- advanced breast cancer after treatment with a CDK4/6 inhibitor plus endocrine therapy. Progression-free survival by blinded independent central review (primary end point) will be assessed in the intention-to-treat population and ESR1 mutation-positive subpopulation. Secondary end points include overall survival, tumor response, safety, pharmacokinetics, patient-reported outcomes, and circulating tumor DNA biomarkers.Clinical trial registration: NCT05654623 (ClinicalTrials.gov).

VERITAC-2 is a clinical trial comparing vepdegestrant, a new drug that degrades estrogen receptors, to an existing treatment called fulvestrant in patients with ER+/HER2- advanced breast cancer: Estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) breast cancer grows in response to estrogen, a hormone in the body, and has low levels or no HER2 protein. People living with ER+/HER2- advanced breast cancer that has grown, spread to another part of the body, or cannot be removed by surgery are often treated with cyclin-dependent kinase (CDK) 4/6 inhibitors and endocrine therapies, but their cancer may get worse on these treatments and new treatments are needed. Fulvestrant, an endocrine therapy that attaches to estrogen receptors, lowers estrogen's effect on tumors and can slow or stop cancer growth. Vepdegestrant, a new medicine being tested for ER+ breast cancer, is a PROteolysis TArgeting Chimera (PROTAC) protein degrader that attaches to estrogen receptors and causes them to be tagged for removal by the cell's natural protein disposal system. By removing estrogen receptors, vepdegestrant may cause tumors to stop growing or shrink.This paper describes the Phase III VERITAC-2 clinical study comparing vepdegestrant versus fulvestrant in people living with ER+/HER2- advanced breast cancer previously treated with a CDK4/6 inhibitor and endocrine therapy.Patients will be randomly assigned to receive vepdegestrant (a pill taken once daily by mouth) or fulvestrant (a shot given into the muscle). The purpose of the study is to find out how long people live without their cancer getting worse with vepdegestrant or fulvestrant. VERITAC-2 will also look at how long people live during the study, side effects people may experience, and the overall well-being of people throughout the study.

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Proteolysis-targeting chimeras (PROTACs) have emerged as a promising technology for inducing targeted protein degradation by leveraging the intrinsic ubiquitin-proteasome system (UPS). While the potential druggability of PROTACs toward undruggable proteins has accelerated their rapid development and the wide-range of applications across diverse disease contexts, off-tissue effect and side-effects of PROTACs have recently received attentions to improve their efficacy. To address these issues, spatial or temporal target protein degradation by PROTACs has been spotlighted. In this review, we explore chemical strategies for modulating protein degradation in a cell type-specific (spatio-) and time-specific (temporal-) manner, thereby offering insights for expanding PROTAC applications to overcome the current limitations of target protein degradation strategy.

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Histone deacetylase 6 (HDAC6) plays a crucial role in the initiation and progression of various cancers, as its overexpression is linked to tumor growth, invasion, migration, survival, apoptosis, and angiogenesis. Therefore, HDAC6 has emerged as an attractive target for anticancer drug discovery in the past decade. However, the development of conventional HDAC6 inhibitors has been hampered by their limited clinical efficacy, acquired resistance, and inability to inhibit non-enzymatic functions of HDAC6. To overcome these challenges, new strategies, such as dual-acting inhibitors, targeted protein degradation (TPD) technologies (including PROTACs, HyT), are essential to enhance the anticancer activity of HDAC6 inhibitors. In this review, we focus on the recent advances in the design and development of HDAC6 modulators, including isoform-selective HDAC6 inhibitors, HDAC6-based dual-target inhibitors, and targeted protein degraders (PROTACs, HyT), from the perspectives of rational design, pharmacodynamics, pharmacokinetics, and clinical status. Finally, we discuss the challenges and future directions for HDAC6-based drug discovery for cancer therapy.

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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.