Proteolysis-targeting chimaeras (PROTACs) as pharmacological tools and therapeutic agents: advances and future challenges

Proteolysis-targeting chimaeras (PROTACs) have been developed to be an emerging technology for targeted protein degradation and attracted the favour of academic institutions, large pharmaceutical enterprises, and biotechnology companies. The mechanism is based on the inhibition of protein function by hijacking a ubiquitin E3 ligase for protein degradation. The heterobifunctional PROTACs contain a ligand for recruiting an E3 ligase, a linker, and another ligand to bind with the protein targeted for degradation. To date, PROTACs targeting ∼70 proteins, many of which are clinically validated drug targets, have been successfully developed with several in clinical trials for diseases therapy. In this review, the recent advances in PROTACs against clinically validated drug targets are summarised and the chemical structure, cellular and in vivo activity, pharmacokinetics, and pharmacodynamics of these PROTACs are highlighted. In addition, the potential advantages, challenges, and prospects of PROTACs technology in disease treatment are discussed.     

Cereblon versus VHL: Hijacking E3 ligases against each other using PROTACs

The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degradation. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compound 14a induced potent, rapid and profound preferential degradation of CRBN over VHL in cancer cell lines. At lower concentrations, weaker degradation of VHL was instead observed. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown.     

Recent advances in IAP-based PROTACs (SNIPERs) as potential therapeutic agents

Proteolytic targeting chimaeras (PROTACs) have been developed as an effective technology for targeted protein degradation. PROTACs are heterobifunctional molecules that can trigger the polyubiquitination of proteins of interest (POIs) by recruiting the ubiquitin-proteasome system, thereby inhibiting the intracellular level of POIs. To date, a variety of small-molecule PROTACs (CRBN, VHL, IAP, and MDM2-based PROTACs) have been developed. IAP-based PROTACs, also known as specific and nongenetic IAP-dependent protein erasers (SNIPERs), are used to degrade the target proteins closely related to diseases. Their structures consist of three parts, including target protein ligand, E3 ligase ligand, and the linker between them. So far, many SNIPERs have been extensively studied worldwide and have performed well in multiple diseases, especially cancer. In this review, we will present the most relevant advances in the field of SNIPERs and provide our perspective on the opportunities and challenges for SNIPERs to become therapeutic agents.     

Selective CDK6 degradation mediated by cereblon,VHL, and novel IAP-recruiting PROTACs

Inhibitors of CDK4 and CDK6 have emerged as important FDA-approved treatment options for breast cancer patients. The properties and pharmacology of CDK4/6 inhibitor medicines have been extensively profiled, and investigations into the degradation of these targets via a PROTAC strategy have also been reported. PROTACs are a novel class of small-molecules that offer the potential for differentiated pharmacology compared to traditional inhibitors by redirecting the cellular ubiquitin–proteasome system to degrade target proteins of interest. We report here the preparation of palbociclib-based PROTACs that incorporate binders for three different E3 ligases, including a novel IAP-binder, which effectively degrade CDK4 and CDK6 in cells. In addition, we show that the palbociclib-based PROTACs in this study that recruit different E3 ligases all exhibit preferential CDK6 vs. CDK4 degradation selectivity despite employing a selection of linkers between the target binder and the E3 ligase binder.     

The rise of covalent proteolysis targeting chimeras

Targeted protein degradation offers several advantages over direct inhibition of protein activity and is gaining increasing interest in chemical biology and drug discovery. Proteolysis targeting chimeras (PROTACs) in particular are enjoying widespread application. However, PROTACs, which recruit an E3 ligase for degradation of a target protein, still suffer from certain challenges. These include a limited selection for E3 ligases on the one hand and the requirement for potent target binding on the other hand. Both issues restrict the target scope available for PROTACs. Degraders that covalently engage the target protein or the E3 ligase can potentially expand the pool of both targets and E3 ligases. Moreover, they may offer additional advantages by improving the kinetics of ternary complex formation or by endowing additional selectivity to the degrader. Here, we review the recent progress in the emerging field of covalent PROTACs.     

嵌合分子(DHT-PROTAC)在前列腺癌研究中的进展

前列腺癌多发于老年男性,已成为老年男性常见肿瘤之一。内分泌治疗目前是晚期前列腺癌主要治疗方法,但仍避免不了前列腺癌最终进展成激素非依赖性前列腺癌,导致内分泌治疗的失败。当前,对前列腺癌细胞株的AR表达的研究,主要集中在DNA水平及mRNA水平,而对AR蛋白翻译后调控的研究较少。近些年来,嵌合分子(DHT-PROTAC)是基于蛋白水平,调控AR蛋白的表达,成为研究前列腺癌转归新的热点。DHT-PROTAC是一种新型人工合成的异型双功能小分子;这种小分子是DHT与泛素连接酶E3识别基团的嵌合体,它不仅能与AR结合,而且能在结合后,诱导AR的泛素化,从而通过泛素-蛋白酶途径降解AR;本文介绍了嵌合分子的作用原理,回顾了近些年前列腺癌的治疗进展,分析了嵌合分子将来在前列腺癌治疗中的应用前景。     

嵌合分子（DHT-PROTAC）在前列腺癌研究中的进展

前列腺癌多发于老年男性,已成为老年男性常见肿瘤之一。内分泌治疗目前是晚期前列腺癌主要治疗方法,但仍避免不了前列腺癌最终进展成激素非依赖性前列腺癌,导致内分泌治疗的失败。当前,对前列腺癌细胞株的AR表达的研究,主要集中在DNA水平及mRNA水平,而对AR蛋白翻译后调控的研究较少。近些年来,嵌合分子（DHT-PROTAC）是基于蛋白水平．调控AR蛋白的表达,成为研究前列腺癌转归新的热点。DHT-PROTAC是一种新型人工合成的异型双功能小分子;这种小分子是DHT与泛素连接酶E3识别基团的嵌合体,它不仅能与AR结合,而且能在结合后,诱导AR的泛素化,从而通过泛素一蛋白酶途径降解AR;本文介绍了嵌合分子的作用原理,回顾了近些年前列腺癌的治疗进展,分析了嵌合分子将来在前列腺癌治疗中的应用前景。     

Hi-JAK-ing the ubiquitin system: The design and physicochemical optimisation of JAK PROTACs

PROTACs have recently emerged as a novel paradigm in drug discovery. They can hijack existing biological machinery to selectively degrade proteins of interest, in a catalytic fashion. Here we describe the design, optimisation and biological activity of a set of novel PROTACs targeting the Janus kinase family (JAK1, JAK2, JAK3 and TYK2) of proximal membrane-bound proteins. The JAK family proteins display membrane localisation by virtue of their association with cytoplasmic tails of cytokine receptors, and there are no reports of a successful PROTAC strategy being deployed against this class of proteins. JAK PROTACs from two distinct JAK chemotypes were designed, optimising the physicochemical properties for each template to enhance cell permeation. These PROTACs are capable of inducing JAK1 and JAK2 degradation, demonstrating an extension of the PROTAC methodology to an unprecedented class of protein targets. A number of known ligase binders were explored, and it was found that PROTACs bearing an inhibitor of apoptosis protein (IAP) ligand induced significantly more JAK degradation over Von Hippel–Lindau (VHL) and Cereblon (CRBN) PROTACs. In addition, the mechanism of action of the JAK PROTACs was elucidated, and it was confirmed that JAK degradation was both IAP- and proteasome-dependent.     

Impact of linker length on the activity of PROTACs

Conventional genetic approaches have provided a powerful tool in the study of proteins. However, these techniques often preclude selective manipulation of temporal and spatial protein functions, which is crucial for the investigation of dynamic cellular processes. To overcome these limitations, a small molecule-based novel technology termed "PROteolysis TArgeting ChimeraS (PROTACs)" has been developed, targeting proteins for degradation at the post-translational level. Despite the promising potential of PROTACs to serve as molecular probes of complex signaling pathways, their design has not been generalized for broad application. Here, we present the first generalized approach for PROTAC design by fine-tuning the distance between the two participating partner proteins, the E3 ubiquitin ligase and the target protein. As such, we took a chemical approach to create estrogen receptor (ER)-α targeting PROTACs with varying linker lengths and the loss of the ER in cultured cells was monitored via western blot and fluorometric analyses. We found a significant effect of chain length on PROTAC efficacy, and, in this case, the optimum distance between the E3 recognition motif and the ligand was a 16 atom chain length. The information gathered from this experiment may offer a generalizable PROTAC design strategy to further the expansion of the PROTAC toolbox, opening new possibilities for the broad application of the PROTAC strategy in the study of multiple signaling pathways.     

Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1

Protein quality control and subsequent elimination of terminally misfolded proteins occurs via the ubiquitin-proteasome system. Tagging of misfolded proteins with ubiquitin for degradation depends on a cascade of reactions involving an ubiquitin activating enzyme (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). While ubiquitin ligases responsible for targeting misfolded secretory proteins to proteasomal degradation (ERAD) have been uncovered, no such E3 enzymes have been found for elimination of misfolded cytoplasmic proteins in yeast. Here we report on the discovery of Ubr1, the E3 ligase of the N-end rule pathway, to be responsible for targeting misfolded cytosoplasmic protein to proteasomal degradation.     

Degradation of huntingtin mediated by a hybrid molecule composed of IAP antagonist linked to phenyldiazenyl benzothiazole derivative

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by aggregation of mutant huntingtin (mHtt), and removal of mHtt is expected as a potential therapeutic option. We previously reported protein knockdown of Htt by using hybrid small molecules (Htt degraders) consisting of BE04, a ligand of ubiquitin ligase (E3), linked to probes for protein aggregates. Here, in order to examine the effect of changing the ligand, we synthesized a similar Htt degrader utilizing MV1, an antagonist of the inhibitor of apoptosis protein (IAP) family (a subgroup of ubiquitin E3 ligases), which is expected to have a higher affinity and specificity for IAP, as compared with BE04. The MV1-based hybrid successfully induced interaction between Htt aggregates and IAP, and reduced mHtt levels in living cells. Its mode of action was confirmed to be the same as that of the BE04-based hybrid. However, although the affinity of MV1 for IAP is greater than that of BE04, the efficacy of Htt degradation by the MV1-based molecule was lower, suggesting that linker length between the ligand and probe might be an important determinant of efficacy.     

A novel DCAF1‐binding motif required for Vpx‐mediated degradation of nuclear SAMHD1 and Vpr‐induced G2 arrest

HIV‐2 and closely related SIV Vpx proteins are essential for viral replication in macrophages and dendritic cells. Vpx hijacks DCAF1–DDB1–Cul4 E3 ubiquitin ligase to promote viral replication. DCAF1 is essential for cell proliferation and embryonic development and is responsible for the polyubiquitination of poorly defined cellular proteins. How substrate receptors recruit the DCAF1‐containing E3 ubiquitin ligase to induce protein degradation is still poorly understood. Here we identify a highly conserved motif (Wx4Φx2Φx3AΦxH) that is present in diverse Vpx and Vpr proteins of primate lentiviruses. We demonstrate that the Wx4Φx2Φx3AΦxH motif in SIVmac Vpx is required for both the Vpx–DCAF1 interaction and/or Vpx‐mediated degradation of SAMHD1. DCAF1‐binding defective Vpx mutants also have impaired ability to promote SIVΔVpx virus infection of myeloid cells. Critical amino acids in the Wx4Φx2Φx3AΦxH motif of SIV Vpx that are important for DCAF1 interaction maintained the ability to bind SAMHD1, indicating that the DCAF1 and SAMHD1 interactions involve distinctive interfaces in Vpx. Surprisingly, VpxW24A mutant proteins that were still capable of binding DCAF1 and SAMHD1 lost the ability to induce SAMHD1 degradation, suggesting that Vpx is not a simple linker between the DCAF1–DDB1–Cul4 E3 ubiquitin ligase and its substrate, SAMHD1.VpxW24A maintained the ability to accumulate in the nucleus despite the fact that nuclear, but not cytoplasmic, mutant forms of SAMHD1 were more sensitive to Vpx‐mediated degradation. The Wx4Φx2Φx3AΦxH motif in HIV‐1 Vpr is also required for the Vpr–DCAF1 interaction and Vpr‐induced G2 cell cycle arrest. Thus, our data reveal previously unrecognized functional interactions involved in the assembly of virally hijacked DCAF1–DDB1‐based E3 ubiquitin ligase complex.     

Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously “undruggable” proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12–186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target “degradability.”     

植物泛素/26S蛋白酶体途径研究进展

泛素/26S蛋白酶体途径是最重要的,有高度选择性的蛋白质降解途径,由泛素激活酶、泛素结合酶、泛素蛋白连接酶和26S蛋白酶体组成,参与调控植物生长发育的多个方面。泛素蛋白酶体途径参与植物体内的众多生理过程,如植物激素信号,光形态建成、自交不亲和反应和细胞周期等。本文就泛素/26S蛋白酶体途径以及在植物生长发育中的作用的研究近况做一综述。     

Autoantigen Ro52 is an E3 ubiquitin ligase

Anti-Ro/SSA antibodies are classic autoantibodies commonly found in patients with Sj?gren's syndrome, a chronic autoimmune disease characterized by dryness of the eyes and mouth. The autoantibodies recognize a RING-finger protein, Ro52, whose function is still unknown. Since many RING-finger proteins have been identified as E3 ubiquitin ligases, this study was designed to determine whether Ro52 functions as an E3 ubiquitin ligase. For this purpose, recombinant Ro52 was purified from bacterial lysate and used to investigate its activity of E3 ubiquitin ligase in vitro. Its enzymatic activity was also tested in HEK293T cells using wild-type Ro52 and its RING-finger mutant. Our results indicated that Ro52 ubiquitinates itself in cooperation with E2 ubiquitin-conjugating enzyme UbcH5B, thereby validating that Ro52 is a RING-finger-type E3 ubiquitin ligase. Importantly, this ubiquitin modification is predominantly monoubiquitination, which does not target Ro52 to the proteasome for degradation.     

Salmonella ubiquitination: ARIH1 enters the fray

Ubiquitination is a post‐translational modification in which ubiquitin, a 76‐amino acid polypeptide, is covalently bound to one or more lysines of a target protein. Ubiquitination is mediated by the coordinated activity of ubiquitin activating (E1), conjugating (E2), and ligating (E3) enzymes. Ubiquitin is widely investigated for its ability to regulate key biological processes in the cell, including protein degradation and host–bacteria interactions. The determinants underlying bacterial ubiquitination, and their precise roles in host defense, have not been fully resolved. In this issue of EMBO Reports, Polajnar et al 1 discover that Ring‐between‐Ring (RBR) E3 ligase ARIH1 (also known as HHARI) is involved in formation of the ubiquitin coat surrounding cytosolic Salmonella. Evidence suggests that ARIH1, in cooperation with E3 ligases LRSAM1 and HOIP, modulates the recognition of intracellular bacteria for cell‐autonomous immunity.     

Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum

The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys(48)-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-alpha and CD3-delta.