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.     

What is the functional role of the thalidomide binding protein cereblon?

It has been found that nonsense mutation R419X of cereblon (CRBN) is associated with autosomal recessive non-syndromic mental retardation. Further experiments showed that CRBN binds to the cytosolic C-terminus of large-conductance Ca⁺⁺ activated potassium channel (BK_Ca) α-subunit and the cytosolic C-terminus of a voltage-gated chloride channel-2 (ClC-2), suggesting that CRBN may play a role in memory and learning via regulating the assembly and surface expression of BK_Ca and ClC-2 channels. In addition, it has also been found that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK) and prevents formation of a functional holoenzyme with regulatory subunits β and γ. Since AMPK is a master sensor of energy balance that inhibits ATP-consuming anabolic pathways and increases ATP-producing catabolic pathways, binding of CRBN with α1 subunit of AMPK may play a role in these pathways by regulating the function of AMPK. Furthermore, CRBN interacts with damaged DNA binding protein 1 and forms an E3 ubiquitin ligase complex with Cullin 4 where it functions as a substrate receptor in which the proteins recognized by CRBN might be ubiquitinated and degraded by proteasomes. Proteasome-mediated degradation of unneeded or damaged proteins plays a very important role in maintaining regular function of a cell, such as cell survival, dividing, proliferation and growth. Intriguingly, a new role for CRBN has been identified, i.e, the binding of immunomodulatory drugs (IMiDs), e.g. thalidomide, to CRBN has now been associated with teratogenicity and also the cytotoxicity of IMiDs, including lenalidomide, which are widely used to treat multiple myeloma patients. CRBN likely plays an important role in binding, ubiquitination and degradation of factors involved in maintaining function of myeloma cells. These new findings regarding the role of CRBN in IMiD action will stimulate intense investigation of CRBN’s downstream factors involved in maintaining regular function of a cell.     

The Casein kinase 1α agonist pyrvinium attenuates Wnt-mediated CK1α degradation via interaction with the E3 ubiquitin ligase component Cereblon

The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN–CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.     

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.     

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.     

Replacing the phthalimide core in thalidomide with benzotriazole

The advent of proteolysis-targeting chimaeras (PROTACs) mandates that new ligands for the recruitment of E3 ligases are discovered. The traditional immunomodulatory drugs (IMiDs) such as thalidomide and its analogues (all based on the phthalimide glutarimide core) bind to Cereblon, the substrate receptor of the CRL4A^CRBN E3 ligase. We designed a thalidomide analogue in which the phthalimide moiety was replaced with benzotriazole, using an innovative synthesis strategy. Compared to thalidomide, the resulting “benzotriazolo thalidomide” has a similar binding mode, but improved properties, as revealed in crystallographic analyses, affinity assays and cell culture.     

The emerging role for Cullin 4 family of E3 ligases in tumorigenesis

As a member of the Cullin-RING ligase family, Cullin-RING ligase 4 (CRL4) has drawn much attention due to its broad regulatory roles under physiological and pathological conditions, especially in neoplastic events. Based on evidence from knockout and transgenic mouse models, human clinical data, and biochemical interactions, we summarize the distinct roles of the CRL4 E3 ligase complexes in tumorigenesis, which appears to be tissue- and context-dependent. Notably, targeting CRL4 has recently emerged as a noval anti-cancer strategy, including thalidomide and its derivatives that bind to the substrate recognition receptor cereblon (CRBN), and anticancer sulfonamides that target DCAF15 to suppress the neoplastic proliferation of multiple myeloma and colorectal cancers, respectively. To this end, PROTACs have been developed as a group of engineered bi-functional chemical glues that induce the ubiquitination-mediated degradation of substrates via recruiting E3 ligases, such as CRL4 (CRBN) and CRL2 (pVHL). We summarize the recent major advances in the CRL4 research field towards understanding its involvement in tumorigenesis and further discuss its clinical implications. The anti-tumor effects using the PROTAC approach to target the degradation of undruggable targets are also highlighted.     

沙利度胺抑制肿瘤细胞分泌VEGF/bFGF机制的探讨*

目的: 探讨Cereblon(CRBN)对沙利度胺抑制人肺癌A549细胞及人肝癌HepG2细胞分泌VEGF/bFGF的影响。方法: 采用慢病毒介导的短发夹RNA(shRNA)干扰技术建立稳定敲低CRBN的A549细胞系(A549_CRBN)及HepG2细胞系(HepG2_CRBN)并通过实时定量PCR(Real-time PCR)和蛋白质印记(Western blot)实验验证。将A549细胞分为阴性对照组(A549_luciferase)、CRBN低表达组(A549_CRBN);HepG2细胞分为阴性对照组(HepG2_luciferase)、CRBN低表达组(HepG2_CRBN),以上细胞按照 3×10⁵ cells/well接种到6孔板中,放入37℃,5%CO2的培养箱中培养24 h,分别加入1 ml含100 μmol/L沙利度胺(thalidomide组)和1 ml 1‰ DMSO(control组)的培养液,继续培养24 h再行后续实验,每组设计3个复孔。MTS法检测沙利度胺对细胞增殖的影响;Real-time PCR检测VEGF、bFGF、c-jun mRNA表达,ELISA法检测VEGF、bFGF蛋白表达。结果: 与对照组比较,沙利度胺在浓度为1、10、50、100 μmol/L 时对A549 及HepG2细胞的增殖能力无显著影响(P＞0.05)。与A549_CRBN或HepG2_CRBN组比较,A549_luciferase及HepG2_luciferase组分泌的VEGF及bFGF均显著降低(P＜0.05)。与A549_luciferase或HepG2_luciferase细胞的对照组比较,沙利度胺可抑制A549_luciferase和HepG2_luciferase细胞的VEGF和bFGF的表达(P＜0.05),而对A549_CRBN和HepG2_CRBN细胞中VEGF和bFGF的表达无显著抑制作用;与HepG2_luciferase细胞的对照组比较,沙利度胺可抑制HepG2_luciferase细胞的c-Jun表达(P＜0.01),而对HepG2_CRBN细胞的c-Jun表达无显著抑制作用。结论: 沙利度胺对A549和HepG2细胞VEGF和bFGF表达的抑制作用可能是通过CRBN介导的,而c-Jun可能是抑制作用的关键转录因子之一。     

Sulfur-containing marine natural products as leads for drug discovery and development

Among the large series of marine natural products (MNPs), sulfur-containing MNPs have emerged as potential therapeutic agents for the treatment of a range of diseases. Herein, we reviewed 95 new sulfur-containing MNPs isolated during the period between 2021 and March 2023. In addition, we discuss that the widely used strategies and the emerging technologies including natural product-based antibody drug conjugates (ADCs), small-molecule-based proteolysis targeting chimeras (PROTACs), nanotechnology-based drug carriers, artificial intelligence (AI)-driven drug discovery have been used for improving the efficiency and success rate of NP-based drug development. We also provide perspectives regarding the challenges and opportunities in sulfur-containing MNPs based drug discovery and development and future research directions.     

Immunomodulatory drugs inhibit expression of cyclooxygenase-2 from TNF-alpha, IL-1beta, and LPS-stimulated human PBMC in a partially IL-10-dependent manner

Immunomodulatory drugs (IMiDs) are potent inhibitors of TNF-alpha and IL-1beta and elevators of IL-10 production in LPS-stimulated human PBMC. They are currently in clinical trials for various diseases, including multiple myeloma, myelodysplastic syndrome, and melanoma. In the present study, we have investigated the effects of thalidomide, CC-5013 and CC-4047 on the expression of COX-2 by stimulated PBMC. Our results show that thalidomide and IMiDs inhibited the expression of COX-2 but not the COX-1 protein in LPS-TNF-alpha and IL-1beta stimulated PBMC and shortened the half-life of COX-2 mRNA in a dose-dependent manner. They also inhibited the synthesis of prostaglandin E2 from LPS-stimulated PBMC. While anti-TNF-alpha or IL-1beta neutralizing antibodies had no effect on COX-2 expression, anti-IL-10 neutralizing antibody elevated the expression of COX-2 mRNA, and protein from treated PBMC. These data suggest that the anti-inflammatory and anti-tumor effects of IMiDs may be due in part to elevation of IL-10 production and its subsequent inhibition of COX-2 expression.     

Stopping the beating heart of cancer: KRAS reviewed

It has taken four decades of research to see the first major breakthrough for KRAS-driven cancers. In particular, the last decade has seen a paradigm shift with the discovery of druggable pockets on KRAS and clinical efficacy with covalent KRAS^G12C inhibitors, culminating in the first approval of sotorasib monotherapy as second-line treatment in KRAS^G12C-driven non–small-cell lung cancer. Nevertheless, 85% of all KRAS-mutated cancers still lack novel agents. In this review, we will outline the structure, function, and post-translational modifications of KRAS and highlight the various approaches being adopted to drug KRAS, ranging from selective to pan concepts. The range of molecular modalities being explored, including PROTACs and glues, will also be described. Finally, an outlook toward the next wave of KRAS drugs and the challenges of resistance will be given.     

Epidermal growth factor receptor PROTACs as an effective strategy for cancer therapy: A review

Epidermal growth factor receptor (EGFR), a transmembrane glycoprotein that mediates cellular signaling pathways involved in cell proliferation, angiogenesis, apoptosis, and metastatic spread, is an important oncogenic drug target. Targeting the intracellular and extracellular domains of the EGFR has been authorized for a number of small-molecule TKIs and mAbs, respectively. However, their clinical application is limited by EGFR catalytic structural domain alterations, cancer heterogeneity, and persistent drug resistance. To bypass these limitations, protease-targeted chimeras (PROTACs) are emerging as an emerging and promising anti-EGFR therapy. PROTACs compensate for the limitations of traditional occupancy-driven small molecules by exploiting intracellular protein destruction processes. Recently, a mushrooming number of heterobifunctional EGFR PROTACs have been created using wild-type (WT) and mutated EGFR TKIs. PROTACs outperformed EGFR TKIs in terms of cellular inhibition, potency, toxicity profiles, and anti-drug resistance. Herein, we present a comprehensive overview of the development of PROTACs targeting EGFR for cancer therapy, while also highlighting the challenges and opportunities associated with the field.     

Proteolysis targeting chimeras (PROTACs) for epigenetics research

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules and allow selective protein degradation by addressing the natural ubiquitin proteasome system. As this new strategy of chemically induced protein degradation can serve as a biological tool and provides new possibilities for drug discovery, it has been applied to a variety of targets including (nuclear) receptors, kinases, and epigenetic proteins. A lot of PROTACs have already been designed in the field of epigenetics, and their synthesis and characterization highly contributed to structural optimization and improved mechanistic understanding of these molecules. In this review, we will discuss and summarize recent advances in PROTAC discovery with focus on epigenetic targets.     

Radioligands targeting purinergic P2X7 receptor

The purinergic P2X7 receptor (P2X7R) is an adenosine triphosphate (ATP) ligand-gated cationic channel receptor. P2X7R is closely associated with various inflammatory, immune, cancer, neurological, musculoskeletal and cardiovascular disorders. P2X7R is an interesting therapeutic target as well as molecular imaging target. This brief digest highlights the radioligands targeting P2X7R recently developed in drug discovery and molecular imaging agent development.     

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.     

Substrate specificity of lymphoid-specific tyrosine phosphatase (Lyp) and identification of Src kinase-associated protein of 55 kDa homolog (SKAP-HOM) as a Lyp substrate

A missense single-nucleotide polymorphism in the gene encoding the lymphoid-specific tyrosine phosphatase (Lyp) has been identified as a causal factor in a wide spectrum of autoimmune diseases. Interestingly, the autoimmune-predisposing variant of Lyp appears to represent a gain-of-function mutation, implicating Lyp as an attractive target for the development of effective strategies for the treatment of many autoimmune disorders. Unfortunately, the precise biological functions of Lyp in signaling cascades and cellular physiology are poorly understood. Identification and characterization of Lyp substrates will help define the chain of molecular events coupling Lyp dysfunction to diseases. In the current study, we identified consensus sequence motifs for Lyp substrate recognition using an "inverse alanine scanning" combinatorial library approach. The intrinsic sequence specificity data led to the discovery and characterization of SKAP-HOM, a cytosolic adaptor protein required for proper activation of the immune system, as a bona fide Lyp substrate. To determine the molecular basis for Lyp substrate recognition, we solved crystal structures of Lyp in complex with the consensus peptide as well as the phosphopeptide derived from SKAP-HOM. Together with the biochemical data, the structures define the molecular determinants for Lyp substrate specificity and provide a solid foundation upon which novel therapeutics targeting Lyp can be developed for multiple autoimmune diseases.     

The thalidomide saga

Over the past 50 years, thalidomide has been a target of active investigation in both malignant and inflammatory conditions. Although initially developed for its sedative properties, decades of investigation have identified a multitude of biological effects that led to its classification as an immunomodulatory drug (IMiD). In addition to suppression of tumor necrosis factor-alpha (TNF-alpha), thalidomide effects the generation and elaboration of a cascade of pro-inflammatory cytokines that activate cytotoxic T-cells even in the absence of co-stimulatory signals. Furthermore, vascular endothelial growth factor (VEGF) and beta fibroblast growth factor (bFGF) secretion and cellular response are suppressed by thalidomide, thus antagonizing neoangiogenesis and altering the bone marrow stromal microenvironment in hematologic malignancies. The thalidomide analogs, lenalidomide (CC-5013; Revlimid) and CC-4047 (Actimid), have enhanced potency as inhibitors of TNF-alpha and other inflammatory cytokines, as well as greater capacity to promote T-cell activation and suppress angiogenesis. Both thalidomide and lenalidomide are effective in the treatment of multiple myeloma and myelodysplastic syndromes for which the Food and Drug Administration granted recent approval. Nonetheless, each of these IMiDs remains the subject of active investigation in solid tumors, hematologic malignancies, and other inflammatory conditions. This review will explore the pharmacokinetic and biologic effects of thalidomide and its progeny compounds.