Discovery of intracellular self-assembly protein degraders driven by a tumor-specific activatable bioorthogonal response (2023)


Proteolysis Targeting Chimera (PROTAC) is a type of heterobifunctional molecule that has been rapidly developed in recent years to degrade an interesting protein through the ubiquitin-proteasome pathway [1,2]. The advantage of PROTAC is not only that the target protein is effectively inhibited, but also that the target protein is rapidly broken down and eliminated. The catalytic amount of the drug can be used to degrade the intracellular target protein, which ensures its high safety, drug resistance and wide application potential. During detailed research, some problems gradually surfaced, which restricted further development. First, the molecular weights of PROTACs are generally in the range of 700 Da-1200 Da, compromising their membrane permeability and oral bioavailability [[3], [4], [5]]. Furthermore, PROTACs lack selectivity for tumor tissues, which can result in off-tissue and on-target effects that have adverse effects on normal cells or tissues [6,7]. Several studies to improve cell permeability or to target tumors of PROTAC have been reported [8]. Klein proposed a strategy to optimize PROTAC cell permeability and cell viability by replacing the amide with an ester [9]. Cell click-formed proteolysis targeting chimeras (CLIPTACs) were successfully constructed by Lebraud to target two important oncological targets [10]. Folic acid-modified PROTAC has been synthesized and can be targeted to cancer cells with high folate receptor α (FOLR1) expression to degrade related proteins [11]. It has also been reported that hypoxia-activated PROTAC shows a stronger knockdown effect in tumor hypoxia than in normoxia [12]. All these strategies have given great impetus to the development of PROTACs.

There are many studies that have shown that the copper content in tumor tissue is significantly higher than in normal tissue [13,14]. Many tumor-specific therapies have been developed to take advantage of this property [15,16]. The copper ion-catalysed 1,3-dipole cycloaddition reaction between alkynes and azides is a high-throughput chemical click reaction that allows efficient binding of the two corresponding molecules [17,18]. Due to its high yield and properties that can be performed in water at room temperature, even in living cells, it is widely used in bioconjugation, chemical synthesis and materials science [19,20].

In this article, the combination strategy of copper-catalyzed azide-alkyne cycloaddition (CuAAC) and PROTAC is applied to improve cellular permeability of PROTAC and reduce side effects. We designed and synthesized two types of precursor molecules with azide and alkyne groups, respectively, which can be converted to PROTAC in situ by click reaction using endogenous copper in tumor tissues. Compared with conventional PROTAC molecules, self-assembled PROTAC based on azide and alkyne offers the following advantages. First, PROTAC is generated by intracellular self-assembly of two precursor molecules, which indirectly reduces molecular weight, which helps to increase cellular permeability. Second, the precursor molecules we design and synthesize are not targetable, but self-assembled PROTACs based on azide and alkyne groups can only be prepared by copper ion catalysis, which is greater in tumor cells than in normal cells. Thus, our self-assembled PROTACs are tumor tissue specific, which means that efficient tumor therapy can be achieved without significant side effects on normal cells or tissues.

Fragments section.

Synthesis of precursor molecules with azide or alkyne

Intracellular self-assembled PROTAC is formed by the click reaction between a precursor molecule containing an azide and a precursor molecule containing an alkyne group, catalyzed by endogenous copper (Fig. 1). These two precursor molecules can bind to the target protein and ubiquitin ligase E3, respectively. Sorafenib is a multi-target kinase inhibitor that can inhibit tumor cell proliferation not only directly but also indirectly by inhibiting the RAF/MEK/ERK mediated cell signaling pathway


In this work, we took advantage of the click reaction between azide and alkyne and the fact that copper ions are significantly higher in tumor tissue than in normal tissue. external catalyst. This strategy not only increases PROTAC's tumor targeting, but also increases cellular permeability by decreasing molecular weight. HPLC and high-resolution ultra-fast mass

general chemical methods

All reagents were purchased from commercial suppliers and used without further purification. Reaction progress was followed by TLC on pre-coated silica gel plates (Kieselgel 60 F254); Stains were detected by UV or other suitable stains.1H NMR spectra (600 MHz) pt13C-NMR spectra (600 MHz) were recorded on a Bruker spectrometer with TMS as an internal standard. Standard mass spectra and high resolution mass spectra were obtained by electrospray ionization mass spectrometry (ESI-MS). O

Declaration of conflict of interest

The authors declare that they are not aware of any competing financial interests or personal relationships that could influence the work described in this article.


This work was supported byNational Science Foundation of China(NSFC, nº.82173742), ofScience Fund for Outstanding Young Scientists of Shaanxi Province(Nee.2022JC-54), it's atMajor Research and Development Projects of Shaanxi Province(Nee.2023-YBSF-131). Major institutions sharing the Xi'an Jiaotong University platform were thanked for supporting NMR research.

(Video) Molecular Dissection of Tumor Biology

Selected Items (6)

  • Research Article

    Discovery of a first-class Aurora-A covalent inhibitor for the treatment of triple-negative breast cancer

    European Journal of Medicinal Chemistry, Band 256, 2023, Artikel 115457

    Aurora kinases, belonging to the serine/threonine protein family, play a crucial role in cell cycle regulation and mitotic spindle assembly. They are often highly expressed in various tumor types and the use of selective Aurora kinase inhibitors has become a potential treatment option for cancer therapy. Despite the development of some reversible Aurora kinase inhibitors, none have been approved for clinical use. In this study, we report the discovery of first-in-class Aurora-A irreversible covalent inhibitors that target a cysteine ​​residue at the substrate binding site. These inhibitors were characterized in enzymatic and cellular assays11cshowed selective inhibition of normal cells and cancer cells, as well as Aurora A and B kinases. The covalent bond of11cversus Aurora A was confirmed by SPR, MS, and enzyme kinetics analysis, and Cys290-mediated covalent inhibition was supported by an ascending analysis of inhibitor-modified targets. In addition, Western blot assays were performed on cells and tissues, and Cellular Thermal Shift Assays (CETSA) were also performed on cells to demonstrate selectivity for Aurora A kinase.11cdemonstrated similar therapeutic efficacy compared to the positive control in an MDA-MB-231 xenograft mouse modelENMD-2076, where only half the dose is neededENMD-2076. These results confirmed that11ccould be a promising drug candidate for the treatment of triple negative breast cancer (TNBC). Our work may offer a new perspective for the design of covalent Aurora kinase inhibitors.

  • Research Article

    Current scenario of pyrazole hybrids with in vivo therapeutic potential against cancer

    European Journal of Medicinal Chemistry, Band 257, 2023, Artikel 115495

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    Chemotherapy drugs play a crucial role in treating many types of cancer, yet cancer prevalence and death rates remain high. Drug resistance and low specificity of currently available chemotherapeutic agents represent major obstacles to effective anticancer chemotherapy and require the development of new anticancer agents. A highly versatile five-membered heterocycle with two adjacent nitrogen atoms, pyrazole has remarkable therapeutic effects and robust pharmacological potency. Pyrazole derivatives, particularly pyrazole hybrids, have been shown to be effectivein vitroEmliveAnti-cancer efficacy through multiple mechanisms including induction of apoptosis, regulation of autophagy and cell cycle arrest. In addition, several pyrazole hybrids such as crizotanib (pyrazol-pyridine hybrid), erdafitinib (pyrazol-quinoxaline hybrid) and ruxolitinib (pyrazol-pyrrolo[2,3-d]pyrimidine hybrid) have already been approved for cancer therapy, the which shows that pyrazole hybrids are useful scaffolds for the development of new anticancer drugs. The aim of this review is to summarize the current scenario of potential pyrazole hybridsliveAnti-cancer efficacy and mechanisms of action, toxicity and pharmacokinetics, with articles published in the last 5 years (since 2018) to further facilitate the rational use of more potent candidates.

  • Research Article

    Discovery of new imidazotetrazinones with potential to overcome tumor resistance

    European Journal of Medicinal Chemistry, 2023, Artikel 115507

    We describe the design, organic synthesis and characterization, including X-ray crystallography, of a series of new analogues of the clinically used anticancer drug temozolomide, along with their ownin vitrobiological assessment. The work led to the discovery of a new series of anticancer imidazotetrazines that have the potential to overcome tumor resistance to temozolomide. The rationally designed compounds, which contain a propargyl alkylating group and a thiazole ring as an isosteric substitute for a carboxamide, are easily synthesized (gram scale), exhibit well-defined rigid structures and enhanced antiproliferative activity against human tumor cell lines, including MGMT -expressing and MMR- Faulty lines, molecular traits that confer tumor resistance. Cell proliferation data were confirmed by clonogenic cell survival assays and DNA flow cytometric analysis was performed to determine the effects of new analogues on cell cycle progression. Detailed1H NMR spectroscopic investigations showed that the new drugs are stable in solution and confirmed their mode of action. The propargyl and thiazole substituents significantly improve potency and physicochemical properties, metabolism and permeability of the drug, suggesting that thiazole13should be prioritized for further preclinical evaluation.

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  • Research Article

    3-Hydroxy-pyridin-4(1H)-ones, such as siderophore-mediated release of isobavachalcone, enhance antibacterial activity against the pathogen Pseudomonas aeruginosa

    European Journal of Medicinal Chemistry, Band 257, 2023, Artikel 115454

    A chalcona naturally prenylated isobavacalcona (IBC) shows good antibacterial activity against gram-positive bacteria but is ineffective against gram-negative bacteria, which is most likely due to the outer membrane barrier of gram-negative bacteria. The Trojan horse strategy has been shown to be an effective strategy to overcome the reduction in outer membrane permeability of gram-negative bacteria. In this study, eight different 3-hydroxy-pyridine-4(1HThe )-Ene-isobavachalcone conjugates were designed and synthesized based on the Trojan siderophore strategy. The conjugates showed minimum inhibitory concentrations (MICs) 8 to 32 times lower and semi-inhibitory concentrations (CI) 32 to 177 times lower.50n) againstPseudomonas aeruginosaPAO1 and clinically multidrug resistant (MDR) strains compared to the parental strainIBCunder iron restriction. Later studies showed that the antibacterial activity of the conjugates was regulated by the bacterial pathway of iron absorption under different conditions of iron concentration. Studies on the antibacterial mechanism of the conjugate1bdemonstrated that it exerts an antibacterial effect by disrupting the integrity of the cytoplasmic membrane and inhibiting cellular metabolism. finally merge1bshowed a lesser cytotoxic effect on Vero cells thanIBCand a positive therapeutic effect in the treatment of bacterial infections caused by PAO1 gram-negative bacteria. In summary, this work demonstrates thatIBCcan be administered to Gram-negative bacteria in combination with 3-hydroxy-pyridin-4(1H)-ones as siderophores and provides a scientific basis for the development of antibacterial agents effective against Gram-negative bacteria.

  • Research Article

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    New antifungal triazoles with alkynylmethoxyl side chains: design, synthesis and evaluation of biological activity

    European Journal of Medicinal Chemistry, Band 257, 2023, Artikel 115506

    Previous work has led to the design, synthesis and rational testing of new triazole antifungal analogues with alkynylmethoxyl side chains. examination ofin vitroshowed antifungal activityCandida albicansSC5314 eCandida glabrata537 reported MIC values ​​of ≤0.125 µg/mL for most compounds. between these connections16,18, Em29demonstrated broad-spectrum antifungal activity against seven species of human pathogenic fungi, two of which were resistant to fluconazoleC. albicansisolated and two multiresistantwhite earsisolated. Additional 0.5 µg/ml16,18, Em29was more effective than 2 µg/ml of fluconazole in inhibiting the fungal growth of the tested strains. The most active connection (16) completely inhibited the growthC. albicansSC5314 at 16 µg/mL for 24 hours affected biofilm formation and disrupted the mature biofilm at 64 µg/mL. SeveralSaccharomyces cerevisiaeStrains overexpressing recombinant Cyp51s or drug efflux pumps are indicated16,18, Em29Cyp51 targeted without being significantly affected by frequent active site mutation, but was susceptible to overexpression and target efflux by MFS and ABC transporters. GC-MS analysis showed that16,18, Em29bothered themC. albicansErgosterol biosynthetic pathway by inhibition at Cyp51. Molecular docking studies have identified binding modes of18with Cyp51. The compounds showed low cytotoxicity, low hemolytic activity and favorable ADMT properties. important, calm16proved to be powerfulliveantifungal activity inG. mellonellainfection model. Overall, this study presents more potent triazole analogues with broad-spectrum activity and low toxicity, which may contribute to the development of new antifungal drugs and help overcome antifungal resistance.

  • Research Article

    Recent advances in small molecule drugs for the clinical treatment of multiple myeloma

    European Journal of Medicinal Chemistry, Band 257, 2023, Artikel 115492

    Multiple myeloma (MM) is a hematological malignancy of plasma cells currently considered incurable. Despite the introduction of new immunomodulators and proteasome inhibitors, MM remains a challenging disease with high rates of relapse and refractory disease. Management of patients with refractory and relapsing MM remains a daunting task, in large part due to the emergence of multidrug resistance. Therefore, there is an urgent need for new therapies to address this clinical challenge. In recent years, much research has been done to discover new therapies to treat MM. Clinical use of the proteasome inhibitor carfilzomib and the immunomodulator pomalidomide was gradually introduced. As basic research advances, new therapeutics including panobinostat, a histone deacetylase inhibitor, and selinexor, a nuclear export inhibitor, have entered testing and clinical applications. The aim of this review is to provide a comprehensive overview of the clinical applications and synthetic routes of selected drugs and provide valuable information for future research and drug development in relation to MM.

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