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Enzyme-Catalyzed Activation of Pro-PROTAC for Cell-Selective Protein Degradation

2022; Chinese Chemical Society; Volume: 4; Issue: 12 Linguagem: Inglês

10.31635/ccschem.022.202101529

2096-5745

Chunjing Liang, Qizhen Zheng, Tianli Luo, Weiqi Cai, Lanqun Mao, Ming Wang,

Peptidase Inhibition and Analysis

Open AccessCCS ChemistryRESEARCH ARTICLE7 Dec 2022Enzyme-Catalyzed Activation of Pro-PROTAC for Cell-Selective Protein Degradation Chunjing Liang, Qizhen Zheng, Tianli Luo, Weiqi Cai, Lanqun Mao and Ming Wang Chunjing Liang Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Qizhen Zheng Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Tianli Luo Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Weiqi Cai Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 , Lanqun Mao College of Chemistry, Beijing Normal University, Beijing 100875 and Ming Wang *Corresponding author: E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 University of Chinese Academy of Sciences, Beijing 100049 https://doi.org/10.31635/ccschem.022.202101529 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Proteolysis targeting chimera (PROTAC) technology is a chemical protein knockdown approach that degrades protein by hijacking the cellular ubiquitin-proteasome system to impede tumor growth. Its therapeutic potential, however, is difficult to define due to the lack of control over the cell selectivity of PROTACs, in particular, if the therapeutic purpose is to be executed in a specific cell type. Herein, we report the design of a Pro-PROTAC and its catalytic activation of the endogenous overexpressed enzyme in cancer cells for cell-selective protein degradation. We demonstrate that the chemical modification of the binding site between PROTAC and E3 ligase with trimethyl-locked quinone efficiently blocks the protein degradation capability of PROTAC. However, NAD(P)H quinone dehydrogenase 1 (NQO1), an enzyme overexpressed in cancer cells, could reduce the trimethyl-locked quinone to remove the chemical modification and activate NQO1-PROTAC for cancer cell-selective protein degradation. Further, we show that NQO1-catalyzed β-Lapachone reduction potentiated cellular oxidative stress to activate aryl boronic acid-caged ROS-PROTAC in living cells for bromodomain-containing protein 4 degradation with enhanced cell selectivity. Collectively, our strategy of designing Pro-PROTAC in response to endogenous species of diseased cells expands the chemical biology toolbox for cell-selective protein degradation and would be of great interest in targeted therapeutics discovery. Download figure Download PowerPoint Introduction Controlling protein degradation in a precise manner is of great importance for studying protein function in biological systems and modulating cell signaling for disease treatment.1–4 Proteolysis targeting chimera (PROTAC) technology is a chemical protein knockdown approach that degrades protein by hijacking the cellular ubiquitin-proteasome system.5,6 In general, a bivalent small-molecule degrader is designed to recruit E3 ubiquitin ligase to the protein of interest (POI), forming a stable ternary complex for proximity-induced ubiquitylation and proteasomal protein degradation.7–11 Compared with the traditional inhibition strategy that consistently occupies the active sites of POI, PROTAC can degrade protein in a sub-stoichiometric manner12 and target disease-causing proteins considered intractable. 12–15 Despite the great success of designing PROTAC for inducing protein degradation,16–19 this technology offers less control or spatiotemporal regulation of protein degradation. Therefore, a systematic application of PROTACs might affect cells and tissues in a non-selective manner, resulting in potential toxicity and compromised clinical translation potency. In this regard, Pro-PROTAC that switches its activity under the control of endogenous signal is highly appreciated for conditional protein degradation and targeted disease treatment. Cell-selective protein degradation could be achieved with Pro-PROTACs that stay "inert" until "activation" by the characteristic profile of the microenvironment of diseased cells, such as the chemical signal or enzyme upregulated in malignant cells. Recent studies have shown the effectiveness of controlling Pro-PROTAC activity using external stimuli20–25; however, the potency of designing Pro-PROTAC in response to the endogenous environment of malignant cells for cell-selective protein degradation remains unknown, despite the high demand. Recently, we have reported that conjugating protein with chemical moieties that are responsive to the intracellular environment of diseased cells could regulate protein activity with cell selectivity.26–28 Herein, we sought to further develop a conditional protein degradation strategy by integrating enzyme-responsive chemistry with a PROTAC degrader (Pro-PROTAC) to switch its activity in a cell-selective manner. To this end, we designed the chemical modification ("caging") of PROTAC degrader, comprising a Von Hippel–Lindau (VHL, a cell division regulator protein) E3 ligase recruiter to control its binding with E3 ligase, with a subsequent PROTAC activity (Figure 1a). It has been reported that the hydroxyproline group of the VHL ligand is essential for recruiting E3 ligase for PROTAC.12,29,30 Therefore, the caging of the hydroxyproline group of VHL E3 ligase recruiter with an enzyme-removable moiety switches off PROTAC activity. However, the Pro-PROTAC could restore its activity once exposed to the intracellular environment, specifically removing the caging chemistry (Figure 1b). As a proof-of-concept study of a successful design of enzyme-activated Pro-PROTAC, we first designed Pro-PROTAC with trimethyl-locked quinone conjugation that could be reduced and removed by NAD(P)H quinone dehydrogenase 1 (NQO1), an enzyme overexpressed in cancer cells31,32 (Figure 1c). We found that NQO1-PROTAC was effective in targeted protein degradation, depending on the intracellular expression level of NQO1, and it preferentially degraded the protein of cancer cells. Further, we demonstrated that by designing NQO1-catalyzed futile reduction of β-Lapachone (β-Lap), a substrate for NQO1 and also a small-molecule anticancer drug could enhance intracellular reactive oxygen species (ROS) level in cancer cells33,34 and activate aryl boronic ester-modified, ROS-responsive ROS-PROTAC for cancer cell-selective protein degradation (Figure 1d). The enzyme-activated Pro-PROTAC is not only effective for selective degradation of Halo-tagged reporter protein,35 but also for degrading endogenous bromodomain-containing protein 4 (BRD4), underscoring its versatility and potential for cell-selective protein degradation, as well as targeted therapeutics discovery. Figure 1 | Rational design of chemically caged PROTAC degraders and their enzymatic activation for cell-selective protein degradation. (a) The design of caged PROTAC degrader to control its binding with E3 ligase. (b) The caged PROTAC restores its activity when exposed to an intracellular environment. (c) The uncaging reaction of enzyme-activated directly Pro-PROTAC. (d) The uncaging reaction of enzyme-activated indirectly Pro-PROTAC. Download figure Download PowerPoint Experimental Methods General All reagents used for chemical synthesis were purchased from 3A chemicals (Beijing, China) and Innochem Technology Co., Ltd. (Beijing, China) and used without further purification. 1H NMR spectra were recorded at room temperature on an AVANCE III 400 HD spectrometer (Swiss Brock, Fällanden, Switzerland). Flow cytometry was performed using Beckman Coulter CytoFLEX (Beckman, Los Angeles, CA, United States). NQO1 was purchased from Abcam (Boston, MA, United States). HeLa and HEK293T cells were obtained from the National Infrastructure of Cell Line Resources (Beijing, China). HeLa-HaloGFP and HEK293T-HaloGFP cells, stably expressing the self-labeling haloalkane dehalogenase tag (Halo-tagged) green fluorescent protein (HaloGFP) have been described in the literature.35 Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Beijing, China) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Life Technologies, New York, NY, United States) at 37 °C in a humidified atmosphere of 5% CO2. Synthesis of Pro-PROTAC PROTAC molecules (1 equiv) and 4-dimethylaminopyridine (DMAP; 5 equiv) were dissolved in dry dichloromethane (DCM), followed by the addition of N,N-Diisopropylethylamine (DIPEA; 10 equiv). The mixture was stirred at 0 °C, then a solution of the caging group anhydrous dichloromethane (DCM; 1.2 equiv) was added dropwise at 0 °C. The mixture was stirred at room temperature for 12 h and then purified by flash chromatography on a silica gel column using DCM/MeOH (20/1) as the eluent. The structures of Pro-PROTACs were verified by 1H NMR and mass spectra. NQO1-triggered HaloPROTAC release from NQO1-HaloPROTAC To demonstrate NQO1 could trigger HaloPROTAC release from NQO1-HaloPROTAC, NQO1-HaloPROTAC (200 μM) was incubated with NQO1 (20 ng/mL) along with nicotinamide adenine dinucleotide (NADH) (0.5 mM) in Dulbecco's Phosphate Buffered Saline (DPBS) at 37 °C for 20 h. At the end of the incubation, the reaction mixture was purified using Amicon Ultra Centrifugal Filters (MWCO 3 kDa; Darmstadt, Germany). The purified filtrate was extracted with DCM, and the combined organic layer was dried using Na2SO4, then concentrated under reduced pressure. The sample was analyzed using electron spray ionization mass spectrometry (ESI-MS). ROS-triggered HaloPROTAC release from ROS-HaloPROTAC To demonstrate that ROS could trigger the release of HaloPROTAC from ROS-HaloPROTAC, ROS-HaloPROTAC (1 mM) was incubated with H2O2 (2 mM) in MeOH for 4 h. At the end of the incubation, the reaction mixture was analyzed using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. To assess the efficiency of the reaction, the reaction mixture of 1 mM ROS-HaloPROTAC and H2O2 (2 mM) dissolved in MeOH were monitored at 37 °C by high-performance liquid chromatography (HPLC; ACQUITYArc System, Waters, Shanghai, China). Cell viability assays Cells were cultured and sub-seeded in 48-well plates at a density of 25,000 cells per well the day before the experiment. Then cells were treated with Pro-PROTAC at varying concentrations, ranging from 0.01 to 20 mM, and incubated for 24 h. Cell viability was determined using Alamar Blue assay and compared with untreated cells. Protein degradation using Pro-PROTAC Cells were cultured and sub-seeded in a 48-well plate at a density of 25,000 cells per well the day before the experiment. Then the cells were treated with Pro-PROTAC at different concentrations, using 1% (DMSO) as co-solvent. After 24 h treatment, the cells were harvested to quantify the HaloGFP and BRD4 expression profile using flow cytometric analysis and western blot assay, respectively. siRNA knockdown of NQO1 HeLa-HaloGFP cells were seeded at a density of 100,000 cells per well in a 6-well plate 24 h prior to siRNA delivery. On the day of the experiment, siNQO1 (antisense-strand: 5′-CCGUACACAGAUACCUUGA-3′) or (160 or 240 pmol) or scrambled siRNA (antisense-strand: 5′-GAACUUCAGGGUCAGCUUG-3′) (siNC, 160 pmol) and ROS-TK-11 (9.6 μg) were mixed with 200 μL sodium acetate buffer (25 mM, pH 5.2) and incubated for 15 min before adding to the cells. The final concentration of siRNA added to cells was 80 or 120 nM. After 58 h of treatment, the cells were harvested for western blot assay to quantify NQO1 expression. Results and Discussion Design and synthesis of NQO1-responsive HaloPROTAC NQO1 is very effective at catalyzing the two-electron reduction of quinones and is recognized as an important approach toward detoxification in mammalian systems.32 The altered expression of NQO1 is associated with the progression of many tumors; therefore, the conditional degradation of disease-causing proteins in response to NQO1 might have the potential for developing targeted cancer therapy. In this study, we first conjugated trimethyl-locked quinone to the hydroxyproline group of HaloPROTAC to impede the formation of a stable ternary complex between NQO1-HaloPROTAC and VHL E3 ligase and to switch off the activity of HaloPROTAC accordingly (Figures 1c and 2a). NQO1-HaloPROTAC was synthesized in a route as described in the Supporting Information. However, the treatment of NQO1-HaloPROTAC with NQO1 catalyzed quinone reduction and subsequent self-immolating cleavage of a trimethyl-locked quinone from NQO1-HaloPROTAC to release HaloPROTAC (Figure 2a). We found that incubation of NQO1-HaloPROTAC (0.2 mM) with NQO1 (20 ng/mL) in DPBS efficiently removed the quinone caging and released HaloPROTAC, confirmed by the ESI-MS analysis of NQO1-HaloPROTAC before and after NQO1 treatment (Figure 2b). Figure 2 | NQO1-triggered NQO1-HaloPROTAC activation. (a) Chemical principle of NQO1-triggered activation of NQO1-HaloPROTAC; (b) MS analysis of NQO1-HaloPROTAC before and after NQO1 treatment. NQO1-HaloPROTAC (0.2 mM) was incubated with NQO1 (20 ng/mL) along with NADH (0.5 mM) in DPBS for 6 h before mass spectrometry analysis. Download figure Download PowerPoint NQO1-HaloPROTAC was highly biocompatible for PROTAC study, evidenced by its low cytotoxicity during the growth inhibition assays of HeLa and 293T cells ( Supporting Information Figure S1). To evaluate the potential of NQO1-HaloPROTAC for enzyme-activated protein degradation, we genetically engineered HaloGFP that could be degraded by HaloPROTAC through covalent conjugation between HaloGFP and the chloroalkane-labeled HaloPROTAC. We found that the treatment of NQO1-upregulated HeLa cells stably expressing HaloGFP treated with NQO1-HaloPROTAC, which resulted in effective GFP depletion (Figures 3a–3c), showing dependency on the concentration of NQO1-HaloPROTAC added to the cells ( Supporting Information Figure S2). For example, the HaloGFP level of HeLa cells treated with NQO1-HaloPROTAC (1 μM) decreased to 47% of the untreated cells, comparable to that of uncaged HaloPROTAC treated cells. Importantly, 2-NP-HaloPROTAC, an NQO1 non-responsive PROTAC degrader in which the hydroxyproline was caged with a 2-nitrophenyl group, failed to show a similar HaloGFP degradation effect as NQO1-HaloPROTAC under the same experimental conditions. Collectively, these studies indicated the effectiveness of designing NQO1-responsive caging chemistry of Pro-PROTAC for enzyme-activated protein degradation. Figure 3 | NQO1-HaloPROTAC treatment efficiently degrades HaloGFP expression of HeLa cells. (a) Schematic diagram of target protein degradation with NQO1-HaloPROTAC. (b) Fluorescence imaging of HaloGFP-stably expressing HeLa cells without (W/O) and with (W/) NQO1-HaloPROTAC treatment (1 μM) for 24 h. scale bar: 20 μm. (c) The normalized HaloGFP expression of HeLa cells treated with 1 μM of different PROTAC degraders as indicated for 24 h. The data are presented as mean ± SD (n = 3). The statistical significance was calculated via one-way ANOVA, ****P < 0.0001. Download figure Download PowerPoint Intracellular NQO1 expression regulates NQO1-HaloPROTAC activity Next, we knocked down the NQO1 expression of HeLa-HaloGFP cells using RNA interference (RNAi) to verify the crucial role of intracellular NQO1 in activating NQO1-HaloPROTAC. To this end, the cells were treated with siNQO1 or scramble siRNA (siNC) using lipid nanoparticle transfection we developed very recently.36 We observed that siNQO1 (120 nM) treatment decreased NQO1 expression to 20% of non-treated or siNC-delivered cells ( Supporting Information Figure S3a). When HeLa-HaloGFP cells with siNQO1 transfection were treated further with NQO1-HaloPROTAC (1 μM), the HaloGFP degradation was significantly prohibited compared with that of HaloPROTAC treatment (Figure 4a). However, siNC transfection did not show an inhibition effect on HaloGFP degradation using NQO1-HaloPROTAC. Moreover, NQO1 silencing prohibited HaloGFP degradation at varying concentrations of NQO1-HaloPROTAC ( Supporting Information Figure S3b), confirming the determinative role of intracellular NQO1 level in activating NQO1-HaloPROTAC in situ to induce protein degradation. Figure 4 | The activity of NQO1-HaloPROTAC for protein degradation is controlled by intracellular NQO1 expression. (a) NQO1 knockdown of HeLa cells decreased the protein degradation efficiency of NQO1-HaloPROTAC. HeLa cells were transfected with 120 nM siNQO1 or scramble siRNA before NQO1-HaloPROTAC (1 μM) treatment. (b) Selective HaloGFP degradation using NQO1-HaloPROTAC. 293T and HeLa cells stably expressing HaloGFP were treated with 1 μM NQO1-HaloPROTAC for 16 h before GFP expression analysis. The data are presented as mean ± SD (n = 3), the statistical significance was calculated via one-way ANOVA, **P < 0.01, ****P < 0.0001. Download figure Download PowerPoint Further, we sought to study the potential of NQO1-HaloPROTAC for cell-selective protein degradation using cells that demonstrate variable NQO1 expression. Accordingly, we genetically engineered both HeLa cells and 293T cells to stably express HaloGFP and treated them with NQO1-HaloPROTAC under the same condition. Indeed, we found that with NQO1-HaloPROTAC (1 μM) treatment, HaloGFP of HeLa cell density decreased to ∼ 48% of the untreated cells (Figure 4b). However, NQO1-HaloPROTAC treatment only slightly degraded HaloGFP of 293T cells, indicating the enhanced protein degradation efficiency of NQO1-HaloPROTAC in cancer cells, making use of the overexpressed NQO1 in these cells to activate PROTAC selectively. Design of ROS-responsive PROTAC To improve cell selectivity of protein degradation through NQO1-regulated PROTAC, we further designed an NQO1-mediated cascade reaction to potentiate and differentiate the intracellular environment of cancer cells to activate Pro-PROTAC. Herein, we chemically caged HaloPROTAC with an aryl boronic ester and generated a ROS-responsive Pro-PROTAC, termed ROS-HaloPROTAC (Figure 5a). We established previously that the chemical modification of an essential residue in a protein (RNase A) with aryl boronic ester showed controlled protein activity in response to intracellular ROS.37 Therefore, we hypothesized that the caging of HaloPROTAC with aryl boronic ester could switch off PROTAC activity, while ROS-triggered biorthogonal aryl boronic ester cleavage could activate ROS-HaloPROTAC for protein degradation (Figure 5a). To this end, we designed NQO1-catalyzed reduction of β-Lap, which generated excessive ROS in living cells to activate ROS-HaloPROTAC (Figure 1d). Compared to NQO1-HaloPROTAC that simply relied on cellular NQO1 to remove trimethyl-locked quinone for Pro-PROTAC activation, NQO1-catalyzed β-Lap reduction differentiated and amplified intracellular ROS in cancer cells to activate ROS-HaloPROTAC with higher efficiency for cell-selective protein degradation. Figure 5 | Intracellular ROS-activated PROTAC for cell-selective protein degradation. (a) Chemical activation of ROS-HaloPROTAC by ROS to release HaloPROTAC. (b) HPLC analysis of ROS-triggered ROS-HaloPROTAC degradation. ROS-HaloPROTAC (1 mM) was incubated with 2 mM H2O2 at 37 °C at the indicated times before HPLC analysis. (c) PMA stimulation enhanced the protein degradation efficiency of ROS-HaloPROTAC. HeLaHaloGFP cells were treated with ROS-HaloPROTAC (1 μM) and PMA (2 μM) for 24 h before GFP expression analysis. (d) β-Lap pre-treatment significantly enhanced the protein degradation efficiency of ROS-HaloPROTAC in NQO1-overexpressing HeLa cells. HeLa-HaloGFP cells and 293T-HaloGFP cells were treated with ROS-HaloPROTAC (1 μM) for 24 h in the absence (W/O) or presence (W/) of β-Lap (1 μM) before GFP expression analysis. The data are presented as mean ± SD (n = 3), the statistical significance was calculated via one-way ANOVA, **P < 0.01, ****P < 0.0001. Download figure Download PowerPoint The ROS-responsive nature of ROS-HaloPROTAC was verified using ESI-MS and HPLC analysis. As shown in Supporting Information Figure S4, mass spectrometry analysis of the reaction mixture, ROS-HaloPROTAC (1 mM) and H2O2 (2 mM) showed MS peaks corresponding to HaloPROTAC. The HPLC analysis also indicated that with an increased incubation time of ROS-HaloPROTAC and H2O2, the percentage of HaloPROTAC in the reaction mixture increased gradually (Figure 5b and Supporting Information Figure S5). More than 85% of ROS-HaloPROTAC was converted to HaloPROTAC after 5 h of H2O2 treatment, confirming the effectiveness of using H2O2 to remove the chemical caging of ROS-HaloPROTAC. ROS-HaloPROTAC showed a minimal negative effect in prohibiting cell growth ( Supporting Information Figure S6); therefore, it was biocompatible for the cell-selective protein degradation study. To verify the caging effect of aryl boronic ester modification on the protein degradation capability of HaloPROTAC, both HeLa-HaloGFP and 293T-HaloGFP cells were treated with ROS-HaloPROTAC. Cellular GFP analysis indicated a very low efficiency of ROS-HaloPROTAC in degrading HaloGFP in both cell lines (Figure 5c and Supporting Information Figure S7), indicating relatively low efficiency of ROS-HaloPROTAC activation by endogenous ROS. However, when the cells were pre-incubated with phorbol 12-myristate-13-acetate (PMA; 2 μM), which stimulated the cells to generate ROS non-selectively,36,37 we observed an enhanced HaloGFP degradation in these cells, compared with a parallel experiment without PMA stimulation (Figure 5c and Supporting Information Figure S7). For example, the HaloGFP level of HeLa cells co-treated with PMA and ROS-HaloPROTAC was reduced to 68% of non-treated cells, confirming the potential of harnessing intracellularly generated ROS to activate ROS-HaloPROTAC for protein degradation. NQO1 and β-Lap co-treatment activated ROS-HaloPROTAC for cell-selective protein degradation Next, we studied whether NQO1-catalzyed β-Lap reduction could activate ROS-HaloPROTAC in living cells for cell-selective protein degradation. Thus, we simultaneously treated HeLa-HaloGFP or 293T-HaloGFP cells with ROS-HaloPROTAC (1 μM) along with β-Lap (1 μM), followed by GFP degradation quantification 24 h post-treatment. Indeed, the GFP intensity of HeLa-HaloGFP cells was reduced to ∼45% of non-treated cells in the presence of ROS-HaloPROTAC and β-Lap, while minimal GFP depletion was observed when the cells were treated with ROS-HaloPROTAC alone (Figure 5d). In comparison, when NQO1 low-expressing 293T-HaloGFP cells were treated similarly with ROS-HaloPROTAC and β-Lap, very low protein degradation efficiency was observed, and there was no significant difference between the HaloGFP degradation and the counterpart cells treated with ROS-HaloPROTAC alone (Figure 5d). Collectively, these studies demonstrated the effectiveness of designing an NQO1-catalyzed β-Lap reduction system to enhance intracellular ROS concentration in cancerous cells, thereby activating ROS-HaloPROTAC for cell-selective protein degradation. Cell-selective BRD4 degradation using ROS-BRD-PROTAC Finally, we sought to study the potential of using NQO1-activated Pro-PROTAC for selective degradation of endogenous protein in cancer cells. To this end, BRD4, a bromodomain and extra-terminal domain (BET) family member, which plays a pivotal role in modulating the expression of essential oncogenes in cancer cell progression, was selected as the target protein.38,39 ROS-BRD-PROTAC connected to JQ1,40 a BRD4-binding ligand with ROS-responsive VHL E3 ligase recruiter (Figure 6a), was designed for cell-selective BRD4 degradation. ESI-MS analysis indicated that ROS treatment removed the aryl boronic ester caging from ROS-BRD-PROTAC to release BRD-PROTAC ( Supporting Information Figure S8). A biochemical assay was performed by treating HeLa and 293T cells with ROS-BRD-PROTAC (1.5 μM) in the absence or presence of β-Lap (1 μM), followed by BRD4 expression assay using western blot analysis. We found that ROS-BRD-PROTAC treatment could barely degrade BRD4 in both cell lines ( Supporting Information Figure S9), consistent with previous findings that ROS-HaloPROTAC could not be activated by endogenous ROS to degrade HaloGFP (Figure 5c and Supporting Information Figure S7). However, ROS-BRD-PROTAC and β-Lap co-treatment of HeLa cells showed a 70% decrease of BRD4 level compared with the untreated cells; the BRD4 degradation efficiency was comparable to BRD-PROTAC without the chemical caging (Figure 6b). In sharp contrast, ROS-BRD-PROTAC showed a minimal BRD4 degradation effect on 293T cells both with and without β-Lap co-treatment (Figure 6c). Figure 6 | NQO1-activated ROS-BRD-PROTAC for cell-selective BRD4 degradation. (a) chemical structure and activation of ROS-BRD-PROTAC for BRD4 degradation; BRD4 expression of HeLa cells (b) and 293T cells (c) treated with ROS-BRD-PROTAC and β-Lap. The two different cell lines were treated with varying concentrations of ROS-BRD-PROTAC as indicated in the absence (W/O) or presence (W/) of β-Lap (1 μM) for 24 h. BRD4 expression was examined using western blot analysis. Download figure Download PowerPoint Therefore, the cancer cell-selective BRD4 degradation using ROS-BRD-PROTAC resulted in a selective antitumor effect. Cell proliferation monitoring of HeLa and 293T cells indicated that ROS-BRD-PROTAC and β-Lap co-treatment decreased the viability of HeLa cells to 40%, compared with the untreated cells, while the treatment of ROS-BRD-PROTAC or β-Lap alone did not show an obvious inhibitory effect on HeLa cell growth (Figure 7). However, ROS-BRD-PROTAC treatment had a negligible impact on inhibiting 293T cell growth in the absence and presence of β-Lap. Importantly, BRD-PROTAC did not show a distinctive effect in inhibiting HeLa and 293T cell growth, highlighting the effectiveness of NQO1-catalyzed β-Lap reduction and subsequent ROS-BRD-PROTAC activation to selectively degrade endogenous BRD4 in cancer cells for potential targeted cancer therapy. Figure 7 | ROS-BRD-PROTAC and β-Lap co-treatment selectively prohibited cancer cell growth. HeLa and 293T cells were co-treated with ROS-BRD-PROTAC (5 μM) and β-Lap (0.7 μM) for 60 h before cell viability measurement using Alamar Blue assay. W/O means without β-Lap co-treatment, W/ means treatment with β-Lap. Download figure Download PowerPoint In principle, we developed Pro-PROTAC that combine VHL ligands with endogenous enzyme-activated caging group and ligands to target either HaloGFP or BRD protein. The modularity of this strategy could be extended to other endogenous target proteins of interest, such as Bruton's tyrosine kinase (BTK), cyclin-dependent kinase 9 (CDK9), the receptor tyrosine kinase MET, and proteins associated with neurodegenerative diseases. In addition, the chemical approach to design enzyme-activated Pro-PROTAC could be applied to molecules derived from thalidomide, the ligands of cereblon (CRBN protein). CRBN, like VHL, is the substrate receptor of two cullin-RING ubiquitin ligase complexes. This provides the possibility for the controlled degradation of various proteins in diseased cells. Moreover, due to the overexpression of a large variety of enzymes or other small molecules, such as glutathione (GSH), in diseased cells, we believe that this strategy of controlling PROTAC activity using endogenous materials overexpressed in diseased cells could expand the chemical biology toolbox for cell-selective protein degradation, and should be of great interest for targeted drug discovery. Conclusion We reported in this study a general chemical approach to design enzyme-activated Pro-PROTAC for cell-selective protein degradation. We have demonstrated that the chemical caging of PROTAC degrader, with enzyme-responsive features, efficiently blocked its capability to recruit E3 ligase for protein degradation, while enzyme-promoted chemical decaging activated Pro-PROTAC in a cell-selective manner. Our approach is effective not only for the selective degradation of the fluorescent reporter protein (HaloGFP) but also for the control of endogenous protein (BRD4). Due to the overexpression of a large variety of enzyme in diseased cells, we believe this strategy of controlling PROTAC activity using endogenous enzyme overexpressed in diseased cells could expand the chemical biology toolbox for cell-selective protein degradation and be of great interest for targeted drug discovery. Supporting Information Supporting Information is available and includes details of the synthesis of PROTACs, the study of NQO1-HaloPROTAC and ROS-HaloPROTAC for protein degradation, mass spectrometry, HPLC characterization of Pro-PROTAC activation by ROS, and cell-selective BRD4 degradation following ROS-BRD-PROTAC treatment. Conflict of Interest There is no conflict of interest to report. Funding Information This research was made possible as a result of a generous grant from the National Key R&D Program of China (grant nos. 2017YFA0208100 to M.W., 2018YFE0200800 to L.M.) and the National Science Foundation of China (grant nos. 21778056 and 22077125 to M.W., 21790390, 21790391, and 22134002 to L.M.). Acknowledgments M.W. and L.M. acknowledge the financial support from the National Key Research and Development Program of China (grant nos. 2017YFA0208100 to M.W. and 2018YFE0200800 to L.M.), the National Science Foundation of China (grant nos. 21778056 and 22077125 to M.W. and 21790390, 21790391, and 22134002 to L.M.). References 1. Brand M.; Jiang B.; Bauer S.; Donovan K. A.; Liang Y.; Wang E. S.; Nowak R. P.; Yuan J. C.; Zhang T.; Kwiatkowski N.Homolog-Selective Degradation as a Strategy to Probe the Function of Cdk6 in AML.Cell Chem. Biol.2019, 26, 300–306. Google Scholar 2. Chamberlain P. P.; Hamann L. G.Development of Targeted Protein Degradation Therapeutics.Nat. 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Google Scholar Previous articleNext article FiguresReferencesRelatedDetails Issue AssignmentVolume 4Issue 12Page: 3809-3819Supporting Information Copyright & Permissions© 2022 Chinese Chemical SocietyKeywordsenzymatic activationcell-selective protein degradationproteolysis targeting chimeraAcknowledgmentsM.W. and L.M. acknowledge the financial support from the National Key Research and Development Program of China (grant nos. 2017YFA0208100 to M.W. and 2018YFE0200800 to L.M.), the National Science Foundation of China (grant nos. 21778056 and 22077125 to M.W. and 21790390, 21790391, and 22134002 to L.M.). Downloaded 2,547 times PDF downloadLoading ...