Proteolysis targeting chimeras (PROTACs) represent a breakthrough therapeutic technology for the selective degradation of proteins of interest (POIs). Structural variations in PROTACs unpredictably affect their protein degradation efficiency, which is primarily assessed by quantifying POI abundance via western blotting. However, this method fails to enable non-invasive monitoring of protein degradation in living cells, let alone directly evaluate degradation efficacy in vivo. To address this, Li et al. introduced and developed an environment-sensitive reporter (ESR) for quantifying protein degradation events triggered by PROTACs in vivo. By simultaneously integrating a POI-targeting ligand and an environment-sensitive fluorophore, the ESR signal exhibits a strong fluorescence correlation with POI levels. This non-invasive monitoring reporter provides a high-throughput and convenient method for screening POIs for targeted degraders and predicting therapeutic outcomes mediated by PROTACs in mouse models. The ESR strategy holds potential as a versatile modular protocol for non-invasive quantification of protein degradation at cancer-relevant therapeutic targets, offering a theoretical foundation and new concepts for effective future cancer treatment and detection.

ESR Design and Mechanism for PROTAC-Mediated Protein Degradation

The ESR is a heterobifunctional molecule comprising three key elements: a POI-targeting ligand, an environment-sensitive fluorophore, and a short linker connecting the two (Figure 1a). In a polar aqueous environment, the ESR molecule rotates freely, releasing excited state energy through non-radiative transitions, resulting in weak fluorescence. However, upon binding to the POI guided by the targeting ligand, the torsional motion of the ESR molecule becomes restricted within the non-polar hydrophobic binding pocket of the POI. This reduces non-radiative transitions and significantly enhances the fluorescence signal (Figure 1b). This mechanism provides a precise method for quantifying POI expression. By integrating the POI-targeting ligand and the environment-sensitive fluorophore, the ESR signal demonstrates a strong fluorescence correlation with POI levels. To validate the method's efficacy and generalizability, the study synthesized two reporters, JQ1-NR and ML-NR, based on the environment-sensitive fluorophore Nile Red scaffold, for non-invasive quantification of cancer-relevant therapeutic proteins, BRD4 and GPX4. Furthermore, the ESR strategy simplifies and accelerates the screening of POI degraders. Utilizing this strategy, the study explored the correlation mechanism between the therapeutic efficacy of PROTACs in vivo and changes in ESR fluorescence, using POI levels as a mediator (Figure 1c). These characteristics position the ESR strategy as a versatile modular framework for non-invasive monitoring of various cancer-relevant therapeutic proteins via fluorescence imaging.

Figure 1: ESR design and mechanism for non-invasive quantification of PROTAC-mediated protein degradation in vivo.

ESR Design and Characterization

To validate the feasibility of the strategy, this study initially measured the spectral responses of various fluorophores, including Methylene Blue (MB), Fluorescein (FL), Rhodamine B (RB), and Nile Red (NR), in different solvents to assess their sensitivity to environmental changes (Figure 2a). Solvent polarity was quantified using the Lippert-Mataga polarity parameter Δf (Figure 2b). The optical properties of MB, FL, and RB were random across solvents. However, the fluorescence intensity of NR decreased with increasing solvent polarity, showing a strong correlation (r = -0.8515, P = 0.0073) between NR fluorescence and solvent polarity (Figure 2c, d). Environmental factors also include pH; NR specifically senses changes in environmental polarity without interference from pH effects, making it an ideal choice for developing an ESR system.

Figure 2: Design and characterization of the ESR

To quantify BRD4 protein expression during degradation, a previously reported BET protein degrader, designated JV8, was initially synthesized (Figure 3a). 4T1 cells were exposed to varying concentrations of JV8 (0-100 nM) for 24 hours, and degradation efficacy was measured using western blotting. Both total cellular and cytoplasmic BRD4 levels decreased in a dose-dependent manner, with minimal degradation observed in the nucleus even at the highest tested concentration (Figure 3b, c). Subsequently, the proteasome inhibitor MG132 was employed to explore the protein degradation mechanism of JV8. This inhibitor effectively blocks the proteolytic activity of the 26S proteasome complex, affecting intracellular protein degradation. The addition of MG132 hindered BRD4 protein degradation, confirming that JV8 operates via the ubiquitin-proteasome system (Figure 3d). Immunofluorescence staining further confirmed that JV8 preferentially degrades BRD4 in the cytoplasm (Figure 3e), consistent with the western blotting results. Moreover, the BRD4 degrader JV8 induced a greater extent of apoptosis compared to the protein ligand JQ1(+) (Figure 3f).

Figure 3: Quantification of PROTAC-mediated protein degradation in vitro using the ESR.

Uptake equilibrium of JQ1-NR in living cells was explored via flow cytometry analysis (Figure 4a, b). The saturation concentration for JQ1-NR was 2 μM, with an optimal incubation time of 1 hour. To determine whether JQ1-NR could be used for non-invasive quantification of BRD4 expression, 4T1 cells were incubated under different conditions (Figure 4c). The presence of the BET protein ligand JQ1(+) resulted in a stronger JQ1-NR fluorescence signal compared to the free NR fluorophore. Pretreatment of 4T1 cells with JV8 significantly reduced the JQ1-NR signal, similar to the free NR fluorophore, due to JV8-induced BRD4 protein degradation. The study further confirmed, via confocal laser scanning microscopy (CLSM), that JQ1-NR signal in the cytoplasm of living cells decreased in a dose- and time-dependent manner upon JV8 addition (Figure 4d, e). To investigate the correlation between BRD4 protein levels in different cellular regions and the JQ1-NR signal, the JQ1-NR signal in whole cells was quantified using flow cytometry (Figure 4f) and linearly analyzed against BRD4 protein quantification results from western blotting of respective cellular fractions (Figure 3c). Across concentration and time gradients, the relationship curve between fractional fluorescence and relative BRD4 expression in the cytoplasm showed a strong positive correlation (Figure 4g-i). These results indicate that the JQ1-NR signal has the potential to serve as an alternative strategy to western blotting for non-invasive quantification of BRD4 degradation mediated by JV8.

Figure 4: Validation of quantification for PROTAC-mediated protein degradation.

Quantification of PROTAC-Mediated Protein Degradation In Vivo Using the ESR

Mice bearing 4T1 tumors were administered BRD4-PROTACs (10 mg/kg) intraperitoneally and allowed to act for 24 hours (Figure 5a). To quantify BRD4 protein degradation in vivo, JQ1-NR (20 mg/kg) was injected intratumorally, and quantitative imaging was performed 4 hours post-injection (Figure 5b). Tumors were then excised for ex vivo fluorescence imaging (Figure 5c). Results showed significantly reduced JQ1-NR signals in the JC2, JV4, and JV8 groups, indicating BRD4 protein degradation within these tumors (Figure 5d). This analysis was validated by detecting BRD4 protein levels in these groups via western blotting (Figure 5e, f). BRD4 protein was completely degraded in the JV8 group and partially degraded in the JC2 and JV4 groups, consistent with trends observed in cellular experiments. Analysis of the relationship between JQ1-NR signal and BRD4 levels across treatment groups revealed a good correlation (r = 0.8558, P = 0.0032) (Figure 5g). JV8 addition significantly reduced JQ1-NR signals in various tumor models, including KPC pancreatic cancer, Hepa1-6 hepatocellular carcinoma, 4T1 breast cancer, CT26 colorectal cancer, and HeLa cervical cancer. These results collectively demonstrate that the polarity-sensitive reporter JQ1-NR can non-invasively monitor protein degradation triggered by BRD4-PROTACs via in vivo fluorescence imaging.

Figure 5: Quantification of PROTAC-mediated protein degradation in vivo using the ESR.

This study developed an environment-sensitive reporter (ESR) strategy for non-invasive quantification of PROTAC-mediated protein degradation in living cells and in vivo. By incorporating a POI-targeting ligand, the activated ESR signal exhibits a strong fluorescence correlation with POI levels. ESR-based fluorescence imaging provides a convenient and high-throughput method for identifying POIs for targeted degraders and for early prediction of therapeutic outcomes mediated by PROTACs. It is anticipated that the ESR strategy can be extended through modular design to develop protein degraders for a wide range of cancer-relevant therapeutic targets. This strategy offers significant insights and tools for studying protein function and developing protein degraders.

Tek Biotech (Tianjin) Co., Ltd. has established a comprehensive PROTAC technology platform. We are dedicated to providing high-quality proteolysis targeting chimera development services to scientists worldwide. Our one-stop services include study design, linker selection, ligand selection, PROTAC synthesis, and downstream in vitro cellular validation and in vivo animal validation (including in vivo animal imaging). By staying at the forefront of technological advances, we aim to develop innovative therapeutic tools targeting热门 disease targets and provide robust support for our clients' research projects.

References

[1] Li, T., Zong, Q., Dong, H. et al. Non-invasive in vivo monitoring of PROTAC-mediated protein degradation using an environment-sensitive reporter. Nat Commun 16, 1892 (2025).