Non-invasive in vivo monitoring of PROTAC-mediated protein degradation using an environment-sensitive reporter

Li, Tao; Zong, Qingyu; Dong, He; Ullah, Ihsan; Pan, Zhenhai; Yuan, Youyong

doi:10.1038/s41467-025-57191-0

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Published: 22 February 2025

Non-invasive in vivo monitoring of PROTAC-mediated protein degradation using an environment-sensitive reporter

Tao Li¹^na1,
Qingyu Zong¹^na1,
He Dong¹,
Ihsan Ullah¹,
Zhenhai Pan¹ &
…
Youyong Yuan ORCID: orcid.org/0000-0003-2784-3780^1,2,3

Nature Communications volume 16, Article number: 1892 (2025) Cite this article

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Abstract

Proteolysis targeting chimeras (PROTACs) represent a groundbreaking therapeutic technology for selectively degrading proteins of interest (POIs). The structural variations in PROTACs unpredictably influence their protein degradation efficiency, which is predominantly assessed by quantifying POIs abundance through western blotting. This approach, however, falls short of enabling non-invasive monitoring of protein degradation within living cells let alone assessing directly the degradation effects in vivo. Herein, we develop an environment-sensitive reporter (ESR) for the quantification of protein degradation events triggered by PROTACs in vivo. By simultaneously integrating POIs targeting ligand and an environment-sensitive fluorophore, the ESR signals exhibit a strong fluorescence correlation with the levels of POIs. This non-invasive monitoring reporter offers a high-throughput and convenient way to screen POIs targeting degraders and predict PROTACs-mediated therapeutic outcomes in mouse models. These properties suggest the potential of ESR strategy as a general modular scheme for non-invasive quantification of protein degradation of cancer-related therapeutic targets.

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Introduction

Proteins are the fundamental building blocks in living organisms, regulating normal metabolism and signal communication within cells by interacting with various biomolecules¹. Interestingly, proteolysis targeting chimeras (PROTACs) have emerged as a groundbreaking therapeutic technology to selectively target proteins of interest (POIs) involved in disease pathogenesis for elimination, especially challenging transcriptional factors^2,3. To date, many PROTACs targeting key oncogenic proteins have progressed to clinical trials for cancer therapy, including ARV-110 (targeting the androgen receptor) and ARV-471 (targeting the estrogen receptor)^4,5. The structural variations in PROTACs unpredictably influence their protein degradation efficiency, which is predominantly assessed by quantifying POIs abundance through western blotting (WB)^6,7. This approach lacks the ability to provide non-invasive monitoring of protein degradation within living cells. Furthermore, the discrepancy in PROTACs in vitro and in vivo protein degradation effects can pose challenges to PROTACs-mediated cancer therapy optimization.

Molecular fluorescence imaging technology offers unique spatiotemporal resolution and visualization merits in the following aspects: (a) detection of disease-specific biological targets; (b) early prediction of therapeutic outcomes; (c) high-throughput screening of therapeutic agents^{8,9,10,11,12,13}. Recently, considerable efforts have focused on labeling POIs to elucidate protein degradation using fluorescence imaging technology. For this purpose, genetically encoded fluorescent proteins (FPs) provide a non-invasive strategy to track POIs localization and quantification through the development of fusion proteins^14,15,16. However, FP-based functional probes face challenges such as limited visible wavelength range, low brightness, poor photostability. Moreover, due to the continuous expression of the FP-fused POI, FP-based probes are difficult to accurately detect protein level changes during degradation processes¹⁷. Alternatively, commercial functional molecules specifically label POIs pretreated with genetically encodable protein tags, such as SNAP-tag, HaloTag, PYP-tag, CLIP-tag, FAP, eDHFR/TMP-tag, and BL-tag^{17,18,19,20,21,22,23,24,25,26,27,28,29}. These genetic technologies are valuable for monitoring intracellular parameters, but they are challenging to use and can give false positive results because of residual labeling probes. Promega corporation have developed the HiBit protein labeling system for monitoring changes in endogenous protein levels³⁰. However, a prerequisite for the HiBiT system is a CRISPR/Cas9 engineered cell line whose generation can take a considerable amount of time. In addition, fluorescent and theragnostic PROTACs have been developed for non-invasive monitoring of protein degradation of POIs, effective only for specific proteins with fluorescent ligands³¹. Unfortunately, the above strategies are still limited to the cellular level, and achieving non-invasive imaging of protein degradation in vivo remains challenging.

Here, we develop an environment-sensitive reporter (ESR) for the non-invasive quantification of the protein degradation of PROTACs in vivo, screening POIs targeting degraders, and predicting the outcomes of PROTACs-based cancer therapies. ESR is a heterobifunctional molecule composed of three key elements: a POIs targeting ligand, an environment-sensitive fluorophore and a short linker facilitating their connection (Fig. 1a). In polar aqueous environments, ESR molecules rotate freely, releasing the energy of the excited state through non-radiative transitions, resulting in weak fluorescence signals. However, when bound to POIs under the guidance of POIs targeting ligand, the twisting motion of ESR molecules is restricted within the nonpolar hydrophobic binding pocket of the POIs, reducing non-radiative transitions and significantly enhancing fluorescence signals (Fig. 1b). This mechanism provides a precise approach for quantifying POIs expression. By simultaneously integrating POIs targeting ligand and environment-sensitive fluorophore, ESR signals exhibit a strong fluorescence correlation with POIs levels. To validate the effectiveness and generality of our approach conceptually, we synthesize two reporters (JQ1-NR and ML-NR) based on an environment-sensitive fluorophore Nile Red skeleton for non-invasive quantification of the expression of cancer-related therapeutic proteins (BRD4 and GPX4). Moreover, the ESR strategy simplifies and expedites the screening of POIs degraders. By leveraging the ESR strategy, we explore the correlation mechanism between the therapeutic efficacy of PROTACs in vivo and the fluorescence variation of ESR using POIs levels as a medium (Fig. 1c). These properties position the ESR strategy as a versatile modular framework for non-invasive monitoring of a diverse range of cancer-related therapeutic proteins by fluorescent imaging.

**Fig. 1: Design and mechanism of ESR for the non-invasive quantification of PROTACs- mediated protein degradation in vivo.**

Results

Design and characterization of ESR

To verify the feasibility of our strategy, we initially measured the spectral responses of various fluorophores in different solvents based on relevant literatures to investigate their sensitivity to environmental changes, including Methylene Blue (MB), Fluorescein (FL), Rhodamine B (RB) and Nile Red (NR) (Fig. 2a)^{32,33,34,35,36}. The solvent polarities were quantified using the Lippert-Mataga polarity parameter Δƒ (Fig. 2b, Supplementary Table 1)^37,38. The optical properties of MB, FL, and RB in various solvents are random, except for NR, whose fluorescence intensity decreases with increasing solvent polarity. We observed a good correlation (r = −0.8515, P = 0.0073) between the fluorescence intensity of NR and solvent polarity (Fig. 2c, d). The alterations in environmental factors also involve acidic-basic status (pH). We tested the spectral responses of the above fluorophores under different pH conditions (Supplementary Fig. 1). The optical properties of FL are greatly influenced by pH (r = 0.9882, P < 0.0001), while NR remains almost unchanged (r = 0.0791, P = 0.8661). These results demonstrated that NR can specifically sense changes in environmental polarity without being influenced by pH interference, making it an ideal choice for the development of ESR systems.

The bromodomain and extra-terminal (BET) family proteins, including BRD2, BRD3, BRD4, and BRDT, play a crucial role as epigenetic readers in cellular biology³⁹. JQ1 (+) is the primary ligand with high affinity for all BET family proteins⁴⁰. Among these, BRD4 is recognized as a promising antitumor target due to its crucial role in gene transcription⁴¹. Furthermore, chemical modifications to the tert-butyloxycarbonyl moiety of JQ1 (+) minimally affect binding within the BRD4 cavity^41,42. Considering these factors, we have selected BRD4 as the model protein for our investigation. We chemically linked JQ1 (+) to the NR skeleton to develop a series of environment-sensitive reporters (JQ1-n-NR, n = 1, 2, 3) by regulating the length of the linker (Fig. 2e, Supplementary Fig. 2). Using JQ1-1-NR as an example, molecular docking simulation was performed to explore the binding mechanism of JQ1-1-NR with BRD4 protein (PDB code: 3MXF) (Supplementary Fig. 3). The result indicated that JQ1-1-NR could enter the nonpolar hydrophobic binding pocket of the BRD4 protein and formed the hydrogen bond with Q85 in the surface groove. Subsequently, to further validate the binding of JQ1-1-NR to BRD4 protein, we conducted the cellular thermal shift assay (CETSA). As illustrated in Fig. 2f, we observed that the BRD4 protein degraded as the temperature increased in the DMSO control group. In contrast, both JQ1 (+) and JQ1-1-NR effectively stabilized the BRD4 protein in 4T1 cell lysates under the same temperature conditions. Notably, like the control group, the free Nile red fluorophore failed to stabilize the BRD4 protein. These results indicated that the JQ1-1-NR can bind to the BRD4 protein under the guidance of JQ1 (+) moiety, whereas the free Nile red fluorophore does not interact with BRD4 protein. In addition, we evaluated the fluorescent changes of environment-sensitive reporters (JQ1-n-NR, n = 1, 2, 3) upon binding to the BRD4 protein. The fluorescence ratio of JQ1-1-NR gradually increased and tended to saturate with increasing concentrations of BRD4 protein (0–12 μM). In contrast, the fluorescence ratios of JQ1-2-NR and JQ1-3-NR increased slowly (Fig. 2g, h). This inconsistency can be attributed to the elongation of the linkers, which causes the NR fluorophore in the probe to move away from the nonpolar hydrophobic binding pocket of the target protein, resulting in reducing twist restriction and decreasing fluorescence of the probe. Therefore, we chose JQ1-1-NR and referred to it as JQ1-NR in subsequent research.

ESR quantifies PROTACs-mediated protein degradation in vitro

To quantify the expression of BRD4 protein during the degradation process, we initially synthesized a previously reported BET protein degrader based on relevant references, termed JV8 (Fig. 3a, Supplementary Fig. 4)^43,44,45. 4T1 cells were exposed to varying concentrations of JV8 (0–100 nM) for 24 h to measure degradation effectiveness using WB. BRD4 levels in both total cells and cytoplasm decreased in dose-dependent manner, with minimal degradation of BRD4 in the nucleus even at the highest test concentration (Fig. 3b, c). Similarly, BRD4 protein degradation followed a time-dependent manner at a constant JV8 concentration (Supplementary Fig. 5a, b). Then we explored the protein degradation mechanism of JV8 using the proteasome inhibitor MG132. This inhibitor effectively blocks the protein hydrolysis activity of the 26S proteasome complex, affecting intracellular protein degradation⁴⁶. The addition of MG132 hindered BRD4 protein degradation, verifying the degradation mechanism of JV8 controlled by the ubiquitin-proteasome system (Fig. 3d). Immunofluorescent staining further confirmed that JV8 preferentially degraded BRD4 in the cytoplasm (Fig. 3e), consistent with the WB findings. In addition, the BRD4 degrader JV8 induced a greater degree of apoptosis than protein ligand JQ1(+) (Fig. 3f).

Furthermore, the uptake equilibrium of JQ1-NR in living cells was explored by flow cytometry analysis (Fig. 3g, h; Supplementary Fig. 5c and Fig. 22). The saturation concentration and optimal incubation time of JQ1-NR were 2 μM and 1 h, respectively. To determine whether JQ1-NR could be used for non-invasive quantification of BRD4 expression, we incubated 4T1 cells with different groups, respectively (Fig. 3i). The presence of BET protein ligand JQ1 (+) resulted in stronger JQ1-NR fluorescent signals compared to free NR fluorophore. Pretreating 4T1 cells with JV8 remarkably decreased the JQ1-NR signals, similar to free NR fluorophore, attributed to BRD4 protein degradation triggered by JV8. We further validated that the JQ1-NR signals in the cytoplasm of living cells decreased in a dose-dependent and time-dependent manner upon the addition of JV8, as observed through confocal laser scanning microscopy (CLSM) (Fig. 3j, k and Supplementary Fig. 5d, e). To explore the correlation between BRD4 protein levels in different cellular regions and JQ1-NR signals, we quantified the JQ1-NR signals of whole cells using flow cytometry (Fig. 3l and Supplementary Fig. 5f) and performed linear analysis with the BRD4 protein quantification results from WB in various cellular regions (Fig. 3c and Supplementary Fig. 5b), respectively. Both concentration gradient and time gradient, a plot of fractional fluorescence as a function of relative BRD4 expression in the cytoplasm presented better positive correlation (r_c = 0.9948, R²_c = 0.9897, P_c < 0.0001; r_t = 0.9845, R²_t = 0.9692, P_t = 0.0004) compared to nuclear charts (r_c = 0.9299, R²_c = 0.8646, P_c = 0.0072; r_t = 0.9203, R²_t = 0.8469, P_t = 0.0093) (Fig. 3m–o and Supplementary Fig. 5g–i). These results demonstrated the potential of the JQ1-NR signal as an alternative strategy to western blotting for non-invasive quantification of BRD4 degradation contents controlled by JV8.

Simple and rapid screening of PROTACs achieved by ESR in vitro

To explore whether JQ1-NR could be applied to screen effective BRD4 protein degraders, we have used JV8 as a reference to synthesize a series of BRD4-PROTACs by regulating the length of the linker and ligand of E3 ligase (Fig. 4a–c and Supplementary Figs. 6, 7). By monitoring the JQ1-NR signals in cells pretreated with various BRD4-PROTACs using flow cytometry, we analyzed the BRD4 protein degradation of PROTACs library in six cell lines, including 4T1, KPC, GL261, B16-OVA, Hepa 1–6 and CT26 cells (Fig. 4d). The heat map created from non-invasive fluorescence quantification illustrated the heterogeneous efficacy of different protein degraders within the same cell line, as well as the variability in effectiveness of the same protein degrader across different cell lines (Fig. 4e). These results may stem from variations in linker length of PROTACs. An excessively short linker may sterically hinder the simultaneous binding of the target protein and E3 ligase to the PROTACs, thereby preventing the formation of ternary complexes. Conversely, an excessively long linker may inadequately position the E3 ligase and target protein for effective ubiquitination. Importantly, any observed differences are contingent upon the specific E3 ligase engaged. Subsequently, the JQ1-NR fluorescence signals in cells pretreated with various BRD4-PROTACs were observed more intuitively through fluorescence imaging (Fig. 4f). To verify the accuracy of the above-mentioned screening results, we characterized the degradation capacity of BRD4-PROTACs in 4T1 cells by WB (Fig. 4g, h). Under the same conditions, JV4 and JV8 induced complete degradation of BRD4 protein and JC2 triggered partial degradation. This is consistent with the fluorescent quantitation analysis in 4T1 cells (Fig. 4d). Furthermore, a strong positive correlation was observed between JQ1-NR signals and degradation trends triggered by BRD4-PROTACs (r = 0.9702, P < 0.0001) (Fig. 4i and Supplementary Fig. 8). Thus, our ESR strategy could simplify and accelerate the screening of POIs degraders.

**Fig. 4: Simple and rapid screening of various PROTACs achieved by ESR in vitro.**

ESR quantifies PROTACs-mediated protein degradation in vivo

The mice bearing 4T1 tumors were pretreated with BRD4-PROTACs (10 mg/kg) via intraperitoneal (i.p.) injection and allowed to act for 24 h (Fig. 5a). For in vivo quantification of BRD4 protein degradation, the mice were intratumorally injected with JQ1-NR (20 mg/kg), followed by quantitative imaging at 4 h post-injection (Fig. 5b) and resected the tumor for ex vivo fluorescence imaging (Fig. 5c). The results showed a significant reduction in JQ1-NR signals for the JC2, JV4 and JV8 groups, indicating BRD4 protein degradation in these tumors (Fig. 5d). This analysis was validated by examining the levels of BRD4 protein in these groups via western blotting (Fig. 5e, f). Complete BRD4 protein degradation was observed for JV8, while partial degradation was noted for JC2 and JV4, consistent with the trend of cell experiment results. A good correlation (r = 0.8558, P = 0.0032) was found when analyzing the relationship between the JQ1-NR signals and the BRD4 levels in various treatment groups (Fig. 5g and Supplementary Fig. 9). Furthermore, the addition of JV8 remarkably decreased the JQ1-NR signals in various tumor models, such as KPC pancreatic carcinoma, Hepa 1–6 hepatocellular carcinoma, 4T1 breast carcinoma, CT26 colorectal carcinoma and HeLa cervical carcinoma models. These findings collectively demonstrated that POIs-labelled reporter JQ1-NR could non-invasive monitoring protein degradation triggered by BRD4-PROTACs through in vivo fluorescence imaging.

**Fig. 5: ESR quantifies PROTAC-mediated protein degradation in vivo.**

ESR predicts the therapeutic efficacy of PROTACs in vivo

Evaluating the therapeutic efficacy of PROTACs in living organisms typically requires completing the entire treatment cycle, which takes several weeks. Herein, we developed an environment-sensitive reporter for non-invasive quantification of protein degradation events triggered by PROTACs in vivo. The mice bearing 4T1 tumors were randomly divided into three groups and treated with different doses of JV8 (0/5/10 mg/kg) via i.p consecutively five times, respectively (Fig. 6a). A dose-dependent tumor suppression effect was observed (Fig. 6b, h) after monitoring tumor volume every other day for 15 days. Meanwhile, tumors were excised and weighted in various groups at 14 days post treatment (Fig. 6c, e). Tumor images and weights also revealed that high-dose PROTACs exhibited better therapeutic efficacy. There were no significant weight loss effects (Fig. 6d). Isothermal titration calorimetric (ITC) analysis was then conducted to investigate the affinities of JQ1-NR and JV8 for the BRD4 protein. As shown in Supplementary Fig. 21, the binding affinities were determined to be K_d = 0.65 μM for JV8, K_d = 0.15 μM for JQ1-NR, respectively. JQ1-NR has a slightly stronger binding affinity for the BRD4 protein compared to JV8, as indicated by its lower K_d value. Compared to JQ1 (+), although JQ1-NR exhibits a 2- to 3-fold weaker binding affinity to the BRD4 protein, it still demonstrates a significantly higher binding affinity than the stereoisomer JQ1 (-), which shows no significant interaction with any bromodomain³⁹. Considering these factors, we further evaluated the potential of using POIs-labelled reporter for early prediction of the therapeutic efficacy of RROTACs, mice were intratumorally injected with JQ1-NR prior to the first JV8 administration and after the second JV8 administration, followed by quantitative imaging at 4 h post-injection, respectively (Fig. 6f). The ΔFL (F₀-F₃) intensity of mice exposed to high doses of JV8 was 16.5-fold higher than those exposed to PBS (Fig. 6g). The variation in ΔFL intensity, as well as the corresponding tumor volume variation, were plotted for each mouse in each group (Fig. 6i and Supplementary Fig. 10a). A negative correlation was observed between tumor volume variation and ΔFL intensity (r = −0.9688, P < 0.0001), indicating that higher ΔFL intensity in tumors represented a smaller relative tumor volume in mice at 14 days post-treatment. Further validating the above conclusion, western blotting was performed to quantify BRD4 levels in tumor tissue receiving various JV8 doses at 14 days post-treatment (Fig. 6j, k). The BRD4 levels of mice exposed to PBS were approximately 4.11-fold higher than those exposed to high doses of JV8. Plotting ΔFL intensity against relative tumor volume and BRD4 levels revealed close linear relationships among the three factors (Fig. 6l and Supplementary Fig. 10a–c). Relative BRD4 expression had a negative correlation to the ΔFL intensity (r = −0.9979, P < 0.0001), while a positive correlation to the relative tumor volume (r = 0.9829, P < 0.0001). The tumor tissue slices also showed similar results (Supplementary Fig. 11). Collectively, the fluorescence signal of POIs-labelled reporter is a suitable indicator for early predicting the therapeutic efficacy of PROTACs in vivo.

The generality of ESR strategy toward GPX4

To validate the generality of our strategy for non-invasive quantification of protein degradation, we selected Glutathione peroxidase 4 (GPX4) protein, a core regulator of ferroptosis, as the target for our experiments^47,48. ML-162 is a covalent inhibitor of cellular GPX4⁴⁹. Similarly, we modified ML162 onto the NR skeleton through a chemical linker to prepare an environment-sensitive reporter ML-NR (Fig. 7a and Supplementary Fig. 12). Molecular docking simulation was conducted to investigate the binding mechanism of ML-NR with GPX4 (PDB code: 2OBI) (Supplementary Fig. 13). The result indicated that ML-NR could enter the apolar hydrophobic binding pocket of GPX4 protein and the GPX4-targeting ligand ML162 moiety formed the hydrogen bonds with W136 and K48 in the surface groove. To further validate the binding of ML-NR to GPX4 protein, we conducted the CETSA (Supplementary Fig. 14). Both the GPX4-targeting ligand ML162 and ML-NR were found to enhance the thermal stability of the GPX4 protein, while the presence of Nile red alone did not demonstrate a significant change compared to the DMSO control group. These results that ML-NR is capable of binding to the GPX4 protein. The uptake equilibrium of ML-NR in cells was determined by flow cytometry analysis to be 2 μM and 4 h (Fig. 7c and Supplementary Fig. 15), respectively. We found that ML-NR incubated 4T1 cells displayed approximately 9.6-fold higher fluorescent signal than that of free NR (Fig. 7d). To explore whether ML-NR could be applied to screen the effective GPX4 protein degraders, we synthesized several GPX4-tagrgeting PROTACs by regulating the length of the linker and ligand of E3 ligase based on previous study⁵⁰ (Fig. 7a, b and Supplementary Fig. 16). We monitoring the ML-NR signals in six cell lines treated with various PROTACs, including 4T1, KPC, GL261, B16-OVA, Hepa 1–6, and CT26 cells (Fig. 7e). According the screening results, the lowest fluorescence signal was observed in all cell lines with MC8. The heat map illustrated the heterogeneous efficacy of the same protein degrader across different cell lines (Fig. 7f). Subsequently, through fluorescence imaging, we more intuitively observed the variation of the ML-NR signals in various cell lines pretreated with GPX4-PROTACs (Fig. 7g). To verify the reliability of the screening results, the degradation capacity of GPX4-related PROTACs in 4T1 cells was assessed by western blotting (Fig. 7h, i). Under the same conditions, MC8 induced complete degradation of GPX4 protein, as observed in the above fluorescent analysis. A strong positive correlation was found between the ML-NR signals variation trend and degradation trends caused by GPX4-PROTACs (r = 0.9935, P < 0.0001) (Fig. 7j and Supplementary Fig. 17), supporting that ML-NR could function as a reliable tool for screening GPX4 protein degraders. With ML-NR reporter, we further explored the quantification of GPX4 degradation triggered by MC8 in living cells. The ML-NR signals showed a dose-dependent decrease along with the addition of MC8 (Fig. 7k). Meanwhile, the GPX4 protein levels of 4T1 cells treated with MC8 were analyzed by WB (Fig. 7l, m). A plot depicting fractional fluorescence of ML-NR as a function of GPX4 levels showed a strong positive correlation (r = 0.9922, P < 0.0001) (Fig. 7n), demonstrating that the ML-NR signals effectively quantified the GPX4 protein levels in living cells following treatment with GPX4-PROTACs.

The mice bearing 4T1 tumors were pretreated with GPX4-PROTACs (10 mg/kg) via i.p. injection and allowed to act for 24 h. For in vivo quantification of GPX4 protein degradation, the mice were intratumorally injected with ML-NR (20 mg/kg), followed by quantitative imaging at 6 h. We observed a significant reduction of the ML-NR signals in the MC8 group compared to the other four groups (Fig. 7p and Supplementary Fig. 18). WB analysis also confirmed almost complete GPX4 degradation in the MC8 group (Fig. 7q and Supplementary Fig. 19). Further analysis revealed a strong positive correlation between the ML-NR signaling variation trend and GPX4 protein levels across groups (Fig. 7r and Supplementary Fig. 20) (r = 0.9657, P = 0.008). Therefore, ML-NR can be a valuable indicator for noninvasive quantification of GPX4 protein degradation in vivo.

Discussion

Visualization of protein degradation not only contributes to elucidating the mechanisms and regulatory processes of protein degradation but also aids in predicting the therapeutic efficacy of POIs-related degraders. The PROTACs-mediated protein degradation is generally evaluated by western blotting. However, this approach falls short of enabling non-invasive dynamic detection of protein degradation within living cells let alone evaluating therapeutic effects in vivo. Early prediction of the therapeutic efficacy of PROTACs-mediated protein degradation would be crucial for guiding personalized patient treatment. Recently, great efforts have focused on labeling POIs for tracing the protein degradation process using fluorescence imaging technology. This includes the use of genetically encoded fluorescent proteins or protein tags/chemical probes to perform different functions. While these strategies have proven valuable for monitoring intracellular parameters, genetic technologies face several issues, including complexity in operation and the nonspecific background caused by residual labeling probes. In addition, fluorescent and theragnostic PROTACs have been developed for monitoring of protein degradation of POIs, effective only for specific proteins with fluorescent ligands. There remains a need to develop general strategies for non-invasive quantification of POIs to enable early prediction of PROTACs therapeutic efficacy in vivo. To tackle the challenge, we simultaneously incorporated both POIs targeting ligand and environment-sensitive fluorophore via a chemical linker to design an environment-sensitive reporter (ESR). The ESR produced fluorescence signals within the binding POIs, thereby lighting up the POIs in living cells without any sample processing.

We chose BRD4 as the model protein to demonstrate the utility of the ESR strategy. We successfully validated the feasibility of using the JQ1-NR signals as an alternative strategy to western blotting for the non-invasive quantification of protein degradation of BRD4-related PROTACs, showing a good correlation of the JQ1-NR signals with BRD4 protein levels. Such non-invasive profiling capacity of JQ1-NR was also applied to screen highly effective degraders from a series of PROTACs. More importantly, quantifying the intracellular POIs levels at the early stage of PROTACs-mediated tumor therapy predicted the therapeutic outcomes at the endpoints, wherein POIs levels had a negative correlation to the ΔFL intensity, while a positive correlation to the relative tumor volume. To show the generality of the ESR strategy, as a core regulator of ferroptosis, GPX4 protein was chosen as a target to build a corresponding reporter ML-NR. ML-NR provides a non-invasive way to accurately map out GPX4 levels. Moreover, we have also demonstrated the potential of ML-NR for screening and evaluating the protein degradation efficacy of GPX4-related PROTACs in animal models.

In summary, an environment-sensitive reporter (ESR) strategy was developed for non-invasively quantifying PROTACs-mediated protein degradation in living cells and in vivo. By incorporating POIs targeting ligands, ESR signals activation shows a strong fluorescence correlation with POIs levels. ESR-based fluorescence imaging offers a convenient and high-throughput method for identifying POIs targeting degraders and early prediction of PROTAC-mediated therapeutic outcomes. We anticipate that the ESR strategy can be expanded to develop protein degraders for a wide range of cancer-related therapeutic targets by following a modular design. Notably, any technique using a small-molecule POI binder to recognize a protein will be heavily reliant on the selectivity of the POI binder. Despite the application of our approach being constrained to proteins with known ligands, we believe it still holds significant value in the visualization of protein degradation. Our approach offers a way to non-invasively monitor PROTAC-mediated protein degradation in living cells and in vivo. We are confident that this strategy can provide insights and tools for protein function studies and the development of protein degraders. Off-target effects and intrinsic “hook effect” hinder the therapeutic behavior of PROTACs. At present, we are working on utilizing molecular imaging technology to monitor on-demand release of PROTACs and evaluation of protein degradation efficiency for spatiotemporally controllable protein degradation.

Methods

We confirm that our research complies with all relevant ethical regulations of the South China University of Technology. All animal protocols were approved by the South China University of Technology Animal Care and Use Committee (Approved number: AEC2023066) and were performed following the guidelines for the use of laboratory animals. If the animal starts showing signs of immobility, a huddled posture, the inability to eat, ruffled fur, or self-mutilation, the animal will be euthanized immediately. The subcutaneous tumor maximum diameter was 20 mm and authorized by the Committees on Animal Research and Ethics and was not exceeded at any time during the experiments.

Materials

Unless specifically stated, all chemicals were purchased from commercial suppliers and used as received without further purification. JQ1 carboxylic acid, VHL, and CRBN ligands were purchased from Bide (Shanghai, China). 3-Hydroxy-N, N-diethylaniline, 1,6-dihydroxynaphthalene, 3-(Boc-amino) propyl bromide, 5-(t-Boc-amino)-1-pentyl bromide, tert-Butyl (7-bromoheptyl)carbamate, 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecan-16-oic acid, Boc-NH-PEG₁-CH₂COOH, N-(tert-Butoxycarbonyl)glycine, Boc-NH-PEG₂-CH₂COOH, N-BOC-ethylenediamine, N-(tert-butoxycarbonyl)-1,4-butanediamine, tert-butyl [2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate, 1,1-dimethylethyl 13-amino-5,8,11-trioxa-2-azatridecanoate, 4-amino-2-chlorophenol, di-tert-butyl decarbonate, 2-thiophene-carboxaldehyde, 2-chloroacetic acid, azidoacetic acid, 11-azido-3,6,9-trioxaundecan-1-amine were purchased from Energy chemical (shanghai, China). Human BRD4 protein was purchased from Dima Biotech (Wuhan, China). Dulbecco’s modified eagle medium (DMEM), trypsin-EDTA, and penicillin-streptomycin were purchased from Gibco BRL (Eggenstein, Germany). Fetal bovine serum (FBS) was obtained from ExCell Biology, Inc (Shanghai, China). Hoechst 33342 was obtained from Life Technologies. Alexa Fluor®488 anti-mouse antibody was acquired from Biolegend (San Diego, CA, USA). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was acquired from Sigma-Aldrich. NE-PER nuclear and cytoplasmic extraction reagents were purchased from Thermo Scientific.

Instruments

Fluorescence spectra were recorded on a F-7100 spectrofluorometer (HITACHI). ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance III 400 MHz magnet with auto-tunable BBFO probe (5 mm). The MS spectra were recorded on Agilent 1260. The fluorescent properties of JQ1-n-NR were conducted on Tecan Infinite M200PRO multifunctional microplate reader. Fluorescence imaging of cells was acquired on a Laser Scanning Microscope LSM880 (Zeiss). Flow cytometry assay was performed on a Fortessa X20 (BD Biosciences).The binding affinities were conducted by Isothermal titration calorimetric (Nano ITC). In vivo animal fluorescence images were taken via an In Vivo Imaging System (IVIS-CT machine, PerkinElmer).

Material synthesis

The synthesis protocols are provided in Supplementary Information.

Environment-dependent optical properties

Methylene Blue (MB), Fluorescein (FL), Rhodamine B (RB), Nile Red (NR), were dissolved in DMSO to produce a 2 mM stock solution, respectively. The samples (2 μM) for testing were placed in a 2 mL cuvette. PMT voltage was 660 V. The slit width was 5 nm.

The binding experiment of JQ1-n-NR (n = 1, 2, 3) to BRD4 Protein

The JQ1-n-NR (10 μM) was incubated with BRD4 protein (0–12 μM) in PBS buffers (10 mM, pH 7.4) at 37 °C for 1 h. Then, the fluorescent properties of JQ1-n-NR were conducted on Tecan Infinite M200PRO multifunctional microplate reader (E_x/E_m = 500/620 nm).

Molecular docking simulation

To investigate the binding mode between JQ1-NR and BRD4, as well as ML-NR and GPX4, molecular docking simulations were performed using Autodock Vina 1.1.2. The 2D structure of JQ1-NR was drawn using ChemBioDraw Ultra 14.0 and converted to a 3D structure using ChemBio3D Ultra 14.0. The 3D structure of BRD4 (PDB ID:3MXF) or GPX4 (PDB ID:2OBI) was downloaded from the RCSB Protein Data Bank (https://www.rcsb.org/). Based on the docking scores obtained from Vina, the docking position with the highest score was selected for visualization analysis using PyMOL 1.7.6 software.

Cellular thermal shift assay (CETSA)

Cellular thermal shift assay was performed to monitor the target engagement of JQ1-NR in 4T1 cell lysates. Briefly, cell lysates from 4T1 cells were collected, diluted in PBS and separated into aliquots. Each aliquot was treated with NR, JQ1, and JQ1-NR (10 μM) or DMSO after incubation at room temperature for 30 min. The compound-treated lysates were heated individually at different temperatures (40 to 65 °C). The heated lysates were centrifuged and the supernatants were analyzed by SDS-PAGE followed by immunoblotting analysis with the indicated antibody: BRD4 (E8V7I) Rabbit mAb (1:800, Cell signaling technology); Recombinant Anti-beta Actin antibody (1:2000, Servicebio); Goat Anti-Rabbit IgG antibody (1:8000, Biosharp).

The operation method for ML-NR is the same. The heated lysates were centrifuged and the supernatants were analyzed by SDS-PAGE followed by immunoblotting analysis with the indicated antibody: GPX4 Monoclonal antibody (1:1000, Proteintech); Recombinant Anti-beta Actin antibody (1:2000, Servicebio); Goat Anti-Rabbit IgG antibody (1:8000, Biosharp) and Goat Anti-Mouse IgG antibody (1:8000, Biosharp).

Isothermal titration calorimetry (ITC)

ITC was used to characterize the binding affinity of JQ1-NR to BRD4 (BD1 and BD2) protein. The experiments were performed on a Nano ITC (TA, American). Both interaction partners were dissolved in PBS. In routine experiments, JQ1-NR (100 μM) was titrated against BRD4 protein (10 μM), consisting of 25 injections of 2 μL JQ1-NR solution at 200 s time intervals. The experiment was carried out at 25 °C while stirring at 300 rpm. Dilution heats associated with JQ1-NR addition into the buffer (determined in separate control experiments) had small constant values that were negligible to measured binding heats. The data was analyzed by NanoAnalyze^TM Software. The operation method for JV8 to BRD4 (BD1 and BD2) is the same.

Cell and animal culture

4T1 breast carcinoma, CT26 colorectal carcinoma, Hepa 1–6 hepatocellular carcinoma, KPC pancreatic carcinoma, B16-OVA melanoma, and GL261 brain glioma cells were obtained from the American Type Culture Collection (ATCC). The cells were maintained in a complete medium (RPMI 1640 or DMEM with 10% FBS and 1% antibiotics) in a 5% CO₂-containing humidified atmosphere at 37 °C.

Female BALB/c nude mice (6 weeks old), and female BALB/c mice (6 weeks old) were purchased from Hunan SJA Laboratory Animal Co., Ltd (Hunan, China). Animals were housed at approximately 22 ± 2 degrees centigrade; humidity 50 ± 10% on a 12 h light/12 h dark cycle. Experimental group sizes were approved by animal welfare regulatory authorities after being defined to balance statistical power, feasibility, and ethical aspects. The maximal tumor burden permitted is 2,000 mm³, and the maximal tumor size was not exceeded in the experiments. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the South China University of Technology Animal Care and Use Committee (Approved number: AEC2023066).

Cellular fluorescence analysis

4T1, KPC, GL261, B16-OVA, Hepa 1–6, and CT26 cells (1 × 10⁵ cells per well) were cultured on 24-well plates for 12 h, then treated with various BRD4-PROTACs (100 nM) or GPX4-PROTACs (0.8 μM) for 24 h. Then the cells were incubated with JQ1-NR (2 μM, 1 h) or ML-NR (2 μM, 4 h). Finally, cells were washed with PBS three times, collected, and analyzed by flow cytometry (PE channel).

4T1 cells (2 × 10⁵ cells per well) were cultured on glass coverslips for 12 h. Cells were then treated with JV8 at the indicated concentrations or times. Then cells were incubated with JQ1-NR (2 μM) for 1 h. After washing with PBS three times, cells were fixed with 4% formaldehyde. The nuclei were stained with Hoechst 33342 for 15 min and then visualized using CLSM. The 2.5D images were obtained through image processing using ZEN software.

Different cells lines (1 × 10⁴ cells per well) were cultured on 96-well black culture plates. Upon incubation with various BRD4-PROTACs (100 nM) or GPX4-PROTACs (0.8 μM) and JV8 or MC8 at the indicated concentrations or times, the cells were incubated with JQ1-NR (2 μM, 1 h) or ML-NR (2 μM, 4 h). After incubation, the cells were washed with PBS three times, and fluorescence images were immediately acquired using an IVIS spectrum imaging system (E_x/E_m = 500/620 nm). The fluorescence intensities in the cells were quantified by applying region of interest (ROI) over the images, using Living Image Software.

Western blotting analysis

4T1 (2 × 10⁶) cells were treated with various BRD4-PROTACs (100 nM) or JV8 at the indicated concentrations or times. For adherent cells, harvest with trypsin-EDTA and centrifuge at 500 g for 5 min. Wash cells by suspending cell pellets in PBS. Transfer 2 × 10⁶ cells to a 1.5 mL microcentrifuge tube and pellet by centrifugation at 500 g for 3 min. Use a pipette to carefully remove and discard the supernatant, leaving the cell pellets as dry as possible. Add ice-cold CER I to the cell pellet (200 μL). Vortex the tube vigorously on the highest setting for 15 seconds to fully suspend the cell pellet. Incubate the tube on ice for 10 min. Add ice-cold CER II to the tube (11 μL). Vortex the tube for 5 s on the highest setting. Incubate the tube on ice for 1 min. Vortex the tube for 5 s on the highest setting. Centrifuge the tube for 5 minutes at maximum speed in a microcentrifuge (16,000 g). Immediately transfer the supernatant (cytoplasmic extract) to a clean pre-chilled tube. Suspend the insoluble (pellet) fraction produced, which contains nuclei, in ice-cold NER (100 μL). Vortex on the highest setting for 15 s. Place the sample on ice and continue vertexing for 15 seconds every 10 minutes, for a total of 40 min. Centrifuge the tube at maximum speed (~16,000 g) in a microcentrifuge for 10 min. Immediately transfer the supernatant (nuclear extract) fraction to a clean pre-chilled tube. Place on ice until use. SDS-PAGE (10%) electrophoresis was used to separate the cellular proteins (30 μg). After transferring onto nitrocellulose filter membranes (Millipore, U.S.A.). Bovine serum albumin solution (4%) was applied to block nonspecific binding sites. The samples were then treated by the monoclonal antibody against GAPDH (1:5,000, Servicebio), Lamin B (1:5,000, Biosharp), BRD4 (1:800, Cell signaling technology), followed by horse radish peroxidase (HRP) conjugated goat anti-rabbit IgG antibody (1:8,000, Biosharp). An enhanced chemiluminescence (ECL) system (Thermo, U.S.A.) was used to detect signals.

4T1 (2 × 10⁶) cells were treated with various GPX4-PROTACs (0.8 μM) or MC8 at the desired concentrations or times. Then the cells were washed three times in PBS, 50 μL of lysis buffer supplemented with Roche’s Complete Protease Inhibitor Cocktail Tablets was used to treat the cells. SDS-PAGE (12%) electrophoresis was used to separate cellular proteins (30 μg). After transferring onto nitrocellulose filter membranes (Millipore, U.S.A.). Bovine serum albumin solution (4%) was applied to block nonspecific binding sites. The samples were then treated with monoclonal antibody against GAPDH (1:2000, Servicebio), GPX4 (1:1000, Proteintech), followed by horse radish peroxidase (HRP) conjugated goat anti-rabbit IgG antibody (1:8000, Biosharp) and goat anti-mouse IgG antibody (1:8000, Biosharp). An enhanced chemiluminescence (ECL) system (Thermo, U.S.A.) was used to detect signals.

Fluorescence imaging of orthotopic breast tumors in mice

4T1 cells (1 × 10⁶) were injected into the right mammary fat pads to establish an orthotopic 4T1 tumor model. After the tumor volume reached 200 mm³, the tumor-bearing mice were randomly divided into nine or five groups (n = 3), intraperitoneally injected with various BRD4-PROTACs (10 mg kg⁻¹) or GPX4-PROTACs (10 mg kg⁻¹) for 24 h. All the mice were imaged at 4 or 6 h after intratumorally injection of JQ1-NR (20 mg kg⁻¹) or GPX4-NR (20 mg kg⁻¹), using an in vivo spectrum imaging system (E_x/E_m = 500/620 nm) to collect body fluorescence images.

In vivo imaging of different subcutaneous tumor models

The subcutaneous tumor models, including KPC pancreatic carcinoma, Hepa 1–6 hepatocellular carcinoma, 4T1 breast carcinoma, and CT26 colorectal carcinoma, were developed with BALB/c mice. The Hela cervical carcinoma was developed with BALB/c nude mice. One month after implantation (tumor volume reached ~100–150 mm³), tumor-bearing mice were used for imaging studies. All the tumor-bearing mice were first intratumorally injected with JQ1-NR (20 mg kg⁻¹, 4 h) for fluorescence imaging. Subsequently, they were intraperitoneally injected with JV8 (10 mg kg⁻¹) for 24 h. After that, JQ1-NR (20 mg kg⁻¹, 4 h) was injected intratumorally again for fluorescence imaging with an in vivo spectrum imaging system (E_x/E_m = 500/620 nm).

Tumor growth inhibition in vivo

To evaluate tumor suppression efficiency, BALB/c mice bearing 4T1 tumors were randomized into three groups (n = 5), intraperitoneal administered PBS, Low (JV8: 5 mg kg⁻¹), and High (JV8: 10 mg kg⁻¹), respectively, and the tumor volume and body weight of each mouse were measured and recorded during treatment. Mice received these treatments every other day, five times in all. Using this formula to calculate the volume of the tumor: V = L × W × W/2 (L, the longest dimension; W, the shortest dimension). Relative tumor volume was calculated as V/V₀, and V₀ was the tumor volume on the first day before treatment. On day 14, the mice were killed and the excised tumors were photographed and weighed.

All the tumor-bearing mice were intratumorally injected with JQ1-NR prior to the first administration and after the second administration of JV8, followed by quantitative imaging at 4 h post-injection, respectively.

Two hundred milligrams of fresh tumor tissues from different groups were homogenized and proteins were extracted for following Western blot analysis. SDS-PAGE (10%) electrophoresis was used to separate cellular proteins (30 μg). After transferring onto nitrocellulose filter membranes (Millipore, U.S.A.). Bovine serum albumin solution (4%) was applied to block nonspecific binding sites. The samples were then treated with monoclonal antibody against GAPDH (1:5,000, Biosharp), and BRD4 (1:1,000, Biosharp), followed by horse radish peroxidase (HRP) conjugated goat anti-rabbit IgG antibody (1:5,000, Biosharp). An enhanced chemiluminescence (ECL) system (Thermo, U.S.A.) was used to detect signals.

Immunohistochemical analysis

The tumors were harvested, 4% paraformaldehyde fixed, paraffin embedded, sliced, and stained with hematoxylin and eosin (H&E). Tumor sections were also stained with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) apoptosis detection kit assay and observed by CLSM.

The tumor-bearing mice were injected with PBS, Low (JV8: 5 mg kg⁻¹) and High (JV8: 10 mg kg⁻¹) 2 times, JQ1-NR (20 mg kg⁻¹) was intratumorally injected for in vivo imaging on day 3. After 4 h, the tumors were harvested, frozen in liquid nitrogen, embedded in optimum-cutting temperature (OCT) tissue compound (Sakura, Tokyo, Japan), and sectioned into 7 μm slices, stained with Hoechst 33342 and observed by CLSM.

The BRD4 levels of the tumor were evaluated by CLSM. The tumor-bearing mice were injected with PBS, Low (JV8: 5 mg kg⁻¹) and High (JV8: 10 mg kg⁻¹) 2 times. Then tumors were harvested, frozen in liquid nitrogen, embedded in optimum-cutting temperature (OCT) tissue compound (Sakura, Tokyo, Japan), and sectioned into 7 μm slices, then blocked with 5% BSA-PBS for 2 h at room temperature. Tumor slices were incubated with a BRD4 monoclonal antibody in 0.1% BSA-PBS at a dilution of 1:300 for 2 h at room temperature. Then tumor slices were washed with PBS and incubated with Alexa Fluor®488-conjugated secondary antibody in 0.1% BSA-PBS at a dilution of 1:500 for 1 h at room temperature in the dark. The nuclei were marked with Hoechst 33342 for 30 min and then imaged by CLSM.

Statistical analysis

All data were expressed as mean ± standard deviation. Statistical differences among experimental groups were analyzed by Student’s t-test (two-tailed). P < 0.05 was considered statistically significant. All statistical calculations were performed using the GraphPad Prism software package (PRISM 9.0, GraphPad Prism Software).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data generated in this study are available within the Article, Supplementary Information, Source Data file, and from corresponding author(s) upon request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Key R&D Program of China (No.2022YFB3804700 Y.Y.), the National Natural Science Foundation of China (Nos. 52373135 Y.Y., 52073101 Y.Y.), Guangdong Provincial Pearl River Talents Program (No. 2019QN01Y088 Y.Y.).

Author information

These authors contributed equally: Tao Li, Qingyu Zong.

Authors and Affiliations

School of Biomedical Sciences and Engineering, Guangzhou International Campus, South China University of Technology, Guangzhou, PR China
Tao Li, Qingyu Zong, He Dong, Ihsan Ullah, Zhenhai Pan & Youyong Yuan
National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou, PR China
Youyong Yuan
Key Laboratory of Biomedical Engineering of Guangdong Province, South China University of Technology, Guangzhou, PR China
Youyong Yuan

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Tao Li
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Qingyu Zong
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He Dong
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Contributions

Y.Y. was responsible for the conception and design strategy of the study. T.L. synthesized all the small molecular compounds and conducted the relevant fluorescence tests. T.L., Q.Z., and H.D. performed the biological experiments and acquired the data. Z.P. provided molecular docking simulation. T.L., Q.Z., I. U., and Y.Y. wrote the manuscript.

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Correspondence to Youyong Yuan.

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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). https://doi.org/10.1038/s41467-025-57191-0

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Received: 05 June 2024
Accepted: 14 February 2025
Published: 22 February 2025
DOI: https://doi.org/10.1038/s41467-025-57191-0