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Rapid and high-throughput screening of proteolysis targeting chimeras using a dual-reporter system expressing fluorescence protein and luciferase

BMC Biology volume 23, Article number: 51 (2025) Cite this article

Abstract

Background

Proteolysis targeting chimera (PROTAC), a novel drug discovery strategy, utilizes the ubiquitin–proteasome system to degrade target proteins in cells. While Western blotting, mass spectrometry, and Lumit Immunoassay have been instrumental in determining protein levels, the rapid screening of PROTACs continues to pose challenges, necessitating the development of alternative methodologies.

Results

We herein reported an alternative high-throughput method for screening PROTACs using a dual-reporter system expressing a Renilla luciferase (RLUC)-fused target protein and enhanced green fluorescent protein (EGFP). EGFP served as an internal reference and RLUC as an indicated target protein degradation. Rapid measurement of EGFP or RLUC light signals was achieved using a fluorescence/luminescence plate-based reader in the endpoint mode. The feasibility of the screening model was tested using ARV110, a clinical trial-stage PROTAC targeting the androgen receptor (AR). In EGFP/RLUC-tAR-expressing modal cells treated with varying concentrations of ARV110, normalized RLUC luminescence decreased dose-dependently, as confirmed via western blotting detection of AR expression. Then the platform was used to practically screen Sirtuin 2 (SIRT2) degraders from a small group of PROTACs that we built. Normalized RLUC luminescence changes in model cells expressing EGFP/RLUC-SIRT2 reflected the degradation efficiencies of PROTACs. Compounds 128 and 129 exhibited the highest degradation efficacies, leading to dose-dependent degradation of endogenous SIRT2 protein in the MCF-7 cell line and inducing cell growth arrest.

Conclusions

The dual-reporter system using both fluorescence and chemiluminescence was successfully constructed. Using this method, we identified effective candidate PROTACs against SIRT2. The dual-reporter system may accelerate drug discovery during PROTAC development.

Background

Proteolytic targeting chimeras (PROTAC) are an emerging approach in drug research and development. They address targets that are beyond the regulation and reach of small molecules and antibodies, respectively, by converting them into forms susceptible to degradation in the ubiquitin–proteasome system (UPS). Unlike traditional small-molecule drugs that block protein function, PROTAC technology aims to convert small molecules for targeted protein degradation [1, 2]. Chemically, PROTAC is composed of three components: a ligand, typically a small-molecule binder or inhibitor of the protein of interest (POI); a ligand for E3 ubiquitin ligase (E3); and a covalently interconnected linker, often comprising 3–15 carbons or other atoms connecting the two ends. Mechanistically, this design ensures that PROTACs simultaneously recruit the POI and E3 ligase complex proteins within the cell and bring the POI into proximity for ubiquitination. Subsequently, the ubiquitinated POI is degraded by the endogenous 26S proteasome [3, 4]. Two PROTACs, ARV110 and ARV471, are enrolled in phase II clinical trials for prostate and breast cancer, respectively (NCT03888612 and NCT04072952 on ClinicalTrials.gov) [1].

The CRL4CRBN E3 complex is one of the most widely used E3 ubiquitin ligases in the design of PROTACs. By connecting different binders of target proteins to the CRBN ligand thalidomide or its analogs through a linker, it achieves the degradation of a variety of proteins, including Bromodomain-containing protein 4 (BRD4), BRD9, BCR-ABL, CDK9, FLT3, BTK, ALK, HDAC6, CDK6, BCL-XL, and MDM2 [5, 6]. CRBN ligands are pioneering the PROTACs field by offering a unique combination of advantages that include precise and potent binding affinity to their target E3 ligases, which has been biologically validated, along with desirable physicochemical properties such as optimal molecular weight, good solubility, balanced lipophilicity, and a low propensity for metabolic instability. Additionally, the binding modes of these ligands are well understood, providing a solid foundation for the design and development of effective PROTACs [7].

Characterization of the degradation and recovery of the POI is crucial for evaluating the efficacy of PROTAC. Western blotting (WB) antibody assays and mass spectrometry are used to investigate the changes in endogenous protein levels in pertinent cellular systems [8]. As the responses of these methods to the degradation molecule require lysis, it is challenging to apply them to high-throughput screening formats. In addition, WB analysis requires multiple experimental steps, is time-consuming, and relies on the accessibility of high-quality and specific antibodies for accurate protein quantification. Moreover, mass spectrometry, although highly quantitative, is expensive and requires specialized instruments and analysts. The Lumit Immunoassay uses NanoBiT technology for quick detection of protein phosphorylation and degradation by combining SmBiT and LgBiT to form a light-emitting complex. It is straightforward, rapid, and sensitive, avoiding complex processes [9]. Challenges include chemical labeling, tag compatibility, steric hindrance, assembly efficiency, and cost. Additionally, it cannot detect protein–protein interactions with affinities lower than the LgBiT-SmBiT interaction threshold [10, 11]. Some studies have demonstrated the suitability of dual-reporter assay in meeting high-throughput screening requirements for detecting various cell functions, including research on protein interactions, intracellular signaling, and microRNA regulation [12,13,14,15,16]. While this method has been documented in some studies, its application in drug screening, particularly in PROTAC screening, remains limited.

Sirtuin 2 (SIRT2), a member of the NAD+-dependent lysine deacetylases of the sirtuin family [17], deacetylates histones and non-histone proteins, playing crucial roles in diverse physiological processes. Our previous review emphasized the considerable association between SIRT2 dysfunction and two human diseases: neurologic disorders and cancer [18]. Although early genetic research indicates that the protein exhibits a tumor-suppressive function, many pharmacological data now suggest that SIRT2 inhibition exerts a broad anticancer effect. This positions SIRT2 as a promising target for pharmacological intervention [19, 20].

The present study provided an alternative high-throughput solution for PROTACs screening using a dual-reporter system, expressing enhanced green fluorescent protein (EGFP) and the target protein fused with Renilla luciferase (RLUC). EGFP served as an internal reference. RLUC is used as an indicator for target protein degradation. This method evaluates protein levels in a short time by detecting distinct light signals. To test the feasibility of the screening model, we used this system to detect the degradation effect of ARV110, a PROTAC targeting androgen receptor (AR). And WB verified the screening results. Besides, we created a small sample PROTAC chemical library and chose TM (benzyl (1-oxo-1-(phenylamino)−6-tetradecanethioamidohexan-2-yl)carbamate) as a ligand for the SIRT2 protein [19], and pomalidomide (POM) or lenalidomide (LENA) as ligands for the CRBN protein, respectively. These PROTACs were then screened utilizing the dual-reporter system and their degradation ability to endogenous SIRT2 protein in the MCF-7 cell line and anti-proliferative activity were investigated. This research enhanced the application of dual-reporter systems for the targeted screening of PROTACs in drug discovery processes and expanded drug development targeting SIRT2.

Results

Construction of the fluorescence and chemiluminescence reporting system

We assumed that PROTACs can degrade the fusion protein formed by the target protein and RLUC, and the degradation efficiency can be determined by the luminescence intensity generated by RLUC-catalyzing coelenterazine. This degradation mode is illustrated in Fig. 1A. To test this hypothesis, we first used AR as the target protein to construct a dual-reporter system. EGFP was added as an internal reference to avoid errors caused by cell proliferation.

Fig. 1
figure 1

Construction of the fluorescence and chemiluminescence dual-reporter system. A The degradation mode of the fusion protein formed by PROTAC. B The engineered gene pattern diagram. C Flow cytometric analysis of EGFP-positive cells after transfection. D The expression of the RLUC-tAR fused protein was detected by tAR antibody via immunoblotting

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AR genes generally comprise four structural domains: a low-conserved N-terminal domain, a highly conserved DNA binding domain, a flexible hinge region, and a highly conserved carboxyl ligand binding domain (LBD) [21]. The LBD region (tail of AR, tAR) can bind to enzalutamide (ENZA) or ARV110 [22, 23]. Thus, we synthesized LBD (624–920 aa) and inserted it into the multiple cloning site downstream of RLUC to form a fusion expression, referred to as RLUC-tAR. The engineered gene pattern is shown in Fig. 1B.

Following transfection with a lentiviral expression vector expressing EGFP and RLUC-tAR, HEK293T cells were screened using puromycin and sorted using a green fluorescent protein. The EGFP positive rate of the sorted cells was approximately 98% (Fig. 1C). Immunoblotting revealed that the sorted cells expressed the RLUC-tAR fused protein (70 kDa), whereas HEK293T cells transfected with the control vector did not express either the RLUC-tAR fusion protein or full-length AR protein (FL-AR). LNCaP prostate cancer cells expressed the FL-AR protein (110 kDa) as a positive control (Fig. 1D).

Next, we examined the catalytic activity of RLUC in EGFP/RLUC-tAR-transfected HEK293T cells (modal cells). As shown in Fig. 2A, as the number of cells increased, cell luminescence gradually increased, showing a positive correlation. In contrast, vector-transfected HEK293T cells did not exhibit luminescence signals. In addition, EGFP underwent synchronous changes with RLUC in both WB and RLUC detection, indicating that EGFP can accurately represent changes in cell number. Subsequently, it can serve as an internal reference protein for optical detection (Fig. 2B and C).

Fig. 2
figure 2

RLUC catalytic activity in EGFP/RLUC-tAR transfected HEK293T cells. A Correlation of luminescence intensity and cells. B Correlation of luminescence intensity and EGFP fluorescence. C The expression levels of RLUC-tAR and EGFP along the cell number changes

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Using the dual-reporter system to detect the degradation function of ARV110

Next, we investigated whether AR PROTAC (ARV110) could degrade intracellular RLUC-tAR. Various drugs including ARV110, ENZA, or POM were added to modal HEK293T cells in a concentration range of 0–400 nM for 8 h. End-point analysis revealed that as the dose of ARV110 increased, RLUC luminescence gradually decreased, reaching its lowest point at 50–400 nM. No significant difference was observed in the EGFP signal (Fig. 3A and B). After normalization with green fluorescence, the standard RLUC ratio remained unchanged (Fig. 3C). The IC50 value was calculated as 51.22 nM utilizing GraphPad Prism’s variable slope dose–response model.

Fig. 3
figure 3

Dual-reporting system for detecting the degradation function of ARV110. A RLUC luminescence of modal HEK293T cells treated with drugs, including ARV110, ENZA, and POM, was measured over a concentration range of 0–400 nM for 8 h. B RLUC ratio of modal HEK293T cells. C The standard RLUC ratio after normalization with green fluorescence (RFU). D–F The expression of RLUC-tAR using an AR C-terminal antibody or RLUC antibody treated with ARV110, POM, or ENZA at eight different concentrations ranging from 0 to 200 nM for 8 h. Three independent biological replicates were performed (n = 3), and data of B and C were presented as mean ± standard deviation (SD), *p < 0.05, **p < 0.01, and ***p < 0.001

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WB analysis using an AR C-terminal antibody or RLUC antibody showed that the RLUC-tAR protein gradually degraded with an increase in ARV110 concentration (Fig. 3D). At 100 nM, the maximum degradation rate (67%) was achieved; however, a marginal recovery was observed at 200 nM. The POM and ENZA did not exhibit any discernible alterations to the protein levels (Fig. 3E and F). We overlaid the reporter quantification (normalized RLU ratio) with immunoblot quantification (gray value ratio of WB) to determine their correlation. The results showed (Additional file 1: Fig. S1) that they had a strong linear relationship and were positively correlated, with a Pearson correlation coefficient (r) of 0.9694. Moreover, the correlation was statistically significant (P < 0.0001). These results are consistent with those of the dual-reporter gene cell model, suggesting that the dual-reporter system can rapidly reflect changes in fused-protein degradation.

To eliminate the possibility of non-specific binding between ARV110 and RLUC, HEK293T cells transfected with EGFP and naked RLUC protein were used for ARV110 treatment. As shown in Fig. 4A, no significant changes were observed in this cell line using the RLUC luminescence assay. This indicated that the degradation of the RLUC-tAR protein caused by ARV110 was not attributed to RLUC targeting.

Fig. 4
figure 4

The validation of the degradation effect of ARV110 detected by the dual-reporter system. A The normalized RLUC luminescence of HEK293T cells transfected with EGFP and naked RLUC protein treated with ARV110 at a range of concentrations of 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 nM for 8 h. Data were presented as mean ± SD of three independent experiments. ***p < 0.001. B and C The normalized RLUC ratio (mean ± SD from three independent experiments) of modal HEK293T cells and RLUC-tAR degradation induced by ARV110 (100 nM), MG132 (5 µM), MLN4924 (5 µM), POM (10 µM), or ENZA (100 nM). For the co-treatment, cells were pretreated with ENZA, POM, MLN4924, or MG132 for 2 h, followed by an additional 8 h of treatment with ARV 110. D The time curve depicts the normalized RLUC ratio of modal HEK293T cells treated with ARV110 (100 nM), ENZA (100 nM), or POM (100 nM) over different time points (1 h, 2 h, 4 h, 8 h, 16 h, 24 h, and 48 h). Data are presented as the mean ± SD of three independent replicates. E and F The expression of wild-type full-length AR (FL-AR) in LNCaP cells treated with ARV110 or ENZA at ten different concentrations ranging from 0 to 200 nM for 8 h

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To confirm that the UPS plays a role in ARV110-induced degradation, we co-treated ARV110 cells with a proteasome inhibitor (MG132) or a pan-CUL neddylation inhibitor (MLN4924). As shown in Fig. 4B, the reduction in ARV110-induced luminescence was restored to the levels of untreated cells. Subsequently, a competitive experiment was conducted. Co-treatment with either POM or ENZA alongside ARV110 resulted in the observed decrease in luminescence being blocked. This effect was also observed using WB (Fig. 4C).

Although this system is not suitable for continuous real-time optical monitoring, we used the endpoint method to independently detect cells at specific time points of interest and subsequently plotted a time curve. Figure 4D showed that with ARV110 (100 nM) treatment, the initial degradation curve sharply declined, reaching its lowest point (maximum degradation) at 8 h. Subsequently, there was a marginal upward trend from 16 to 48 h, indicating the level of target recovery.

To further confirm the effect of ARV110 detected using the dual-reporter system, we used prostate cancer cells (LNCaP) expressing wild-type FL-AR for ARV110 treatment. The WB assay showed that ARV110 dose-dependently degraded FL-AR, reaching its minimum at 100 nM, consistent with the degradation pattern observed in the dual-reporter system (Fig. 4E). And the correlation between the dual-reporter assay data (normalized RLU ratio) with immunoblot quantification (gray value ratio of WB) was relatively strong and positive with a Pearson correlation coefficient (r) of 0.9517. Moreover, the correlation was significant (P < 0.0001) (Additional file 1: Fig. S2). ENZA exhibited no effects (Fig. 4F).

In summary, these data demonstrated the expected physiological and PROTAC responses of the exogenous dual-reporter gene system.

Using the dual-reporter system to screen the ideal PROTACs for SIRT2

Considering that the dual-reporter system successfully detected RLUC-tAR degradation, we attempted to use this system for screening PROTACs targeting other proteins, such as SIRT2. Sirtuins are a highly conserved class of NAD+-dependent lysine deacetylases. Within this protein family, SIRT2 is the only isoform predominantly located in the cytosol, capable of moving in and out of the nucleus [24]. Curiosity regarding whether SIRT2 can become a therapeutic target for human cancer remains. SIRT2-nude mice tend to develop tumors earlier than wild-type mice [25]. Nevertheless, several pharmacological studies have identified potent and selective inhibitors of SIRT2 and demonstrated that the protein is an ideal anticancer target [26].

Among SIRT2 inhibitors, TM is a 14-carbon thioacyl lysine compound developed by Jing et al. [19]. Chemically, TM contains a lysine backbone with a Cbz group in the Nα-terminus and an aniline condensed with the C-terminus, with the Nε-terminus connected to a thioxotetradecyl group. The thioacyl lysine groups can react with NAD+ in the sirtuin active site, generating a stable intermediate that inhibits SIRT2. The IC50 value for SIRT2 inhibition was 0.028 mM. TM exhibits anti-proliferative activity against various cancer cells by decreasing c-Myc levels. By substituting thalidomide for the Cbz group using two or four repeating polyethylene glycol linker units, Hong et al. developed a SIRT2 PROTAC (i.e., TM-P4-Thal). TM-P4-Thal can efficiently destroy SIRT2 levels in a range of breast cancer cells and achieve stronger anti-proliferative effects than inhibitors [27].

In this study, a series of TM-based SIRT2 degraders were designed and synthesized to confirm the utility of the constructed screening platform (Additional file 1: Scheme 1) [19, 27, 28]. The designed PROTACs have different linkage sites compared with the existing PROTAC (TM-P4-Thal). In addition, we replaced the C-terminus of aniline with the E3 ligase ligand LENA, which has alkyl and ether linkers. As a positive control, TM-P4-Thal was synthesized and named compound 129. Nine compounds 121–129 (Fig. 5) were synthesized and screened using the dual-reporter system.

Fig. 5
figure 5

The chemical structures of the SIRT2 PROTACs (121–129)

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Unexpectedly, HEK293T cells highly expressed endogenous SIRT2 (Fig. 6A), which competed with the exogenous RLUC-SIRT2 fusion protein for PROTAC binding, thereby interfering with the efficacy of the dual-reporter system. Considering the unavailability of SIRT2-negative cells, we selected the lung cancer cell line A549, characterized by low SIRT2 expression, as the parent cell to minimize the impact of endogenous SIRT2 on the PROTAC screening. Following transfection with the dual-reporter system, the exogenous RLUC-SIRT2 fusion protein was expressed at significantly higher levels than endogenous SIRT2 (Fig. 6A).

Fig. 6
figure 6

SIRT2-PROTAC screening using the dual-reporter system. A SIRT2 protein levels in HEK293T, MCF-7, A549, and transfected A549 cells. B The RLUC luminescence of transfected A549 cells after treatment with compounds 121 − 129, LENA, POM, and TM at a range of concentrations of 0.01, 0.1, 1, and 10 µM for 24 h. C and D The normalized RLUC luminescence of transfected A549 cells after treatment with compounds at the concentrations of 0.1 and 1 µM for 24 h, data are presented as the mean ± SD of three independent replicates. E Western blotting analysis of RLUC-SIRT2 protein in transfected A549 cells treated with indicated compound at a concentration of 0.1 µM for 24 h

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Next, this model was used for screening SIRT2-PROTACs at four different gradient concentrations. At a concentration of 0.1 µM, different compounds exhibited distinct effects (Fig. 6B). Compounds 121, 128, and 129 exhibited the best degradation effects (degradation efficiency, 60–70%). Compounds 124 and 127 demonstrated a moderate degradation efficiency of 40%, whereas other compounds exhibited a degradation efficiency of no greater than 30% (Fig. 6C).

At a concentration of 1 µM, compounds 121, 128, and 129 induced a marginal reduction in SIRT2 degradation (Fig. 6D), and the protein level moderately recovered at 10 µM, which may be owing to the PROTAC’s hook effect. PROTACs with high concentrations are prone to forming a binary complex with the E3 ligase or the protein target, thereby preventing the formation of the ternary complex required for efficient target protein degradation [29]. Other compounds did not show a substantial degradation to protein at 1 µM and 10 µM (Fig. 6B). These results suggested that compounds 121, 128, and 129 are potential SIRT2-PROTAC candidates. Immunoblotting also demonstrated the SIRT2 degradation abilities of compounds 121, 128, and 129 (Fig. 6E).

Subsequently, we validated the efficacy of the candidate PROTACs in MCF-7 cells expressing endogenous SIRT2. MCF-7 cells are ideal for the effective functioning of TM and TM-P4-Thal. These cells were subjected to treatment with the candidate compounds at gradient concentrations. As shown in Fig. 7A–C and Additional file 1: Fig. S3, compounds 128 and 129 exhibited degradation in a dose-dependent manner, whereas the inhibitor TM exhibited no degradation effect. At concentrations of 0.1 and 1 µM, both compounds reached their maximum degradation efficacy. In particular, compound 128 initiated evident degradation at 0.01 µM and exhibited a weaker hook effect at 10 and 20 µM than that of 129. Neither compound exhibited a degradation effect on SIRT1 or SIRT3, thereby confirming their specificity.

Fig. 7
figure 7

The degradation ability of SIRT2-PROTACs in MCF-7 cells expressing endogenous SIRT2. Three independent biological replicates were performed (n = 3). (A-D) The expression of SIRT1, SIRT2, and SIRT3 in MCF-7 cells treated with compounds (128, 129, TM, 121, and 122) at gradient concentrations from 0.001 to 20 µM for 24 h. (E and F) SIRT2 degradation induced by 128 (0.1 µM), 129 (0.1 µM), MG132 (5 µM), MLN4924 (5 µM), POM (10 µM), or LENA (10 µM), and TM (10 µM). For the co-treatment, cells were pretreated with TM, POM, LENA, MLN4924, or MG132 for 2 h, followed by an additional 24 h of treatment with compound 128 or 129

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Notably, the degradation induced by compound 121 was much weaker than that induced by compounds 128 and 129, with a maximum degradation efficiency at 10 µM (Fig. 7D). Compound 121 did not exhibit a degradation efficacy as it did in the dual-reporting system, possibly owing to conformational differences between the exogenous fusion proteins and endogenous proteins.

To further confirm that SIRT2 degradation occurs via the UPS, we co-treated candidate PROTACs with the proteasome inhibitor (MG132) or a pan-CUL neddylation inhibitor (MLN4924). As shown in Fig. 7E and F, this co-treatment rescued PROTAC-induced SIRT2 degradation. Subsequently, a competitive experiment was conducted. When co-treating a CRBN ligand (POM or LENA) or SIRT2 inhibitor (TM) with candidate PROTACs, SIRT2 degradation was attenuated. In addition, we created a negative control compound (130) to validate that PROTAC-induced protein degradation requires CRBN binding. It has been shown that the introduction of a methyl group on the amino group in the LENA moiety blocks the binding of thalidomide analogs to CRBN [30, 31]. The WB result showed that compound 130 did not induce degradation of SIRT2 (Additional file 1: Fig. S4), indicating that the degradation capability disappears after the introduction of a methyl group on the amino group in the LENA moiety, confirming that the degradation capability is dependent on the binding of CRBN.

To delve deeper into the selectivity of proteome degradation, we conducted a DIA-based proteomic study to identify proteins with altered expression in MCF-7 cells following a 24-h exposure to compounds 128 (0.1 μM) and 130 (1.0 μM). As shown in Additional file 1: Fig. S5 of Supplementary material, in the SIRT family, compound 128 exerted a significant degradation effect on SIRT2 protein, while no degradation was observed for SIRT1, SIRT5, SIRT6, and SIRT7. Moreover, the control compound 130 did not show any degradation effects on the entire SIRT family. SIRT3 was not detected in all samples, which may be due to its low abundance in total proteins. The changes in all proteins detected by mass spectrometry can be found in the Attached file 3.

Collectively, these data validate the degradation effect of the two compounds (128 and 129) screened using the dual-reporter system, confirming the effectiveness of this system.

SIRT2 PROTACs inhibited deacetylation and improved cytotoxicity

Next, we assessed the inhibitory effects of PROTACs on SIRT2 deacetylation. After treating MCF-7 cells with PROTACs at concentrations ranging from 0.01 to 10 µM for 6 h, immunoblotting was performed to evaluate the acetylation of α-tubulin, a SIRT2 deacetylation substrate that was previously identified [32]. Upon degradation of SIRT2 protein, the acetylation level is expected to increase owing to the treatments. In the concentration range of 0.01–10 μM, both compounds 128 and 129 considerably increased α-tubulin acetylation. The expression levels of SIRT2 and ac-tu were negatively correlated with Pearson correlation coefficients (r) of − 0.8989 and − 0.9373 for compounds 128 and 129, respectively (P < 0.05) (Additional file 1: Fig. S6). Furthermore, at a concentration of 0.01 μM, the acetylation of α-tubulin proteins was approximately equivalent to the efficiency of TM at a concentration of 10 μM (Fig. 8A and B). These data suggest that SIRT2 deacetylation inhibition by PROTACs is stronger than that by TM at the same concentration.

Fig. 8
figure 8

SIRT2-PROTACs inhibited deacetylation and improved cytotoxicity. Three independent biological replicates were performed (n = 3). A and B The acetylation level of α-tubulin after treatment with compounds 128, 129, and TM at concentrations ranging from 0.01 to 10 µM for 6 h. C The antiproliferative effect of compounds 128, 129, TM, and LENA against MCF-7 cells treated for 72 h (mean ± SD from three independent experiments)

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After demonstrating that PROTACs degrade the SIRT2 protein and inhibit its activity, we evaluated their effect on the proliferation of MCF-7 cells (Fig. 8C). As expected, after 72 h of incubation, the PROTACs exhibited stronger cytotoxicity than TM. In addition, compound 128 exhibited marginally stronger killing activity than compound 129 at high concentrations, possibly owing to the weaker hook effect of compound 128 compared with compound 129.

Collectively, using cells with endogenous SIRT2, we further validated the cellular functional changes after SIRT2 degradation by PROTACs, suggesting that the two compounds serve as candidate PROTACs for SIRT2.

Discussion

Despite the recent successful development of numerous PROTACs, the high-throughput screening of initial compounds through functional characterization remains challenging [8]. WB, the most commonly used method, can visually and quantitatively evaluate the degree of protein loss; however, achieving high-throughput screening is difficult owing to its cumbersome and laborious steps. Furthermore, the use of spectrometry in this field is limited owing to the need for expensive instruments and specialized analysts. However, when PROTACs are screened successfully and precise functional evaluation is required, WB or mass spectrometry may be more appropriate.

Lumit Immunoassay uses NanoBiT technology to detect analytes by bringing together SmBiT and LgBiT, two components of the NanoLuc enzyme, to form an active complex that emits light. This method is simple, fast, and sensitive, eliminating the need for complex procedures. Lumit technology in PROTAC screening monitors ternary complex formation by juxtaposing target proteins with E3 ligases and assessing numerous PROTACs. But challenges include complex chemical labeling, tag compatibility affecting protein functions or interactions, steric hindrance with tag-fused proteins, fusion protein efficiency, and increased costs. It also cannot detect protein–protein interactions below the LgBiT and SmBiT affinity threshold [10, 11].

The dual-reporter system has been extensively used for high-throughput screening, involving gene regulation, cell signaling, ligand–protein interactions, and novel drug development [15, 33,34,35]. Yen et al. created a cell-based system that uses a retroviral construct with a single promoter to express two fluorescent proteins (DsRed and EGFP) to investigate protein stability due to cellular events [36, 37]. Sievers et al. used an eGFP/mCherry reporter to screen for protein degradation in the human zinc finger proteome [38]. Hsia et al. conducted CRISPR screens for BRD4 degradation using a dual-fluorescence reporter, sorting cells based on fluorescence ratios after exposure to BET degraders, to identify factors essential for IBG1 activity [39]. Those systems typically employ a combination of two distinct fluorescent proteins or luciferases in conjunction with fluorescence-based assays. A co-transfected “control” reporter serves as an internal control and the baseline response, whereas another reporter, referred to as the “experimental” reporter, is associated with the impact of particular experimental settings. Preventing experimental variability due to variations in cell viability can be achieved by normalizing the activity of the experimental reporter to that of the internal control.

In this study, we selected a combination of fluorescent (EGFP) and bioluminescent (RLUC) reporters to prevent mutual interference from similar spectra. Selecting a single luciferase from reporters aims to simplify the operation by adding only one substrate. Additionally, cells expressing fluorescence offer the advantage of sorting positive cells after transfection.

Various luciferases are available as “experimental” reporters. Firefly luciferase has a relatively large molecular weight (61 kDa) and the fused protein it tags usually exceeds 100 kDa [40]. The excessive molecular weight of the tag protein may affect the conformation of the fused target protein. Nanoluc is a luciferase with a minimum molecular weight of 19.1 kDa, and is ideal as a reporter [41, 42]. Moreover, it has the highest brightness and is particularly suitable for fusion proteins with low expression levels. RLUC has a moderate molecular weight of 36 kDa and is suitable for the fusion expression of proteins at approximately 60 kDa [43, 44]. Although the inherent brightness of RLUC is 10–100 times lower than that of Nanoluc, it is sufficient for detecting protein degradation induced by PROTACs. As observed in this study, RLUC was used to fuse the tAR or SIRT2 proteins, resulting in a 60–80 kDa fusion protein that offered the appropriate light intensity for protein degradation.

Riching et al. [45] presented a screening strategy using a split reporter (Nanoluc) based on endogenous proteins. They used CRISPR/Cas9 technology to endogenously tag target proteins with HiBiT subunits. In cells expressing target proteins, HiBiT binds with high affinity to LgBiT subunits in the cytoplasm, forming Nanoluc-target protein complexes. This enables the monitoring of target protein loss via luminescent measurements after treatment with degrader compounds. The advantage of this method is that endogenous proteins do not interfere with overall cell function and real-time kinetic protein level measurements are achieved with stabilized luciferase substrates. However, a single reporter cannot monitor changes in cell activity; therefore, protein loss cannot be uncoupled from loss due to cell death. Furthermore, the platform lacks a negative control, making it challenging to eliminate the possibility of the interaction between PROTAC and Nanoluc, potentially resulting in the nonspecific attenuation of the fusion protein, particularly during initial compound screening. The combination of HiBiT technology with fluorescent reagents could achieve a dual-system detection of protein levels, but there are limitations such as the stability of the peptide tag, the cost and availability, and ensuring the cellular permeability of the LgBiT complementary fragment to effectively penetrate the cell membrane [46].

The benefit of using exogenous fusion proteins in our study was that we only needed to express functional portions rather than the entire target protein with the advantages of high expression efficiency, simplified purification, and low cost. For example, the AR PROTAC selectively binds to the active domain (LBD, tAR) at the AR tail [21]. Subsequently, the fusion protein formed by RLUC and tAR can be used for PROTAC screening. In addition, two reporter genes were constructed in one viral vector, enabling the expression of both reporters via distinct promoters following a single transfection. Fluorescent proteins can also be used for positive cell sorting.

In the screening of PROTACs, a cell-free system treated with PROTACs was employed as a background control to eliminate potential fluorescence interference from the drug itself. This strategy ensured that the detected signals were specifically attributable to the action of PROTACs on cells and their interactions with target proteins. While this approach effectively ruled out fluorescence originating from the drug itself, it could not account for fluorescence arising from interactions between PROTACs and other cellular components, which are unlikely given the high selectivity of the drug and the relatively low fluorescence intensity compared to that of luciferase. Consequently, if the selectivity of the drug is low, it may be more appropriate to use cells expressing only the vector and treated with PROTACs as a background control to enable a more accurate assessment.

Additionally, this study employed an endpoint method to independently measure fluorescence intensity at specific time points, rather than continuous real-time optical monitoring. This approach was chosen due to the use of coelenterazine, a widely available and cost-effective substrate. However, this substrate has poor chemical stability in aqueous solutions and a short half-life [47, 48]. For extended periods of continuous monitoring within living cells, the cell-compatible Nano-Glo Live Cell Substrate, a NanoLuc substrate, needs to be utilized. This single-addition, non-lytic assay reagent is specifically designed for detecting the bioluminescence of NanoBiT or NanoLuc in living cells [49, 50]. To capture degradation at various time points using the dual-reporter system, it is possible to establish multiple experimental groups. Alternatively, if continuous monitoring is to be achieved, it is necessary to develop a long-lasting substrate that can bind with Renilla luciferase without adversely affecting cell function.

The restriction of exogenous fusion proteins may be owing to the effect of tags on the conformation of target proteins, despite using a sufficiently long linker. As observed in SIRT2 PROTAC screening in this study, compound 121 performed well in dual-reporter screening. However, its function in MCF-7 cells with endogenous SIRT2 protein was weakened. To minimize this conformational interference, we recommend establishing fusion expression proteins with different luciferase labels and individually screening PROTACs. Furthermore, following initial screening using the dual-reporter technique, the candidate PROTACs should be further identified via WB or mass spectrometry using wild-type cells. Additionally, it is essential to validate their mechanisms of action and selectivity.

To assess the screening capability of the dual-reporter system for suitable PROTACs, we designed a series of new SIRT2 PROTACs based on previous research. This validation confirmed that modifications to the N- and C-termini of the mercaptoformyl lysine structure could effectively bind SIRT2 [51]. Therefore SIRT2 PROTACs with different attachment sites and linkers were developed, providing a complementary aspect to the structural relationship of SIRT2 PROTACs.

As shown in this study, the effective SIRT2 PROTACs could be successfully screened using a dual-reporter system. The standardized luminescence reflected the extent of SIRT2 degradation. Attaching inhibitors via different sites and varying the length of the linkers affects the structure–activity relationship of the PROTACs, resulting in differential degradation [52]. Compared with compound 129, compound 128 uses LENA instead of thalidomide as a CRBN ligand, showing a weaker hook effect and stronger inhibition of cell proliferation, making it an improved SIRT2 PROTAC. Structural analysis reveals that compound 128 differs from compounds 121–127 in its linker; specifically, compound 128 contains a polyethylene glycol (PEG) linker, which has better physicochemical properties, such as good solubility, and the difference in the linker may affect their binding affinity to the target protein [52]. Compounds 128 and 129 share the same linker, while compound 128 incorporates LENA in place of thalidomide as the E3 ligase ligand, which is present in compound 129. Additionally, the conjugation site with the ligand for compound 128 is at the C-terminus of the SIRT2 inhibitor TM. These structural variations might account for the observed differences in degradation efficiency and antitumor activity in those compounds. Future research will involve structural optimization and further cellular function testing for these PROTACs.

The study provided an additional validation of the dual-reporter system’s efficacy and practicality specifically for high-throughput screening of PROTACs. Despite the limited sample size, this dual-reporter system demonstrated high-throughput screening performance. The screening of large sample sizes of PROTACs can be done in the same way. However, in addition to the sample size limitation, the dual-reporter system also requires the selection of appropriate model cells. If the model cells highly express endogenous target proteins, these proteins will compete with the exogenous RLUC fusion protein for binding to PROTACs. This competition will interfere with the efficacy of the dual-reporter system and disrupt the degradation process of PROTACs, resulting in less pronounced changes in luminescence intensity. Therefore, when selecting model cells that express exogenous proteins, it is essential to test the expression levels of the target proteins within those cells.

Conclusions

PROTAC is a powerful tool to address the challenges associated with difficult-to-treat conditions and drug resistance issues. The discovery and functional characterization of PROTACs currently face several obstacles such as cell permeability, formation of ternary complexes, and specificity. A comprehensive chemical series is typically constructed based on the composition of the linkers and the recruitment handle of the E3 ligase complex. This maximizes the chances of identifying an active molecule. In this study, a dual-reporter system using both fluorescence and chemiluminescence was successfully constructed. The screening function of PROTACs was evaluated using the EGFP/RLUC-tAR system and ARV110. We successfully utilized the EGFP/RLUC-SIRT2 system to screen for SIRT2-PROTACs. The dual-reporter system will facilitate the rapid screening of these compounds, providing a structure–activity relationship to guide further structural optimization efforts. These screens can be performed on live cells, a difficult task to achieve with techniques such as mass spectrometry and WB. The dual-reporter system may accelerate drug discovery during PROTAC development.

Methods

Expression plasmids

Clones expressing N-terminal RLUC fusions of human tAR or SIRT2 were obtained from Hanbio Biotechnology Co., Ltd (Shanghai, China). This was prepared by inserting target protein genes into expression vectors for dual reporter genes.

Reagents and cell culture

HEK293T, LNCaP, A549, and MCF-7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). They were cultured in either IMDM (Gibco Company, MT, USA) for HEK293T cells or RPMI 1640 (Gibco Company, Montana, USA) for LNCaP, A549, and MCF-7 cells, supplemented with 10% fetal bovine serum (FBS; Avantor Seradigm, Jiangsu Kangcheng Biotechnology, Jiangsu, China) at 37 °C with 5% CO2. ARV110, ENZA, POM, LENA, MG132, and MLN4924 were purchased from Selleck (Shanghai, China) and used as received.

Generation of the RLUC-tAR or RLUC-SIRT2 stable cell line

For the preparation of lentivirus, a three-plasmid lentiviral system was utilized, including a dual-reporter gene plasmid with the target gene (pCDH-EGFP/RLUC-tAR or pCDH-EGFP/RLUC-SIRT2), the packaging plasmid pSPAX2 (Addgene, 12,260, MA, USA), and the envelope plasmid pMD2.G (Addgene, 12,259, MA, USA). The constructed lentiviral vector, along with the packaging vector, was transfected into HEK293T cells that had been cultured overnight. The components of the liposome transfection complex were as follows: pSPAX2 (10 μg), pMD2.G (5 μg), the dual-reporter gene plasmid (10 μg), and Lipofiter™ (Hanbio, HB-TRLF-10000, Shanghai, China) (75 μL). After mixing the liposome transfection complex uniformly, it was incubated at room temperature for 15 min and then was slowly added to the HEK293T cells and cultured in an incubator at 37 °C with 5% CO2. After transfection for 16 h, the medium was replaced with a fresh complete medium containing 10% fetal bovine serum. Viral supernatants were collected at 48 h and 72 h post-transfection; the viral supernatant was centrifuged (4 °C, 2000 × g, 10 min) to remove cell debris. The supernatant containing the virus was then transferred to an ultracentrifuge tube and centrifuged (4 °C, 82,700 × g, 120 min). The virus was resuspended in a complete medium, aliquoted into sterilized virus tubes, and stored at − 80 °C.

HEK293T and A549 cells in the logarithmic growth phase were digested and resuspended. After being seeded at a density of 1 × 105/mL onto 6-well plates, the cells were incubated for 12 h. The culture medium was aspirated and subsequently replaced with fresh medium. The prepared lentivirus solution, diluted with a concentration gradient of phosphate buffer solution (PBS) was added, mixed well, and incubated. After 24 h, the lentivirus-containing medium was aspirated and a complete medium which contained 1.5 μg/mL puromycin and 10% FBS was added. Incubation continued for 48–72 h. The medium was subsequently changed to a normal medium, and cultivation continued. After 1 week, characterization of RLUC, tAR, EGFP, and SIRT2 expression was conducted in the infected cells.

Western blotting

Protease inhibitor-containing RIPA buffer was used to harvest and lyse the cells. After being separated using a 10% SDS–PAGE Criterion X-gel (Bio-Rad), the cell lysates were transferred to a polyvinylidene fluoride membrane. QuickBlock™ Blocking Buffer (Beyotime, P0231, Shanghai, China) was used to block the membranes, and a primary antibody against RLUC (1:1000, Abcam, ab185925, MA, USA), EGFP (1:1000, CST, 4267, MA, USA), SIRT2 (1:1000, Abcam, ab211033, MA, USA), or tAR (1:1000, CST, 54,653, MA, USA) was used to probe them overnight at 4 °C. GAPDH (1:1000, CST, 2118, MA, USA) or the anti-β-actin antibody (1:1000, CST, 4967, MA, USA) was used as the control. After incubation with the secondary antibody (CST, 7074, MA, USA), signals were displayed using an ECL detection reagent (Pierce, 32,106, Beijing, China). The membrane was placed on the sample stage of the chemiluminescence imaging system (LI-COR Odyssey® CLx, LI-COR Biosciences, NE, USA) and the exposure time and sensitivity were set using the appropriate software to acquire and save the image. Original western blots shown in the figures are provided in Additional file 2. Grayscale values were analyzed using ImageJ software (NIH, Bethesda, MD, USA). The protein residual rate was calculated using the following formula:

$$\mathrm{Target\;protein}(\mathrm{TP})\mathrm{residual\;rate}=(\mathrm{TP_{experimental\;group}}/\mathrm\beta-\mathrm{actin_{experimental\;group}})/(\mathrm{TP_{DMSO\;group}}/\mathrm\beta-\mathrm{actin_{DMSO\;group}})\times100\%.$$

Flow cytometric analysis

Transfected cells were harvested in cold PBS. EGFP-positive cells were sorted and collected using flow cytometry on a BD Verse flow cytometer (BD Bioscience, NJ, USA).

Bioluminescence imaging

Cells (20 k/40 k/60 k/80 k/100 k) with the dual-reporter gene system were inoculated into 96-well black-walled plates. Green (excitation wavelength, 488 nm; emission wavelength, 510 nm) or red fluorescence (excitation wavelength, 580 nm; emission wavelength, 610 nm) was first detected using a multifunctional enzyme labeler (Biotek Synergy H1, USA), and the RFUexperimental group values were measured. At the end of the assay, 10 µL of coelenterazine (7.2 µM, Selleck, S7777, TX, USA) was added to each well, and bioluminescence was detected using a multifunctional enzyme labeling instrument to measure the RLUexperimental group value.

PROTACs screening of dual-reporter gene expression system

The screening function of the dual-reporter gene expression system is illustrated using the example of PROTACs targeting the androgen receptor (AR). Cells expressing EGFP/RLUC-tAR were digested and counted; subsequently, 1 × 105 cells/well were seeded in 96-well black-walled plates, with some reserved cell-free wells. Drugs including various concentrations of ARV-110 and ENZA were added to the wells, reaching a final concentration of 0 − 400 nM. The incubation was prolonged for 8 h at 37 °C, 5% CO2. Green fluorescence was detected using a multifunctional microplate reader. The wells of PROTACs were measured as the RFUexperimental group, those with DMSO as the RFUDMSO group, and those with PROTACs and no cells as the RFUdrug background. Wells containing DMSO were measured for RFUDMSO background. After detection, 10 µL of coelenterazine (7.2 µM) was added to each well and bioluminescence was detected on the multifunctional enzyme labeling apparatus. Similar to the above RFU values, the measured RLU values were named as RLUexperimental group, RLUDMSO group, RLUdrug background, and RLUDMSO background. The equations are as follows:

$$\mathrm{RLU}\;\mathrm{ratio}\;=\;\lbrack(\mathrm{RLU_{experimental\;\mathrm{group}}}\;-\;\mathrm{RLU_{drug\;\mathrm{background}}})/(\mathrm{RFU_{experimental\;\mathrm{group}}}\;-\;\mathrm{RFU_{drug\;\mathrm{background}}})\rbrack/\lbrack(\mathrm{RLU_{DMSO\;\mathrm{group}}}\;-\;\mathrm{RLU_{DMSO\;\mathrm{background}}})/(\mathrm{RFU_{DMSO\;\mathrm{group}}}\;-\;\mathrm{RF_{UDMSO\;\mathrm{background}}})\rbrack\;\times\;100\%.$$

Degradation efficiency = 100% − RLU ratio.

Proteomics assay

MCF-7 cells were seeded at 1 × 106 cells in plates. Compounds 128 (0.1 µM) and 130 (1.0 µM) were added and incubated for 24 h. Proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, 89,901, MA, USA), and concentration was quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23,227, MA, USA). Data Independent Acquisition (DIA) quantitative proteomics was used to quantify the amount of protein in the sample. Briefly, after the cysteines were reduced and alkylated, protein precipitation was achieved by introducing 6 volumes of ice-cold acetone, followed by an incubation period of 24 h at − 20 °C. The precipitate was collected via centrifugation (4 ℃, 10,000 × g, 10 min), and subsequently, the protein pellet was left to dry. After proteolytic digestion (1:20 enzyme: protein, 37 ℃, 16 h) and desalination, the sample detection was conducted by the employment of liquid chromatography-tandem mass spectrometry (LC–MS/MS) (Q Exactive HF-X Hybrid Quadrupole) in Beijing Biotech Pack Scientific Co., Ltd. The raw quantitative values (Intensity) of all proteins are listed in Additional file 3.

Determination of the antitumor activity of SIRT2-PROTACs

In 96-well plates, cells (1 × 104 cells/well) in the logarithmic growth phase were planted. FBS (10%, 100 μL) was added and the plates were incubated at 37 °C in a 5% CO2 cell culture incubator for 24 h. Drug-containing medium (200 µL) was added to the test wells (final concentrations of the drug were 40, 20, 10, 5, 2.5, and 1.25 µM). Three replicate wells were established for each concentration. Incubation of the plates lasted 72 h at 37 °C and 5% CO2. The medium from the drug-containing wells was aspirated and washed once with PBS. Thereafter, each well received 100 µL of media with 10% cell counting kit-8 (CCK-8) reagent (MCE, HY-K0301, NJ, USA), and the plates were incubated for 2 h at 37 °C in an incubator with 5% CO2. OD values were determined using an enzyme-labeling instrument at a wavelength of 450 nm. The OD value of each test well was subtracted from the background OD value (blank group). The mean ± SD was calculated for f three independent replicates. Cell viability (%) was calculated as follows: (OD of spiked cells/OD of control cells) × 100% and a concentration-viability curve was constructed using GraphPad, plotting the concentration of the compounds against the corresponding viability.

Statistical analysis

Experiments and processing were performed using the Prism software (version 7.0, GraphPad Software Inc., San Diego, CA, USA). All data are presented as the mean ± SD. Specific replicate numbers are described in the figure legends for each set of data, and individual data points are listed in Additional file 4. A one-way analysis of variance was used to determine significant differences. Student’s t-test was used to compare two groups, and data were considered significant at *p < 0.05, **p < 0.01, and ***p < 0.001.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files or are available from the corresponding author upon reasonable request. The synthesis, characterization data, and spectra of SIRT2-PROTACs were contained in Additional file 1. Original western blots for all figures are provided in Additional File 2. The raw quantitative values (Intensity) of all proteins detected in the mass spectrometry and individual data values are provided in Additional file 3 and Additional file 4, respectively.

Abbreviations

PROTAC:

Proteolysis targeting chimera

RLUC:

Renilla luciferase

EGFP:

Enhanced green fluorescent protein

AR:

Androgen receptor

SIRT2:

Sirtuin 2

UPS:

Ubiquitin-proteasome system

POI:

Protein of interest

WB:

Western blotting

LENA:

Lenalidomide

POM:

Pomalidomide

ENZA:

Enzalutamide

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Acknowledgements

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Nos.:U20A20375, 82372779), Tianjin Municipal Science and Technology Project (Grant No.: 20JCYBJC00110), Tianjin Municipal Education Commission Foundation (Grant No.: 2019KJ187), Tianjin Key Medical Discipline (Specialty) Construction Project (Grant No.: TJYXZDXK-009A), CAMS Innovation Fund for Medical Sciences (Grant Nos.: 2021-I2M-1–015, 2021-I2M-1–052), and Tianjin Medical University Cancer Hospital “14th Five-Year Plan” Summit Discipline Support Project–Outstanding Potential Discipline (Grant No.: 7–2-11–2).

Author information

Authors and Affiliations

  1. Department of Pharmacy, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin’S Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin, 300060, China

    Shuai Meng, Bole Li & Jie Zhang

  2. Department of Immunology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin’S Clinical Research Center for Cancer, Key Laboratory of Cancer Immunology and Biotherapy, Tianjin, 300060, China

    Yuan Meng, Xuena Yang, Wenwen Yu, Xiubao Ren & Lin Zhang

  3. Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, 300192, China

    Tianjun Liu

  4. Department of Biotherapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin, 300060, China

    Xiubao Ren

  5. Haihe Laboratory of Cell Ecosystem, Tianjin, 300060, China

    Xiubao Ren

Authors
  1. Shuai Meng
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  2. Yuan Meng
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  3. Xuena Yang
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  4. Wenwen Yu
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  5. Bole Li
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  6. Tianjun Liu
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  7. Jie Zhang
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  8. Xiubao Ren
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  9. Lin Zhang
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Contributions

S.M.—methodology, validation, formal analysis, investigation, data curation, writing—original draft preparation, reviewing and editing, visualization; Y.M.—formal analysis, data curation, visualization; X.Y.—methodology, validation, formal analysis; W.Y.—methodology, reviewing and editing, formal analysis; B.L.—reviewing and editing, visualization; T.L.—formal analysis, methodology, resources; J.Z.—conceptualization, project administration, funding acquisition; X.R.—conceptualization, methodology, validation, formal analysis, investigation, data curation, writing—reviewing and editing, visualization; L.Z.—resources, writing—original draft preparation, reviewing and editing, supervision, funding acquisition. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jie Zhang, Xiubao Ren or Lin Zhang.

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Supplementary Information

12915_2025_2153_MOESM1_ESM.pdf

Additional file 1: Chemistry of compounds 121-129 including synthesis, characterization data, and spectra. Scheme 1. The synthesis of SIRT2-PROTACs 121-128. Fig. S1. The correlation of the reporter quantification (Normalized RLU Ratio) with immunoblot quantification (Gray Value Ratio of WB) in modal HEK293T cells treated with ARV110 at concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100, 200 nM for 8 h. Fig. S2. The correlation of the reporter quantification (Normalized RLU Ratio) with immunoblot quantification (Gray Value Ratio of WB) in LNCaP cells treated with ARV 110 at concentrations of 0, 0.73, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 200 nM for 8 h. Fig. S3. The expression of SIRT2 in MCF-7 cells treated with SIRT2-PROTACs, TM, LENA, and POM at a concentration of 0.1 µM. Fig. S4. The structure of negative control compound 130 (A) and the expression of SIRT2 in MCF-7 cells treated with compounds 130 at concentrations from 0.001 to 20 µM for 24 h. Fig. S5. Degradation selectivity of compound 128. Data-independent acquisition (DIA)-based proteomic analysis of MCF-7 cells treated with DMSO, compound 128 (0.1 μM), and 130 (1.0 μM) for 24 h. The bar graphs represent the raw quantitative values (Intensity) of each protein detected in the mass spectrometry. Fig. S6. The correlation of SIRT2 expression levels with ac-tu expression levels in MCF-7 cells treated with compounds 128 and 129 at concentrations ranging from 0.01 to 10 µM for 6 h.

Additional file 2: Original western blots shown in the figures.

Additional file 3: The raw quantitative values (Intensity) of all proteins detected in the mass spectrometry.

12915_2025_2153_MOESM4_ESM.xlsx

Additional file 4: Individual data values of Fig.  3B, Fig.  3C, Fig.  4A, Fig.  4B, Fig.  4D, Fig.  6C, Fig.  6D, and Fig.  8C.

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Meng, S., Meng, Y., Yang, X. et al. Rapid and high-throughput screening of proteolysis targeting chimeras using a dual-reporter system expressing fluorescence protein and luciferase. BMC Biol 23, 51 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12915-025-02153-7

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Keywords

BMC Biology

ISSN: 1741-7007

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