Synthesis of selective BCL-XL PROTAC and potent antitumor activity in glioblastoma

doi:10.21203/rs.3.rs-4599552/v1

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Synthesis of selective BCL-XL PROTAC and potent antitumor activity in glioblastoma

https://doi.org/10.21203/rs.3.rs-4599552/v1

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Glioblastoma (GBM), the most aggressive and treatment-resistant form of brain cancer, is significantly influenced by GBM stem cells (GSCs), which contribute to tumor initiation and recurrence. In this study, we introduce two novel proteolysis-targeting chimeras (PROTACs), AN-1 and AN-2, engineered to degrade BCL-X_L, a critical anti-apoptotic protein in the BCL-2 family. These PROTACs are optimized from ABT-263 and uniquely utilize MDM2 as an E3 ligase, a strategy not previously employed in GBM therapy. Our approach leverages the high expression of MDM2 in GSCs versus its low expression in platelets, enhancing therapeutic specificity and reducing the risk of thrombocytopenia—a major side effect of direct BCL-X_L inhibition. Both in vitro and in vivo studies demonstrate that AN-1 and AN-2 effectively inhibit GSC proliferation, promote apoptosis, and substantially improve survival rates without inducing thrombocytopenia. This work not only highlights the potential of targeting underutilized E3 ligases for cancer therapy but also suggests a highly selective approach for treating GBM that might overcome the current limitations of existing therapies.

Biological sciences/Cancer/CNS cancer

Biological sciences/Cancer/Cancer therapy/Drug development

Glioblastoma

Glioblastoma Stem Cells

Proteolysis-targeting chimeras

BCL-XL

Glioblastoma (GBM), a rapidly growing and highly invasive cancer, stands as the most common and lethal malignant primary brain tumor, characterized by a median survival of merely 14 months and 2-year survival rates below 10%[1, 2]. Currently, standard treatment for GBM involves surgical resection followed by radiation therapy and adjuvant temozolomide chemotherapy[3]. Despite these efforts, the prognosis remains far from optimistic, and recurrence post-treatment is prevalent. A major contributor to this recurrence is the presence of glioblastoma stem cells (GSCs)[4, 5]. GSCs are an independent cell subpopulation displaying robust self-renewal, differentiation, and sphere-forming capabilities. It is reported that GSCs are resistant to conventional radiotherapy and chemotherapy, thereby resulting in GBM recurrence[6, 7]. Researchers are actively exploring various treatments, including CAR-T therapy, oncolytic virus therapy, and others[8]. However, the therapeutic efficacy of these approaches is hampered by the intricate nature of GBM[8, 9]. Consequently, the focal point should be on devising strategies to effectively eliminate GSCs, addressing a singular target as the foundation of GBM treatment.

Apoptosis, a programmed cell death, serves not only as a physiological mechanism ensuring normal cell development and tissue homeostasis but also as a therapeutic pathway capable of disrupting organ function and inducing local or systemic inflammation[10, 11]. It can activate the intrinsic apoptotic pathway by Bcl-2-mediated mitochondrial cytochrome c release or initiate the extrinsic apoptotic pathway through the binding of death receptor ligands. Subsequently, caspase cleavage of downstream signaling pathways is promoted, culminating in apoptosis[11–13]. The Bcl-2 family encompasses anti-apoptotic proteins such as BCL-2, BCL-X_L, MCL-1, and BCL-W, as well as pro-apoptotic proteins like Bax and Bak[14, 15]. Additionally, there are pro-apoptotic "activators" (BIM, BID, PUMA) and "sensitizers" (BAD, NOXA, BIK, etc.)[16]. In cancer cells, strategies involve up-regulating anti-apoptotic proteins like BCL-2 and BCL-X_L while down-regulating or inactivating pro-apoptotic components such as Bax and Bak to evade apoptosis and enhance resistance to radiotherapy and chemotherapy[17, 18]. Targeting an important regulator of the Bcl-2 protein family has proven effective in enhancing radiation therapy and overcoming apoptosis resistance in various cancers[19, 20]. This has led to significant pharmaceutical interest in developing inhibitors for BCL-2 family proteins, with the FDA-approved BCL-2 inhibitor venetoclax finding wide application in B-cell and myeloid malignancies[21, 22]. However, the clinical utility of the BCL-2/BCL-X_L dual inhibitor, ABT-263, is constrained by its targeting specificity and thrombocytopenic toxicity. Platelets, being entirely dependent on BCL-X_L for survival, limit the use of ABT-263 due to its impact on thrombocytopenia. Thrombocytopenia can result in bleeding in the gastrointestinal tract, urinary tract, conjunctiva, and mucous membranes, as well as significant postoperative bleeding. In severe cases, it can cause spinal or intracranial hemorrhage, as well as lower limb paralysis or intracranial hypertension, posing a threat to life. Consequently, while BCL-X_L stands out as a promising cancer target, its exploration has been hampered by thrombocytopenic toxicity[23, 24]. In this context, we present the application of PROTAC to selectively target BCL-X_L, aiming to mitigate platelet-related toxicity and on-target.

PROTAC represents a cutting-edge protein degradation technology that triggers the degradation of disease-causing target proteins, offering innovative avenues for drug development[25, 26]. A PROTAC is a heterobifunctional molecule comprising three essential components: a ligand binding to the target protein (Protein of interest), a ligand binding to the E3 ligase, and a linker connecting the two ligands[25, 27]. These PROTAC molecules engage both the target protein and E3 ligase simultaneously, forming a ternary complex. This complex facilitates the transfer of ubiquitin from the E3 ligase to the target protein, leading to its ubiquitination[28–31]. The ubiquitinated target protein is subsequently recognized and degraded into amino acid fragments by the 26S proteasome within the cell[32]. Currently, widely utilized E3 ligases include von Hippel-Lindau (VHL), mouse double minute 2 homolog (MDM2) and cereblon (CRBN)[28, 33]. Studies have indicated that employing VHL as a PROTAC for E3 ligases can mitigate the hematological toxicity associated with ABT-263[34]. Notably, the E3 ligase MDM2 selectively binds to the P53 site on the surface of MDM2, concurrently stabilizing the P53 protein and degrading target proteins, so that enhanced anti-tumor activity[35, 36]. Nutlin-1 is the first-in-class effective and selective MDM2 inhibitor that functions as a ligand for MDM2[37].

In this study, we performed bioinformatics analysis by integrating published literature databases, found that MDM2 is highly expressed in GBM while being lowly expressed in platelets[34]. For this reason, utilizing MDM2 to recruit PROTAC for BCL-X_L degradation theoretically reduces its platelet toxicity, resulting in effective in vivo therapeutic effects. By analyzing the structure of ABT-263 and Nutlin-1, their derivatives were synthesized. And then these two small molecules were connected by using an appropriate linker to synthesize PROTAC with targeted specific degradation of BCL-X_L. Through retrosynthetic analysis of the structures of ABT-263 and Nutlin-1, rational derivatives were synthesized, and suitable linkers were employed to connect the two small molecules into PROTACs, AN-1 and AN-2. These PROTACs exhibited exceptional specificity and potent efficacy in degrading BCL-X_L with GSCs, demonstrating their excellent in vivo tumor-targeting capability, potent anti-tumor effects, and reduced platelet toxicity on normal organs (Fig. 1). These findings demonstrated the potential of AN-1 and AN-2 as safe first-in-class BCL-X_L-targeting antitumor agents. Moreover, our results clarified a novel general strategy to transform anti-tumor agents with target-specificity and dose-limiting toxicities into tumor-selective, less toxic PTOTACs by utilizing specific E3 ligase.

ABT-263 regulates GSC tumorigenesis

GBM is a common and highly lethal glioma, characterized by rapid progression and recurrence[38, 39]. Oral alkylating agent temozolomide is the first-line chemotherapy drug for GBM patients following surgical resection[40, 41]. Increased expression of anti-apoptotic BCL-2 proteins has been reported in various solid tumors and is often associated with resistance to conventional chemotherapy[42–44]. Inhibiting these BCL-2 family proteins with small molecules has been widely explored as a therapeutic strategy for cancer[45, 46]. ABT-263 is a dual inhibitor of BCL-2 and BCL-X_L, but its clinical application is limited due to its low targeting and toxicity such as thrombocytopenia[34, 47].

To investigate the relationship between BCL-2 proteins and clinical outcomes in tumor patients, we initially analyzed the expression of BCL-X_L and BCL-2 in the REMBRANDT database. Compared to non-tumor brain tissue, BCL-X_L mRNA expression was significantly upregulated in GBM patients (Fig. S1A). Among the 398 patients in the glioma cohort, patients who had higher BCL-X_L expression exhibited significantly shorter survival compared to those with lower expression (Fig. S1B). Consistently, in the CGGA database, Higher BCL-X_L expression predicted adverse prognosis in glioma patients (Fig. S1C and D). However, the mRNA expression of BCL-2 showed no obvious difference between non-tumor and GBM patients. Additionally, there was no impact on the survival period of glioma patients no matter what expression of BCL-2 in the REMBRANDT database (Fig. S1E and F). The above findings were consistent with the CGGA database (Fig. S1G and H). These findings collectively reveal a significant association between BCL-X_L mRNA expression and poor prognosis in glioma patients. These indicate that BCL-X_L might be considered as a target for treating GBM. Subsequently, to further validate the feasibility of BCL-X_L as a target, we collected samples of normal astrocytes (ASCR), glioblastoma cells (U87, U251), and GSCs (X01, 83, 448). Through immunoblotting, the results showed that BCL-X_L was overexpressed in X01, 83, and 448 cells relative to ASCR (Fig. S2A). Following this, the expression of BCL-X_L was down-regulated while there was no distinction in BCL-2 since we used BCL-2/BCL-X_L inhibitor, ABT-263, consistent with previous findings[34](Fig. S2B). Thus, it concluded that ABT-263 could inhibit specifically the expression of BCL-X_L, which laid a solid foundation for further research.

To explore the effects of ABT-263, we selected 83 and X01 GSCs to carry out the following research. Compared to the control group, ABT-263 inhibited the expression of BCL-X_L in a concentration-dependent manner, with maximum inhibition achieved at 1 µM in X01 and 83 GSCs. The presence of GSC suggests the capability for indefinite proliferation and demonstrates a high degree of plasticity, thus contributing to the observed intratumor heterogeneity in GBM^[47]. To explore the role of ABT-263 in maintaining GSC tumorigenicity, we first tested the proliferation of the live floating cells in a culture medium. Data showed that with increasing concentrations of ABT-263, its ability to inhibit cell (X01 left, 83 right) growth was significantly enhanced (Fig. 2A). Furthermore, ABT-263 significantly decreased the sphere-forming ability of X01 and 83 GSCs in limiting dilution assays (LDAs), a widely used method for determining the self-renewal capacity of stem cells (Fig. 2B and C). Based on these findings, we further explored whether this inhibitory effect is related to programmed cell death in GSCs. To evaluate this phenomenon, we examined the expression of the core apoptotic effectors PARP1 and caspase3. The results showed that the specific inhibition of BCL-X_L initiated cell apoptosis (Fig. 2D). Using flow cytometry with membrane-associated protein (Annexin V) and 7-AAD staining, we found that X01 and 83 GSCs death increased in a dose-dependent manner with a total of 47.29%and 43.1% (Q2 + Q3) cell death in 1µM (Fig. 2E). These results indicated that BCL-X_L inhibition had a better anti-GBM ability.

AN-1 and AN-2 induce proteasomal degradation of BCL-X_L

As reported, ABT-263 is not approved because inhibition of BCL-X_L induces on-target and dose-limiting thrombocytopenia[24, 48, 49]. To overcome these difficulties, researchers have designed a selective BCL-X_L PROTAC, that targeted BCL-X_L to the Von Hippel-Lindau (VHL) E3 ligase for degradation[34]. These studies offer a promising avenue for the design of PROTACs targeting BCL-X_L, potentially enhancing anti-GBM therapeutic effects. Actually, the vast majority of the approximately 600 E3 ligases in humans remain unexplored, presenting significant opportunities for developing degraders that target oncoproteins with tissue, tumor, and subcellular selectivity. Currently, widely utilized E3 ligases include VHL, MDM2 and CRBN[32, 35]. Next, we analyzed the abovementioned typical E3 ligases. From the REMBRANDT database, MDM2 exhibited higher expression in GBM, whereas VHL displayed no obvious differences and CRBN showed lower expression in GBM compared with non-tumor, suggesting BCL-X_L targeting VHL PROTAC may not working in GBM[34] (Fig. S1I). Besides, we also detected the expression of MDM2 in GSCs. Surprisingly, MDM2 showed higher expression in GSCs (Fig. S1I). Thus, we developed BCL-X_L targeting MDM2 PROTAC for GBM-targeting therapy. To mitigate the toxicity of ABT-263, through the design and synthesis of derivatives from Nutlin-1 (Scheme 1) and ABT-263 (Scheme S1-S3), linked together by two different types of linkers, we created the PROTACs, AN-1 and AN-2, targeting the degradation of BCL-X_L. The synthesis of PROTACs, AN-1 and AN-2, are outlined in Scheme 1. Firstly, compound N1 reacts with iodomethane to obtain N2, and then reacts with iodoisopropane to obtain N3. Compound N3 reacts with (1R, 2S)-1,2-bis (4-chlorophenyl) ethane-1,2-diamine under the catalysis of NBS to obtain compound N4. Compound N4 reacts with triphosgene under the catalysis of TEA, and then with Boc-piperazine to obtain compound N5. Then, the Boc protecting group was removed by TFA to give compound N6. Compound N6 reacts with succinic anhydride to obtain compound N7, which was further condensed with intermediate compound D10 (Scheme. S3) under the conditions of TBTU and DIPEA to obtain AN-1. Compound N7 was condensed with tert butyl 3- (2- (2-aminoethoxy) ethoxy) propanoate under the conditions of TBTU and DIPEA to obtain compound N8. Then, the tBu protecting group was removed by TFA to give compound N9. Compound N9 was condensed with compound D10 under the conditions of HBTU and DIPEA to obtain AN-2.¹H NMR and ¹³C-NMR spectroscopy was used to confirm the structures of key intermediates.

PROTAC is a bivalent small molecule which could bind the target protein (BCL-X_L) and E3 ligase (MDM2) to form a ternary complex, and subsequently tag the target protein. In this case, ubiquitinated proteins are recognized and degraded by the proteasome (Fig. 3A). Subsequently, we validated the synthesized AN-1 and AN-2's ability to degrade BCL-X_L protein at the cellular level. At a concentration of 5 µM, immunoblotting results demonstrated that over time, AN-1 and AN-2 exhibited an enhanced capability to degrade BCL-X_L (Fig. 3B and C). To confirm whether the degradation of BCL-X_L by AN-1 and AN-2 occurs through the typical proteasomal pathway, we used the proteasome inhibitor MG132 for blockade. Immunoblot results indicated that MG132, inhibiting proteasomal activity, could abolish the BCL-X_L degradation induced by AN-1 and AN-2, suggesting that AN-1 and AN-2 degrade BCL-X_L through the ubiquitin-proteasome pathway, consistent with the typical PROTAC mechanism (Fig. 3D).

Effects of AN-1 and AN-2 on GSCs level

Next, we assessed the activity of AN-1 and AN-2 against GSCs. Initially, we detected GSC proliferation. Results indicated a dose-dependent growth inhibition on the second day of treatment with AN-1 and AN-2 in GSCs, highlighting the significant capability of AN-1 and AN-2 in inhibiting GSC growth (Fig. 4A, B and Fig. S3A). Additionally, we evaluated the impact of AN-1 and AN-2 on GSCs' ability to form spheres. Representative images of the GSC clones were presented (Fig. 4C and Fig. S3B left). Quantification data also demonstrated that AN-1 and AN-2 are more powerful than the vehicle group in inhibiting the GSC sphere clone formation (Fig. 4D and Fig. S3B right). The previous results indicated that AN-1 and AN-2 significantly inhibit GSC proliferation and sphere clone formation. Furthermore, we aimed to understand whether this inhibitory effect is related to programmed cell death in GSCs. Immunoblotting results showed that at a concentration of 0.1 µM, core apoptotic effectors PARP and caspase3 were cleaved, indicating that AN-1 and AN-2 initiate cell apoptosis, and the cleavage effect becomes more pronounced with increasing concentrations (Fig. 4E and Fig. S3C). Meanwhile, flow cytometry results demonstrated dose-dependent cell death in GSCs. In X01 cell, the apoptosis rate for AN-1 at 0.1 µM was 21.72% (Q2 + Q3), and for AN-2, it was 26.12% (Q2 + Q3). And also, in 83 and 448 GSCs, the apoptosis rate for AN-1 at 0.1 µM was 22.8% (Q2 + Q3), 24.52% (Q2 + Q3), and for AN-2, it was 24.93% (Q2 + Q3), 27.92% (Q2 + Q3). In comparison, the apoptotic effects of AN-1 and AN-2 were superior to ABT263, with AN-2 showing better apoptotic effects than AN-1 (Fig. 4F and Fig. S3D). These data suggest that AN-1 and AN-2 possess stronger anti-GSC capabilities and may require further validation in vivo.

AN -1 and AN-2 are more potent and safer antitumor agent than ABT263 in vivo

The preceding research data indicated that AN-1 and AN-2 effectively inhibit GSC proliferation and sphere formation at low concentrations while promoting apoptosis without comparing with ABT-263. To better highlight the advantages of AN-1 and AN-2 over ABT-263, we further aimed to assess the inhibitory effects of AN-1, AN-2, and ABT-263 on BCL-X_L degradation, cell viability, and sphere-forming ability at low concentrations. Immunoblotting results demonstrated that at 0.1µM, AN-1 and AN-2 induce BCL-X_L degradation, while the degradation ability of ABT-263 on BCL-X_L is relatively less pronounced (Fig. S4A). GSC proliferation results showed that AN-1, AN-2, and ABT-263 all inhibit cell growth, with AN-1 and AN-2 outperforming ABT-263, and AN-2 is better than AN-1 (Fig. S4B). Furthermore, we assessed the sphere-forming ability of GSCs. Representative images of the GSC clones were presented, and quantification data also demonstrated the AN-1 and AN-2 are more powerful than ABT-263 treatment in inhibiting the GSC sphere clone formation (Fig. S4C). Subsequently, we tested the in vivo anti-GBM potential of the AN-1 and AN-2 using mouse xenograft models from the 83 GSC (Fig. 5A). After implanting tumor cells, mice were randomly divided into four groups (Vehicle, ABT-263, AN-1 and AN-2). Starting intraperitoneal injection administration one week later. On the 11th day, fluorescence imaging was performed (Fig. 5B), and mouse weights were measured every other day after administration. Compared to the vehicle and ABT-263 groups, the AN-1 and AN-2 groups showed significantly extended survival (Fig. 5C). Moreover, the tumor weight of the AN-1/AN-2 group showed a significant decrease compared with the control group (Fig. 5D). After finishing treatment, the brains of these mice were harvested for detailed slice observation. As expected, the results show that AN-1 and AN-2 treatment leads to decreased Ki67 levels accompanied by increased Cleaved-Caspase3 and TUNEL, proving strong proliferation inhibition, and effective tumor cell ablation by apoptosis induced by AN-1 and AN-2 (Fig. 5E). These data suggest that AN-1 and AN-2, as BCL-X_L-targeting degraders, offer potential for both degradation and anti-GBM efficacy.

BCL-X_L is one of the most critical and validated cancer targets to date, and there is currently a lack of safe and effective treatments. Inhibiting BCL-X_L can induce targeted and dose-limiting thrombocytopenia. Therefore, we further evaluated the toxicity of AN-1, AN-2, and ABT-263 in a mouse model. Mice were intraperitoneally administered every other day, and blood samples were taken after two administrations. The data showed significant platelet toxicity with ABT-263, while no apparent toxicity was observed with AN-1 and AN-2. Compared to the control group, red blood cells remained largely unchanged, and although lymphocytes and white blood cells showed slight decreases in the treatment groups, the trends in reduction with AN-1 and AN-2 were lower than with ABT-263 (Fig. 5F). These findings suggested that at therapeutic equivalent doses, the platelet toxicity of AN-1 and AN-2 was lower than that of ABT-263. These results indicated that AN-1 and AN-2 were safer and more effective anti-tumor drugs than ABT-263, with the potential to be used as a single-agent therapy for tumors relying solely on BCL-X_L.

Here, we present a novel strategy aimed at enhancing the specificity of targeting and reducing the systemic toxicity of anti-tumor drugs using PROTAC technology. Specifically, we transform the BCL-2 family inhibitor ABT-263, known for its high platelet toxicity, into a BCL-X_L-specific PROTAC (AN-1 and AN-2) with markedly reduced side efficacy. The AN-1 and AN-2 show superior inhibition of GSCs proliferation and demonstrate significant anti-GBM efficacy in vivo without inducing thrombocytopenia, a common side effect associated with ABT-263.

ABT-263, also recognized as Navitoclax, serves as a potent inhibitor of BCL-2 family proteins, pivotal in regulating apoptosis, the programmed cell death process[50, 51]. Its significance lies in its potential as an anti-cancer agent, particularly in treating hematological malignancies and solid tumors. By inhibiting BCL-2 and related proteins, ABT-263 induces apoptosis in cancer cells, leading to their death[52]. However, ABT-263 also presents significant limitations and challenges[53]. One major concern is its potential to induce thrombocytopenia, characterized by low platelet levels in the blood. BCL-2 inhibition by ABT-263 can trigger platelet apoptosis, potentially leading to heightened bleeding risks and dose-limiting toxicities[53]. This adverse effect has prompted efforts to develop next-generation BCL-2 inhibitors with improved safety profiles[54]. To overcome these challenges, we linked the BCL-2 and BCL-X_L binding moiety from ABT-263 to MDM2 with an optimized linker. By applying MDM2 as an E3 ligase, AN-1 and AN-2 have emerged as BCL-X_L-targeting anti-GBM drugs, eliminating the drawbacks of low targeting specificity and high toxicity. Compared with ABT-263, they have excellent antitumor activity while reducing platelet toxicity. This is the first time to use of MDM2 as an E3 ligase in GBM therapy, capitalizing on its high expression in GBM and low in platelets, thereby enhancing both specificity and safety.

GBM poses formidable challenges in comprehending its biological mechanisms, devising effective therapies, and enhancing patient prognosis[55, 56]. Temozolomide serves as a clinical treatment for GBM; however, its vulnerability to drug resistance frequently contributes to GBM recurrence[57, 58]. In comparison to traditional inhibition techniques, PROTAC offers the potential to eliminate target proteins rather than merely inhibiting their activity, potentially resulting in more profound and durable therapeutic effects. Moreover, PROTAC depends on E3 ligases to trigger protein degradation. Based on this premise, it becomes feasible to target E3 ligases with high expression specifically in the target cells, without affecting other cellular components. In fact, scientists have explored a PROTAC named DT2116, which targeted BCL-X_L rather than BCL-2, utilizing VHL as an E3 ligase. VHL is scarcely expressed in platelets but highly expressed in tumor cells (breast cancer, liver cancer, prostate cancer, colorectal cancer, renal cell carcinoma, acute lymphoblastic leukemia). This effectively reduces the risk of thrombocytopenia caused by PROTACs. DT2116 efficiently suppresses the growth of aforementioned tumors either as a standalone treatment or when used in combination with other chemotherapeutic agents. Importantly, it does not induce appreciable thrombocytopenia[34]. And also, another study in the same team, it was found that DT2116 have an important role in inhibiting tumor growth (non-small cell lung cancer, colorectal cancer and pancreatic cancer) when combined with Sotorasib (a covalent inhibitor of KARS^G12C)[59]. Meanwhile, Tan et al. reported a selective BCL-X_L PROTAC degrader (SIAIS361034), targeting BCL-X_L to CRBN E3 ligase for degradation. CRBN is moderately expressed in platelets, but its expression level is significantly lower compared to tumor cells. SIAIS361034 could remarkably suppress the growth of medulloblastoma tumor carrying W539J mutation in Smoothened[60]. In this study, we chose to utilize MDM2 as the E3 ligase primarily due to its elevated expression in GBM while being relatively lower in platelets. This strategic choice effectively mitigates the issue of platelet toxicity. Meanwhile, it exhibits outstanding anti-GBM efficacy and significantly prolongs the survival of mice.

In summary, the synthesis of selective BCL-X_L PROTACs (AN-1 and AN-2) holds great promise for targeted therapy in GBM. By exploiting the unique vulnerabilities of GSCs and harnessing underutilized E3 ligases, this approach offers new avenues for improving therapeutic outcomes in this challenging malignancy. Further preclinical and clinical studies are warranted to validate the efficacy and safety of this approach and to facilitate its translation into clinical practice.

Chemical Synthesis and Structural Characterization of Intermediates and Target Compounds

¹H NMR and ¹³C NMR spectra were recorded on Bruker AVANCE Ⅲ spectrometer (Bruker Company, Germany), using TMS as an internal standard CDCl₃ and DMSO-d₆ as solvents. Chemical shifts are given in ppm (δ). TLC analysis was carried out on silica gel plates GF254 (Qingdao Haiyang Chemical, China). Silica gel column chromatography was performed with Silica gel 60 G (Qingdao Haiyang Chemical, China). Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification.

Cell culture

Patient-derived GBM stem cells (X01, 83, and 448) were maintained in DMEM/F-12 supplemented with B27 (Invitrogen), EGF (10 ng mL^− 1, R&D Systems), and bFGF (5 ng mL^− 1, R&D Systems). ASCR, U87, and U251 cells were purchased from the American Type Culture Collection (ATCC). These cells were maintained in DMEM (dulbecco′s modified eagle medium) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin solution. All cells were cultured under CO₂ (5%) atmosphere at 37°C.

Cell proliferation

GSCs were plated at a density of 1000 cells per well in 96-well plates in 80 µL of the medium., containing DMEM/F-12 medium supplemented with B27, epidermal growth factor 24 (EGF; 10 ng mL^− 1), and basic fibroblast growth factor (bFGF; 5 ng mL^− 1). After that, DMSO was added to the control group, and the drug was added to the experimental group, so that the volume of each well was 100µL, with 3 wells in each group. Cell counting kit-8 (CCK-8) (Beyotime) was used to identify the antiproliferative activity of compounds, and plates were read at 450 nm using a Bio-Tek Synergy 2 microplate reader. Data were analyzed using GraphPad Prism 7.0 Software.

Sphere formation assay

GSCs were seeded in 24-well plates with 800 cells per well. Three wells were repeated in each group, and the volume of culture medium in each well was 500 µL. After 12 h, DMSO was added to the control group and drugs were added to the experimental group so that the volume of each well was 1 mL. The 24-well plate was transferred to the incubator to observe the growth state of cells, and photos were taken under an inverted fluorescence microscope to calculate the number of cell pellets in each well.

Flow cytometry analysis of cell apoptosis

After different treatments, X01, 83, and 448 cells were resuspended in BD Pharmingen staining buffer (cat# 554657; BD Biosciences). Cells were incubated with FC block (cat# 553141; BD Biosciences) for 20 minutes. Subsequently, cells were washed and resuspended in 100 µL of BD Pharmingen staining buffer and incubated with APC Rabbit Anti-Mouse CD86 (cat# 560582; BD Biosciences), FITC Rabbit Anti-Mouse CD206 (cat# 565250; BD Biosciences) on ice for 20 minutes. Cells were washed three times with a staining buffer and resuspended in the staining buffer. Cells were centrifuged and resuspended in staining buffer for FACS analysis (FACS Celesta; BD Bioscience, San Jose, USA). After different treatments, Astrocytes and GSCs were resuspended in 0.9% NaCl buffer. Half the cells of the monochromatic component were scalded to death at 80 degrees. Subsequently, cells were incubated with Annexin V-FITC and propidium Iodide on ice for 10 minutes. Then all samples were examined by flow cytometry. All FACS data analysis was performed using FLOWJOTM Software.

Immunoblotting

The cells were seeded in six-well plates at an individual density for 24 or 48 h and then treated with dimethyl sulfoxide (DMSO) or compounds at the indicated concentrations at various times. After being treated with the indicated concentrations of compounds for a specific time, the cells were harvested and washed once with phosphate-buffered saline (PBS) and then lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, #P0013C) according to the directions. The suspension was centrifuged at 13 000 rpm for 15 min to remove insoluble material, and a bicinchoninic acid (BCA) assay was employed to determine the concentration of soluble proteins (Beyotime, #P0011). The lysate protein (20 − 40 µg) was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to poly (vinylidene difluoride) (PVDF) membranes (Millipore, #IPVH00010). The membranes were blocked with 5% nonfat milk or 5% bovine serum albumin (BSA) for 1 h and then incubated with the specific primary antibodies with a 1:1000 dilution, respectively, at 4°C overnight. The primary antibodies and secondary antibodies were purchased from Abclonal and Biosharp, respectively. Membranes were imaged by the Tanon-5200 Chemiluminescent Imaging System with ECL luminescence reagents (Meilunbio, #MA0186).

In vivo experiments

All animal experiments follow standard protocols approved by Henan University Laboratory Animal Center and Ethics Committee (HUSOM2022-450). Female BALB/c nude mice aged 5 weeks were purchased from SPF (Beijing) BIOTECHNOLOGY Co., Ltd., and housed under specific pathogen-free conditions. Mice were group-housed in ventilated cages under controlled temperature and humidity with a 12-hour light-dark cycle. Every animal was randomized by body weight before the experiments. For the orthotopic mouse model, GSCs were first resuspended and then transplanted into the left striatum of 6-week-old female BALB/c nude mice via stereotactic injection. The injection coordinates were 2.2 mm to the left of the midline and 0.2 mm posterior to the bregma at a depth of 3.5 mm. The mice were sacrificed with carbon dioxide when a 20% reduction in weight or severe neurological symptoms were observed, and the brain of each mouse was harvested and fixed in 4% paraformaldehyde for 24 hours at 4°C for further use. Their survival was analyzed using GraphPad PRISM software (version 7; GraphPad PRISM, La Jolla, CA, USA).

Histology and tissue staining

For histological observations, the brains were removed, fixed with 4% paraformaldehyde for 24 h at 4°C, and sectioned at a thickness of 4 µM using an essential microtome (Leica RM2125 RTS). For histological observations, hematoxylin and eosin (H&E) stains were carried out with 1% hematoxylin (DaKo) and 0.25% eosin (Merck). Stained sections were dried and mounted in an organic mounting medium. Before immunohistochemical (IHC) staining for Ki67(1:100, R&D beyotime), Cleaved-Caspase3(1:200, Cell Signaling Technology), the sections were subjected to an antigen retrieval process using citrate buffer (Ph = 6.0), and endogenous peroxidase was blocked by incubating with 3% hydrogen peroxide. The tissue sections were then incubated overnight at 4°C in a humidified chamber with the primary antibody, diluted with antibody diluent buffer (IHC World). The tissue sections for 3,3’-diaminobenzidine (DAB) staining were developed using DAB (Vector Laboratories) as the chromogen.

Statistical analysis

Three independent experiments were applied in this study. Data were obtained as means ± standard deviation (SD). Values of P < 0.05 were considered as statistically significant.

Conflicts of interest

There are no conflicts to declare.

Funding

This work was supported by the Henan Provincial Technology R&D Plan Joint Fund (232301420017).

Author Contributions

B. S., M. L. and J. L. designed the experiments. B. S. and M. L. performed the synthesis, characterization, and assays. R. N. and X. Y. assisted with the animal experiments. All authors contributed to the analysis and discussion of the results. B. S. and M. L. wrote the manuscript. J. W., B, Y., and J. L. conceived and supervised this research.

Acknowledgements

The authors thank BioRender app for applying cartoon.

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Scheme1. Chemical synthesis of AN-1 and AN-2.

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Synthesis of selective BCL-XL PROTAC and potent antitumor activity in glioblastoma

Status:

Version 1

Abstract

Figures

Introduction

Results

ABT-263 regulates GSC tumorigenesis

AN-1 and AN-2 induce proteasomal degradation of BCL-X_L

Effects of AN-1 and AN-2 on GSCs level

Discussion

Experimental Methods

Chemical Synthesis and Structural Characterization of Intermediates and Target Compounds

Cell culture

Cell proliferation

Sphere formation assay

Flow cytometry analysis of cell apoptosis

Immunoblotting

Histology and tissue staining

Statistical analysis

Declarations

Conflicts of interest

Funding

Author Contributions

Acknowledgements

References

Scheme

Additional Declarations

Supplementary Files

Status:

Version 1

Synthesis of selective BCL-XL PROTAC and potent antitumor activity in glioblastoma

Status:

Version 1

Abstract

Figures

Introduction

Results

ABT-263 regulates GSC tumorigenesis

AN-1 and AN-2 induce proteasomal degradation of BCL-XL

Effects of AN-1 and AN-2 on GSCs level

Discussion

Experimental Methods

Chemical Synthesis and Structural Characterization of Intermediates and Target Compounds

Cell culture

Cell proliferation

Sphere formation assay

Flow cytometry analysis of cell apoptosis

Immunoblotting

Histology and tissue staining

Statistical analysis

Declarations

Conflicts of interest

Funding

Author Contributions

Acknowledgements

References

Scheme

Additional Declarations

Supplementary Files

Status:

Version 1

AN-1 and AN-2 induce proteasomal degradation of BCL-X_L