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. 2025 Sep 3;8(9):3160–3169. doi: 10.1021/acsptsci.5c00321

Development of PROTAC-Based Strategies for Cannabinoid Receptor Type 1 (CB1R) Degradation in Cancer

Aseel Kashkush 1, Kisanet Tadesse 1, Judith Furth-Lavi 1, Raphael I Benhamou 1,*
PMCID: PMC12441849  PMID: 40969876

Abstract

The cannabinoid receptor type 1 (CB1R), a key component of the endocannabinoid system (ECS), has been implicated in various oncogenic processes. Its overexpression in breast cancer has been associated with tumor progression and metastasis, primarily through regulation of the cell cycle. Given its role in cancer biology, CB1R represents a promising therapeutic target. In this study, we utilized Proteolysis Targeting Chimera (PROTAC) technology to design and synthesize a series of bifunctional small molecules capable of selectively degrading CB1R in cancer cells. These compounds were specifically engineered to avoid central nervous system (CNS) penetration, thereby minimizing adverse effects linked to the parent compound, Rimonabant. Several of the synthesized molecules effectively induced CB1R degradation. The most promising lead compound not only reduced CB1R-associated downstream signaling but also suppressed cancer cell proliferation and promoted apoptosis, highlighting its therapeutic potential. Importantly, in a 3D spheroid cancer model, the lead compound significantly reduced tumor growth compared to the known CB1R antagonist Rimonabant, demonstrating superior efficacy in targeting both individual cancer cells and complex tumor architecture. Consistent with its design, in vivo evaluation confirmed that the compound does not significantly penetrate the blood–brain barrier, supporting its peripheral selectivity. Overall, our findings establish targeted CB1R degradation via PROTACs as a viable and innovative strategy for cancer therapy, paving the way for the development of next-generation, precision-targeted therapeutics.

Keywords: cannabinoid receptor type 1 (CB1R), PROTAC, targeted degradation, breast cancer, drug design

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The cannabinoid receptor type 1 (CB1R) is a key component of the endocannabinoid system (ECS), primarily expressed in the central nervous system (CNS), with the highest densities located in the brain and spinal cord. − However, it is also expressed in various endocrine and peripheral tissues, including the liver, skeletal muscles, and the cardiovascular system. As a member of the G-protein-coupled receptor (GPCR) family, CB1R is distinguished by its seven transmembrane helices, which facilitate signal transduction and cellular responses to cannabinoid ligands. These cannabinoid ligands can be classified into two main types. The first type includes endogenous ligands, which are naturally produced in the body and derived from arachidonic acid, then metabolized into 2-arachidonoylglycerol (2-AG) and anandamide (AEA). These ligands play a crucial role in modulating various physiological processes by binding to CB1R and other cannabinoid receptors, such as cannabinoid receptor type 2 (CB2R). The second type consists of exogenous ligands, such as Δ9-tetrahydrocannabinol (THC), a plant-derived cannabinoid found in Cannabis sativa, which can also interact with CB1R and influence its signaling pathways. −

Over the past few decades, research has revealed that the ECS, including the CB1R, plays a significant role in various physiological processes and is implicated in multiple diseases, such as chronic liver disease, cancer, , and neurological disorders. , Moreover, CB1R has been shown to regulate several metabolic pathways. Studies have reported that CB1R overactivation is frequently observed in cancers including breast cancer, where it is associated with tumor progression and clinical outcomes. This occurs through mechanisms that enhance cancer cell proliferation, invasion, and migration, while simultaneously inhibiting apoptosis. , CB1R activation in breast cancer cells triggers multiple downstream signaling pathways that collectively promote cell survival, proliferation, and resistance to apoptosis. One major pathway involves the phosphorylation and activation of AKT (AKT serine/threonine kinase), which enhances the transcription and stability of antiapoptotic proteins such as BCL2, thereby supporting tumor cell survival. In parallel, CB1R stimulation can also activate the ERK (extracellular signal-regulated kinase) pathway, leading to ERK phosphorylation (pERK), which further drives oncogenic signaling by enhancing cell survival and suppressing apoptotic responses. This pathway may become hyperactivated in CB1R-overexpressing breast cancers, further amplifying pro-survival signals and contributing to tumor progression. Additionally, CB1R activation may regulate the expression of MCM5, a gene essential for regulating DNA replication and maintaining proper cell cycle progression. MCM5 overexpression has been associated with increased proliferation and has been implicated in the development of various cancers, further underscoring the role of CB1R in supporting tumorigenic cellular behaviors (Figure A).

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(A). CB1R signaling pathway in breast cancer and its role in disease progression. (B). PROTAC strategy utilized in this study for CB1R degradation.

The CB1R plays a central role in the endocannabinoid system, regulating a wide range of neurophysiological processes such as mood, cognition, appetite, motor control, and coordination. − Given its widespread influence on these essential functions, modulating CB1R activity has become a focus of pharmacological research, leading to the development of compounds designed to either enhance or suppress its effects. CB1R-targeting compounds fall into two main categories: agonists, such as THC and Dronabinol, which activate the receptor, and antagonists or inverse agonists, such as Rimonabant and Taranabant, which inhibit its activity. − Since CB1Rs are primarily concentrated in the CNS, the blood–brain barrier (BBB) plays a vital role in regulating the entry of these compounds, affecting their bioavailability and pharmacodynamics. Most CB1R agonists and antagonists are highly lipophilic, enabling them to cross the BBB and exert central effects. However, excessive activation or inhibition of CB1R has been linked to severe psychiatric side effects, including depression and suicidal ideation. A notable example is Rimonabant, a selective CB1R antagonist originally developed for the treatment of obesity and metabolic disorders, with minimal or no affinity for other receptors. Although it demonstrated efficacy in weight management, it was later withdrawn from the market due to severe psychiatric side effects. , These adverse effects were attributed to the hydrophobic nature of the molecule, allowing it to cross the BBB and impact central CB1R, ultimately leading to the neurological complications associated with its use. Beyond the CNS, CB1R is also expressed in peripheral tissues, and researchers seek to develop peripherally selective CB1R-targeting compounds that do not cross the BBB. These compounds reduce CNS-related adverse effects while offering therapeutic potential for cardiovascular, metabolic, and cancer-related conditions. By selectively targeting CB1R activity in the periphery, these novel drugs could provide safer and more effective treatments for various diseases while minimizing the risks associated with central CB1R modulation.

A promising strategy for targeted cancer therapy involves hijacking the cell’s natural protein degradation machinerythe ubiquitin–proteasome system (UPS). This tightly regulated pathway relies on a cascade of enzymes, including E1, E2, and E3 ligases, that work together to tag unwanted proteins with ubiquitin, marking them for recognition and degradation by the 26S proteasome. Proteolysis-targeting chimeras (PROTACs) have emerged as an innovative tool to exploit this system. These heterobifunctional molecules consist of a ligand that binds the protein of interest, a ligand that recruits an E3 ligase, and a linker connecting the two. Upon binding, they promote the formation of a ternary complex which facilitate selective ubiquitination and subsequent degradation of the target protein (Figure B). − Though PROTACs were initially developed for intracellular proteins, several recent studies have extended this concept to G protein-coupled receptors (GPCRs)a class of integral membrane proteins that pose unique structural challenges for degradation. One example involves, a PROTAC targeting the α1A-adrenergic receptor (α1A-AR), overexpressed in prostate cancer, was developed by linking prazosin (a known α1A-AR antagonist) to pomalidomide (a CRBN ligand). This molecule led to a dose- and time-dependent degradation of α1A-AR in HEK293 cells, however the exact mechanism remains unclear due to the extracellular binding site of the ligand and the intracellular localization of CRBN. Another example targets the G protein-coupled estrogen receptor (GPER) and estrogen receptor (ER) for breast cancer therapy. These PROTACs induce proteasome-dependent degradation of both receptors and inhibite breast cancer cell growth, yet the mechanism of E3 ligase recruitment remains unresolved. In contrast, a PROTAC targeting CCR9 binds to an intracellular allosteric site and recruits the VHL E3 ligase, leading to VHL- and ubiquitin-dependent degradation. This example demonstrates a more rational design strategy for degrading GPCRs by targeting intracellular binding sites compatible with E3 ligase recruitment. This approach offers a significant advantage over traditional inhibitors, particularly for challenging targets like the CB1R, which is overexpressed in various cancers, including breast cancer. While several CB1R antagonists have shown therapeutic promise, their long-term use is limited by resistance mechanismssuch as receptor upregulation and activation of compensatory pathwaysas well as the need for continuous dosing. To address these limitations, we developed PROTACs designed to degrade CB1R, enabling sustained downregulation of the receptor and prolonged therapeutic activity. In addition, the molecular design of these degraders may help limit blood–brain barrier penetration, thereby reducing the risk of central nervous system side effects. Notably, this is the first study to explore PROTAC-mediated degradation of CB1R, offering a novel and potentially transformative strategy for targeting CB1R-driven oncogenic signaling in breast cancer.

Results and Discussion

Synthesis of CB1R PROTAC Degraders

As a first step, we selected Rimonabant, the CB1R antagonist, as our CB1R binder. As previously mentioned, Rimonabant was originally developed for the treatment of obesity but was later withdrawn from the market due to severe psychiatric side effects. To conjugate Rimonabant with the E3 ligase recruiter we modified its piperidine ring by converting it into a carboxylic acid functional group. Thalidomide was chosen for its high efficiency in recruiting CRBN, which serves as the substrate receptor within the CRL4-CRBN E3 ubiquitin ligase complex. It was purposefully selected due to its lower molecular weight compared to other E3 ligase recruiters, providing a significant advantage in drug development by enhancing the design of CB1R degraders. The optimization process involved modifying both the type and length of the linker connecting the CB1R binder to the CRBN recruiting moiety to achieve optimal efficacy. Aliphatic chains ranging from 2 to 8 carbons were synthesized to identify the optimal length to direct CB1R ubiquitination (Pro-CB1 to Pro-CB4; highlighted in green, Table and Scheme S1). To introduce rigidity and restrict linker flexibility, we designed and synthesized linkers incorporating heterocyclic rings, such as piperidine and piperazine (Pro-CB5 to Pro-CB7; highlighted in pink, Table and Scheme S1). Finally, to diversify the type of heterocyclic linker, we utilized a click reaction instead of amide formation, incorporating a triazole ring between the CB1R binder and the CRBN recruiter (Pro-CB8 to Pro-CB10; highlighted in blue, Table and Scheme S2). The attachment to thalidomide was achieved through an aniline moiety. Another type of E3 ligase recruiter, an amine-functionalized VHL032 ligand was synthesized to recruit the Von Hippel-Lindau (VHL) protein into the E3 ligase complex for coupling purposes with the CB1R binder. VHL is more effective and selective for certain protein families, such as hypoxia-inducible factor (HIF-1α). Additionally, the VHL-recruiting ligand is larger than the previously synthesized thalidomide-based recruiter (Scheme S3), which may limit molecular penetration, hinder ternary complex formation required for ubiquitination, and ultimately reduce the efficiency of CB1R targeting and degradationa central goal in our E3 ligase comparison. We synthesized two CB1R degraders using the VHL recruiter. In the first degrader, the VHL recruiter is directly coupled to the CB1R binder (Pro-CB11; highlighted in gray, Table ), while in the second, the E3 ligase recruiter and CB1R binder are connected via a linker containing a triazole ring (Pro-CB12; highlighted in gray, Table ). In both cases, the VHL binder was attached via an amid bond.

1. CB1R PROTAC Derivatives .

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Four families of PROTACs were synthesized, differing in both linker type and E3 ligase recruiter. The linkers include aliphatic chains (highlighted in green), heterocyclic linkers (highlighted in pink), and triazole linkers (highlighted in blue). PROTACs containing the VHL recruiter are highlighted with a gray background.

Drug-like Properties

The physicochemical characteristics of druglikeness of all synthesized compounds were assessed using the SWISSADME platform. This tool provides a graphical representation for each compound, evaluating six essential parameters: lipophilicity, size, polarity, solubility, flexibility, and saturation (Table ). The shaded region on each plot indicates the ideal range for drug-like compounds, as established by the criteria of Lovering et al. and Ritchie et al. However, none of the synthesized PROTAC derivatives fell within this defined range (Figure S1). This aligns with the common challenge that PROTACs face in meeting Lipinski’s criteria due to their large size and bifunctional design. , To further assess druglikeness, we utilized the ADMETLab3.0 web server. Interestingly, despite not fitting traditional druglikeness parameters, all synthesized PROTAC derivatives were classified as drug like according to Pfizer’s expanded criteria (Table ), which accommodate the unique properties of hybrid drugs advancing into clinical development. ,, Moreover, according to both SWISSADME and ADMETLab3.0, none of the PROTAC derivatives exhibited BBB permeability (Table ). These findings highlight that the PROTAC derivatives are potentially peripherally restricted and should not penetrate the CNS. This characteristic provides a significant therapeutic advantage, as it reduces the risk of psychotropic side effects commonly associated with CB1R modulation in the CNS.

GLuc-CB1R Reporter Assay

To evaluate the effects of CB1R degraders in cells using a high-throughput approach, a GLuc-FLAG-CB1 reporter assay was employed. In this assay, the CB1R was fused with Gaussia Luciferase (GLuc), allowing for real-time monitoring of its expression through luminescence intensity. A decrease in GLuc luminescence indicates CB1R degradation (Figure A). HEK293 (Human Embryonic Kidney) cells were transfected with the GLuc-FLAG-CB1 plasmid, enabling expression of the fusion protein. The CB1R degraders were then administered at a single concentration of 10 μM for 24 h. We observed a decrease in luminescence intensity upon the addition of several tested compounds, indicating CB1R degradation to varying degrees. Among them, Pro-CB8 exhibited the most significant reduction in luminescence intensity (∼60%), suggesting it was the most effective CB1R degrader (Figure B). In contrast, Rimonabant, the CB1R antagonist, showed minimal luminescence reduction, indicating a negligible effect on CB1R expression. Based on these results, Pro-CB8 was selected for further evaluation due to its superior CB1R degradation activity compared to the other tested compounds.

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GLuc-CB1R reporter assay. (A). Schematic representation of the GLuc-tagged CB1 reporter. (B). Single dose experiment of all designed PROTACs at 10 μM for 24 h. Data are presented as the mean ± SD (n = 3). * Represents p ≤ 0.1, ** represents p ≤ 0.01 and *** represents p ≤ 0.001 as determined by Student t-test compared to vehicle.

Protein Expression

Following the initial cellular screening, we proceeded to evaluate our approach and derivative compounds using the MCF-7 (Michigan Cancer Foundation-7) breast cancer cell line, which is known to overexpress CB1R, a feature associated with its oncogenic characteristics. This makes MCF-7 an ideal model for evaluating the efficacy of our CB1R-targeting PROTACs. First, to confirm cellular uptake of the compound in this cell line and ensure that ubiquitination occurs intracellularly, we exploited the molecule’s intrinsic fluorescence. Using fluorescence microscopy at 3, 8, 24, and 48 h post-treatment, we tracked its accumulation. Intracellular fluorescence became detectable after 3 h, peaked at 8 h, and remained robust through 48 h, demonstrating efficient and sustained cellular penetration (Figure S2). We assessed the most promising compounds from our initial screen in an endogenous cellular assay, focusing on their ability to reduce CB1R protein levels. MCF-7 cells were treated with the selected degraders at 10 μM for 48 h, and CB1R expression was measured by Western blot. Consistent with the reporter assay results, Pro-CB8 significantly reduced CB1R levels by approximately 80% (Figure A). Interestingly, the most potent PROTAC, Pro-CB8, contained a linker featuring a triazole ring, suggesting that increased linker rigidity and the triazole ring contribute to ternary complex formation and inducing more potent degradation. To investigate degradation kinetics, Pro-CB8 was tested in a time-dependent assay at 10 μM, demonstrating a progressive, time-dependent reduction in CB1R levels with the most potent effect observed after 48 h of treatment (Figure B). Based on these findings, Pro-CB8 was further tested in MCF-7 cells for 48 h, in a dose response manner across a concentration range of 1 μM to 20 μM. Pro-CB8 significantly reduced CB1R protein levels, achieving approximately 75% degradation at the highest concentrations with a calculated degradation concentration (DC50) of 3.37 μM (Figures C and S3A). We next aimed to determine whether CB1R degradation was cell line dependent. To do this, we utilized MDA-MB-231 cells (Metastatic Differentiated Adenocarcinoma of the Breast), which are also known to overexpress CB1R. We selected Pro-CB8, the most effective PROTAC in MCF-7 cells, and performed a dose-dependent treatment over 48 h using three concentrations ranging from 1 μM to 10 μM. Pro-CB8 induced a significant reduction in CB1R levels, with a maximum decrease of approximately 90% at 10 μM (Figure S3B). These results suggest that CB1R degradation by Pro-CB8 is not limited to a specific cell line, supporting the broader applicability of our approach.

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Evaluation of CB1R protein degradation. (A). CB1R degradation efficacy with three different degraders. MCF-7 cells were incubated with 10 μM of the degraders for 48 h. Western blot analysis was performed and compared to the housekeeping protein vinculin. (B). Time-dependent treatment for Pro-CB8 in MCF-7, 10 μM dose. (C). Dose-dependent treatment for Pro-CB8 in MCF-7 for 48 h. (D). Mechanistic study of CB1R degradation using Pro-CB8 Treatment was done in the presence of the proteasome inhibitor MG132 at 0.2 μM for 48 h.

To confirm that CB1R degradation was mediated via the ubiquitin-proteasome system (Ups), MCF-7 cells were treated with 0.2 μM MG132, a proteasome inhibitor. , Co-treatment of MG132 with Pro-CB8 prevented CB1R degradation, confirming that proteasome inhibition blocked the effect, and that the observed CB1R reduction relied mainly on E3 ligase recruitment and proteasomal activity (Figure D). To further validate the mechanism of action, siRNA targeting the E3 ligase CRBN was used in MCF-7 cells. Notably, cotreatment with siRNA and Pro-CB8 in MCF7 cells showed no diminution in CB1R protein levels, confirming that CB1R degradation is specifically mediated through the E3 ligase subunit CRBN (Figure S3C,D). As further validation, MCF-7 cells were treated individually with the parental CB1R binder and the CRBN E3 ligase-recruiting moiety (Figure S3E). Neither treatment led to a significant decrease in CB1R levels, reinforcing that receptor degradation was not due to inhibition, but rather to targeted proteasomal degradation driven by PROTAC-mediated ternary complex formation. As part of the specificity validation, the effect of Pro-CB8 on CB2R was assessed. Although CB2R protein levels are challenging to detect reliably using antibody-based methods, we employed a recombinant rabbit monoclonal antibody against CB2R from Invitrogen, which detected a band at the expected molecular weight. The results showed no impact on CB2R expression, supporting the selective degradation of CB1R by Pro-CB8 (Figure S3F).

Effect on Downstream Pathways

To investigate the downstream effects of our most potent degrader, Pro-CB8, we first assessed CB1R signaling by measuring phosphorylation of AKT and ERK (Figure A). Consistent with CB1R’s known coupling to the PI3K/AKT and MAPK/ERK cascades, Pro-CB8 treatment markedly decreased levels of pAKT and pERK by approximately 70% (Figure A). Phosphorylated AKT drives both transcriptional upregulation and stabilization of the antiapoptotic protein BCL-2, thereby blocking apoptotic pathways, while activated ERK promotes oncogenic signalingenhancing cell survival, suppressing apoptosis, and inducing expression of key proliferation factors such as MCM5, which is essential for cancer cell growth and viability. ,, Accordingly, we measured BCL2 and MCM5 mRNA levels in MCF-7 cells following treatment with Pro-CB8 or the parental CB1R ligand, Rimonabant. Our results revealed a reduction in the expression of both genes at 10 μM Pro-CB8, with BCL2 also showing decreased expression at 1 μM. In contrast, Rimonabant treatment did not cause noticeable change in the mRNA levels of either gene at the tested concentrations, emphasizing the enhanced efficacy of targeted protein degradation over mere antagonism (Figure B,C). In addition, BCL2 protein levels were evaluated in MCF-7 cells treated with 10 μM Pro-CB8 in a time-dependent manner, revealing a 56% reduction after 48 h (Figure D). Moreover, we assessed the downstream effect of Pro-CB8 in MDA-MB-231, where it also significantly reduced BCL2 and MCM5 expression by approximately 50%. Again, Rimonabant showed no notable impact (Figure S4A,B). These findings highlight the superior efficacy of Pro-CB8 in downregulating key cancer-related genes and proteins, underscoring its potential as a promising therapeutic candidate for breast cancer treatment.

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Evaluation of CB1R oncogenic downstream pathway. (A). p-ERK, p-AKT and CB1 expression in MCF7 cells treated with Pro-CB8, 10 μM dose for 48 h. (B). Abundance of MCM5 in MCF-7 cells treated dose-dependently with Pro-CB8 and Rimonabant for 24 h, measured by RT-qPCR. (C). Abundance of BCL2 in MCF-7 cells treated dose-dependently with Pro-CB8 and Rimonabant for 24 h, measured by RT-qPCR. (D). BCL2 expression in MCF7 cells treated in a time-dependent manner with Pro-CB8, 10 μM dose. Data is presented as the mean ± SD (n = 9), * represents p ≤ 0.05, ** represents p ≤ 0.01 as determined by a one-way ANOVA comparison relative to untreated (UT).

Inhibition of Cancer-Associated Cellular Behaviors

To further evaluate the cellular functional consequences of Pro-CB8 on cancer-associated cellular behaviors, we conducted proliferation, cytotoxicity, and apoptosis assays in two breast cancer cell lines. Cell viability was first assessed following treatment with Pro-CB8 and Rimonabant at three concentrations (0.1 μM, 1 μM, and 10 μM). To assess cytotoxicity, crystal violet staining was performed to label DNA in adherent viable cells, enabling measurement of their total biomass without altering protein levels in the surviving cells. The CB1R degrader, Pro-CB8, demonstrated clear cytotoxic activity across all tested concentrations in MCF-7 cells, in contrast to Rimonabant, which exhibited a comparatively weaker effect (Figures A,B and S5A). To further validate the viability assay, healthy fibroblast cells were treated with both Rimonabant and Pro-CB8, and no significant decrease in cell viability was observed (Figure S5A,B). These findings emphasize the advantage of targeted degradation in reducing cancer cell viability. Furthermore, a significant reduction in cell proliferation was observed following Pro-CB8 treatment compared to Rimonabant (Figure C). To explore whether the observed decrease in cell viability was associated with apoptosis, we employed the Caspase-Glo 3/7 assay, which measures caspase activity through the luminescent detection of aminoluciferin following substrate cleavage. Consistent with the downregulation of the antiapoptotic protein BCL2, treatment with Pro-CB8 resulted in a robust increase in apoptosis at 10 μM, indicating that the CB1R degrader Pro-CB8 actively induces apoptotic cell death (Figure D). These findings were further validated in the MDA-MB-231 cell line. In alignment with the MCF-7 results, Pro-CB8 significantly reduced proliferation at 1 μM and 10 μM, while also inducing a dose dependent marked increase in apoptosis (Figure S5D,E). Notably, the inhibition of cancer-associated cellular behaviors was more evident at the 48 h mark, consistent with the catalytic degradation of the CB1R. This sustained degradation likely disrupts the expression of key survival proteins, inducing cumulative cellular stress, decreased proliferation, and increased cytotoxicity. Overall, at 1 μM and 10 μM, Pro-CB8 consistently suppressed multiple cancer-associated cellular behaviorsproliferation, viability, and apoptosisdemonstrating a significantly enhanced effect compared to the CB1R antagonist Rimonabant. This superior activity is likely attributed to the catalytic mechanism of the PROTAC degrader, supporting its promise as a potential therapeutic strategy in breast cancer treatment.

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Evaluation of cancer-associated cellular behaviors. (A). Viability assay of MCF-7 cells treated with a single dose (10 μM) of Pro-CB8 and Rimonabant, measured using crystal violet. (B). Quantification of cell viability across treatment with three different concentrations. (C). Proliferation assay of MCF-7 cells treated with two different concentrations of Pro-CB8 and Rimonabant for 48 h. Data is presented as the mean ± SD (n = 3), **** represents p ≤ 0.0001 as determined by a one-way ANOVA comparison relative to 0. (D). Apoptosis assay of MCF-7 cells treated with Pro-CB8 and Rimonabant for 48 h at 10 μM. Data is presented as the mean ± SD (n = 3), *** represents p ≤ 0.001, **** represents p ≤ 0.0001 as determined by a one-way ANOVA comparison relative to 0.

Anticancer Activity in 3D Cell Models

For further validation, we developed 3D cancer spheroid cultures using two breast cancer cell lines, MCF-7 and MDA-MB-231. Spheroids provide a more physiologically relevant model for anticancer drug screening by mimicking the 3D architecture, cell–cell interactions, and microenvironmental gradients found in solid tumors. Importantly, they enable the evaluation of drug penetration and resistance mechanisms, thereby providing more predictive insights into therapeutic efficacy. Following spheroid formation and treatment administration with Pro-CB8, we observed signs of tumor cell death, evidenced by the accumulation of dead cells and disrupted spheroid morphology. Continued cell death overtime led to a decrease in spheroid size and compromised cell–cell adhesion. This progressive loss of structural integrity ultimately caused the disintegration of the spheroids into smaller cellular fragments (Figure A). Notably, treatment with the CB1R degrader Pro-CB8 resulted in pronounced and sustained disintegration of both MCF-7 and MDA-MB-231 spheroids, beginning as early as day three and persisting throughout the observation period (Figures B,D and S6, S7). In contrast, untreated spheroids and those treated with the control compound Rimonabant displayed only an inhibition of the tumor growth in MCF-7 spheroids and no significant observable effect in MDA-MB-231 spheroids (Figure C,E). These findings underscore the robust and consistent anticancer activity of Pro-CB8 across both 2D and 3D experimental models, reinforcing its therapeutic potential. In conclusion, the ability of Pro-CB8 to induce spheroid disintegration highlights its efficacy in a more physiologically relevant system that closely mimics the in vivo tumor microenvironment. These promising results support translational potential of this approach.

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Anticancer effect of Pro-CB8 on 3D breast cancer models. (A). Schematic representation of morphological changes in 3D tumor spheroid following Pro-CB8 treatment. (B). Morphological changes in MDA-MB-231 spheroid after 10 μM doses of Rimonabant and Pro-CB8. (C). Time-course evaluation of spheroid area using imageview software. (D). Morphological changes in MCF-7 spheroid after 10 μM doses of Rimonabant and Pro-CB8. (E). Time-course evaluation of spheroid area using imageview software. Data is presented as the mean ± SD (n = 2), *** represents p ≤ 0.001, **** represents p ≤ 0.0001 as determined by a one-way ANOVA comparison relative to untreated.

In Vivo Blood–Brain Barrier Penetration

To experimentally evaluate the blood–brain barrier permeability of the CB1R-targeting PROTAC Pro-CB8, we performed an in vivo brain penetration assay in mice. Mice were administered an intraperitoneal (IP) injection of Pro-CB8 at a dose of 5 mg/kg. One hour post-treatment, the animals were sacrificed, and their brains were dissected, extracted, and analyzed using LC/MS to quantify compound levels. The experiment was performed in triplicate. In all three samples, the brain concentrations were below 10 ng/g, respectivelylevels considered indicative of minimal CNS exposure. The average brain concentration of the degrader Pro-CB8 was 6.43 ng/g, with a standard deviation of ±3.6 ng/g (Table S2). These experimental results are consistent with our earlier in silico predictions using the SWISSADME and ADMETLab 3.0 platforms, which also indicated that Pro-CB8 is unlikely to cross the blood–brain barrier. These findings not only demonstrate that Pro-CB8 achieves significant degradation of CB1R, but also highlight its favorable pharmacokinetic profile, showing poor brain penetration and confirming its peripheral restriction. This dual profilepotent, selective target degradation combined with limited CNS exposure, positions Pro-CB8 as a promising therapeutic candidate with a potentially improved safety profile compared to traditional CB1R inhibitors.

Conclusion

The CB1 Receptor is increasingly recognized as a compelling therapeutic target in oncology due to its role in promoting tumor progression and resistance to apoptosis. Despite the potential of CB1R antagonists, their clinical utility has been limited by central nervous system (CNS) side effects and the emergence of resistance mechanisms. In this study, we addressed these challenges by designing and synthesizing a novel series of CB1R-targeting PROTACs that offer a dual advantage: selective degradation of CB1R and minimized CNS penetration. Using Rimonabant as the CB1R-binding moiety and thalidomide- or VHL-based ligands as E3 ligase recruiters, we generated a diverse library of bifunctional molecules with varied linker compositions and architectures. Several PROTACs effectively induced CB1R degradation, with the most promising candidates demonstrating potent antiproliferative and pro-apoptotic effects in cancer models, including 3D spheroids. Importantly, druglikeness and ADMET profiling confirmed favorable properties for further development, despite the inherent physicochemical complexity of PROTACs. To complement these in silico predictions, in vivo assessment of blood–brain barrier permeability demonstrated that our lead compound, Pro-CB8, exhibits no significant CNS exposure. These experimental results confirm the peripheral restriction of Pro-CB8, reinforcing its potential as a safer and more selective therapeutic approach for CB1R-driven cancers. Together, these findings establish targeted CB1R degradation as a viable and innovative strategy for cancer therapy, potentially overcoming the limitations of traditional inhibitors and paving the way for the next generation of cannabinoid-based therapeutics.

Supplementary Material

pt5c00321_si_001.pdf (2.2MB, pdf)

Acknowledgments

The authors were funded by the Israel Cancer Research Fund (ICRF, 22- 201-RCDA), the Israeli Centers for Research Excellence from the Council for Higher Education, the Israel Ministry of Innovation, Science and Technology (0004943), and the Israel Science Foundation (ISF, 1925/22).A.K.’s fellowship is funded by the Neubauer fellows in the Sciences Scholarship. We thank Dr. Gali Umschweif-Nevo for providing the mice used in the in vivo experiments. We are also grateful to Laila Agbariya for performing the compound injections and assisting with brain dissections. Special thanks to Dr. Alina Nemirovski for conducting the LC/MS run and data analysis. Primary fibroblasts were provided by Dr. Myriam Grunewald and Orly Hasltuck from Hadassah Organoid Center.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.5c00321.

  • Primers used for RT-qPCR; antibodies used for Western blotting; luminescence reporter assay details; Western blot images for CB1R, Bcl-2, AKT, p-AKT, MAPK, p-MAPK, CB2R, Vinculin, and GAPDH; proteasome inhibitor (MG132) treatment results; crystal violet cell viability assay images and quantification; caspase-3/7 apoptosis assay data; cell proliferation assay data (Resazurin/WST-8); relative mRNA expression levels of Bcl-2 and MCM5; spheroid formation assay images and surface area measurements; in vivo blood–brain barrier penetration LC–MS chromatograms; in silico ADMET predictions for synthesized compounds; 1H NMR and MALDI-TOF spectra for compound characterization (PDF)

R.I.B. directed the study, conceived the idea, and designed experiments. A.K. designed experiments, conducted biochemical and cellular studies, K.T. synthesized compounds, J.F.-L. conducted qPCR experiments and cellular studies.

The Institutional Animal Care and Use Committee of the Hebrew University (AAALAC accreditation #1285; Ethics approval number MD 24-17449-5) approved the experimental protocol used. Animal studies were reported in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals guidelines.

The authors declare no competing financial interest.

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