PROTAC with Folate Targeting

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Proteolysis targeting chimeras (PROTACs) is an emerging biotechnology that treats cancer by selectively degrading specific proteins within cancer cells. Although PROTACs have shown great potential in targeting "undruggable" proteins, they face many challenges in clinical application, including high molecular weight, low bioavailability, poor cell membrane permeability, inadequate targeting, and inefficiency in vivo. The potential toxicity of PROTAC in normal cells limits its clinical application due to off-target effects. Precisely controlling the targeted degradation activity of PROTAC in a tissue-selective manner can minimize potential toxic side effects, considering that folate receptor alpha (FOLR1) is overexpressed in many cancer cells, such as ovarian, breast, kidney, and colorectal cancers, while very low or no expression is found in normal tissues or cells. The authors used folate conjugating strategies to specifically deliver PROTAC to cancer cells to achieve targeted degradation of the target protein, thereby eliminating potential toxicity to normal tissue.

Folate-caged PROTACs

The working principle of the folate-PROTAC system designed in this study is shown in the following. Folate-PROTAC is preferentially transported to cancer cells with high FOLR1 expression. Upon entry into cancer cells, folate-PROTAC is disconnected by endogenous hydrolases and the folate portion is released. The remaining PROTAC molecules recruit endogenous VHL-E3 ubiquitin ligase, which ubiquitinates the target protein and degrades it by the 26S proteasome.

(A) Schematic representation of the folate-PROTAC strategy. (B) Schematic illustration of the activation of folate-ARV-771 by endogenous hydrolase. (C, D) Western blot analysis of BRD protein levels from HeLa or HFF-1 cells treated with the indicated doses of folate-PROTAC for 12 h. (E, F) Cell viability of HeLa or HFF-1 cells after treatment with folate-PROTAC for 72 h. (Liu, J., 2021)

Design and synthesis of folate-ARV-771

First, the researchers bonded folic acid to the hydroxyl group of ARV-771 via an ester bond, resulting in the folate-ARV-771 molecule. Among them, ARV-771 is a well-studied PROTAC molecule that can efficiently degrade BRD protein. The folate-ARV-771 molecule consists of three main components: a warhead for binding to the target protein BRD, a ligand for recruiting VHL-E3 ubiquitin ligase, and folate for targeted delivery to tumor cells. The author also designed and synthesized the negative control folate-ARV-771N, and replaced the ester bond in folate-ARV-771 molecule with an amide bond that could not be cut by hydrolase, so that the active PROTAC could not be released in the cell. The results showed that folate-ARV-771 and ARV-771 had similar degradation ability in HeLa cells with high FOLR1 expression. In HFF-1 cells with low expression of FOLR1, the degradation ability of folate-ARV-771 to BRD protein was much weaker than that of AVR-771. In both types of cells, the amine-linked folate-ARV-771N molecule was not effective at degrading BRD protein. The overall results showed that folate-ARV-771 specifically enriched and degraded the target protein in cancer cells compared to normal cells.

Folate-ARV-771 preferentially degrades BRD in cancer cells

Next, the researchers further explored whether the degradation of the target protein BRD by folate-ARV-771 was related to the folic acid receptor FOLR1. First, the use of additional free folic acid, which binds to FOLR1 on the cell surface, is expected to have an antagonistic effect. As expected, free folic acid caused folate-ARV-771 to fail to degrade the target protein BRD4. The authors then silenced endogenous FOLR1 in HeLa cells, showing that FOLR1 knockdown prevented folate-ARV-771 from degrading BRD4 protein. Notably, pretreatment with free folic acid increased the IC50 of folate-ARV-771 from 365 nM to 1.5 μM, but still showed some cytotoxicity, possibly because the molecule caused cell death through its inhibitory effect on BRD protein. The authors further evaluated the ability of folate-ARV-771 to degrade BRD4 protein in cell lines with different FOLR1 expression levels, and the results showed that the higher the FOLR1 expression level, the more obvious the degradation effect of BRD4 protein. The overall results suggest that folate-ARV-771 is likely to degrade the target BRD protein in cancer cells in a FOLR1-dependent manner.

Folate-PROTAC targets other proteins

The researchers then explored whether this folate-Protac system could also be used to target other proteins. Based on the PROTAC previously developed by our research group, we synthesized two other folate-ProTACs: folate-MS432 (targeting MEK1/2 protein) and folate-MS99 (targeting ALK protein), which were used to target the degradation of MEK1/2 and ALK proteins in tumor cells, respectively. Similar to folate-ARV-771, they both degrade the target protein in a FOLR1-dependent manner.

Chemical structure of folate-MS432 and folate-MS99. (Liu, J., 2021)

In summary, the series of folate-PROTAC systems developed in this study can degrade multiple target proteins (BRDs, MEKs, and ALK) in a folate receptor-dependent manner in cancer cells, and this design can be applied to all VHL recruited PROTACs, providing a universal strategy for achieving targeted degradation of target proteins in cancer cells.

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Folate-PEG-PROTAC

Researchers at Huazhong University of Science and Technology have made important progress in the field of cancer treatment, successfully designing and preparing a novel folate-PEG-PROTAC micelle for improving the specific targeting of tumor cells and the anti-tumor effect in vivo. The study, published in April 2024, marks an important advance for PROTACs technology in the design of drug delivery systems.

Design strategies for MPRO micelles, including diagrams of the MPRO strategy via GSH-sensitive disulfide bonds, and the process by which PEG is cleaved by GSH to release active PROTACs to degrade target proteins after entry into tumor cells mediated by folate. (Ma, J., 2024)

The research team designed and prepared a novel folate-PEG-SS-PROTAC micelle (MPRO) using folic acid's targeting of cancer cells. The micelle utilizes folate as a targeting ligand, improves water solubility via PEG (polyethylene glycol), and is linked to EGFR-targeted PROTAC via reduction-responsive disulfide bonds. This design enables MPRO to self-assemble into nanoparticles with enhanced tumor cell targeting capabilities and responsive release properties in the tumor microenvironment.

The research team verified the stability, uniformity and cell uptake efficiency of MPRO through a series of in vitro experiments. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) results show that MPRO has a uniform spherical structure with a diameter of about 50 nm. Under reductive conditions, MPRO can release EGFR-PROTAC efficiently. In addition, it was observed by flow cytometry and fluorescence microscopy that MPRO micelles can be effectively taken up by tumor cells via folate receptor-mediated pathways. At the cellular level, it was found by Western blot that MPRO showed an efficient ability to degrade EGFR protein. After free folic acid blocked the folate receptor on the surface of tumor cells, the degradation effect of MPRO on EGFR was weakened, which was consistent with the results of uptake experiment. The in vitro anti-tumor activity of MPRO was further tested, and the test results showed that MPRO had superior cytotoxicity to MPRO-C in HCC-827 and PC-9 cells and the pro-apoptosis effect of PC-9 was comparable to that of free EGFR-PROTAC.

The distribution and anti-tumor effect of MPRO were further investigated in vivo. In the established nude mouse model of HCC-827 tumor cells, the accumulation of fluorescein (Dir) in tumor tissues was gradually increased under the EPR effect by injecting the tail vein MPRO@Dir, and reached a peak within 24 hours, indicating that MPRO has a strong ability to target tumor tissues. Compared with the control group, tumor growth was significantly inhibited and EGFR protein levels in tumor tissue were significantly reduced in the MPRO treatment group. These results indicate that MPRO can target tumor in vivo and inhibit tumor growth by degrading EGFR protein level in tumor cells.

The development of MPRO not only overcomes the common problems of poor water solubility and low bioavailability of traditional PROTAC drugs, but also greatly enhances the anti-tumor effect in vivo, showing a good therapeutic potential and clinical application prospect. This study provides new ideas for the design of PROTACs and is expected to promote the development of more drugs targeting protein degradation.

References:

1. Liu, J., et al. Cancer selective target degradation by folate-caged PROTACs. Journal of the American Chemical Society. 2021, 143(19): 7380-7387.
2. Ma, J., et al. Folate‐PEG‐PROTAC micelles for enhancing tumor‐specific targeting proteolysis in vivo. Advanced Healthcare Materials. 2024: 2400109.