Emergence of Research in Pro-PROTACs

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The application value of proteolysis targeting chimera (PROTAC) technology in tumor treatment has become well-established. The molecular core of this technology involves bifunctional molecules capable of recruiting both E3 ubiquitin ligase (E3) and the target protein (POI). Under the action of these bifunctional drugs, termed PROTAC molecules, a ternary complex POI-PROTAC-E3 is formed. This complex triggers ubiquitination of POI and induces subsequent degradation of POI mediated by the ubiquitin-proteasome system (UPS).

Why Develop Pro-PROTACs?

Despite the numerous advantages of PROTAC, it still faces limitations. One major issue is that PROTACs often fail to comply with Lipinski's Rule of Five (Ro5), leading to poor drug permeability, solubility, and oral bioavailability. Another limitation is the resistance to PROTAC-induced degradation, possibly due to poor expression of recruited E3 ligase or mutations. The third problem is the toxicity resulting from the targeting of normal proteins.

The development of prodrug PROTACs (Pro-PROTACs) has emerged as a promising strategy to precisely release active PROTACs into tumors and minimize off-target toxicity to normal tissues.

Mechanism of action of Pro-PROTACs. Fig. 1 Mechanism of action of Pro-PROTACs. (Chen, 2023)

Receptor-Mediated Internalization of Pro-PROTACs

Various membrane protein receptors on the cell surface can specifically bind to extracellular ligands. Pro-PROTACs exploit receptor-mediated internalization to achieve biological functionality.

For receptor-mediated internalization of Pro-PROTACs, improving cell penetration through endocytic pathways and optimizing pharmacokinetics through intravenous injection can enhance their performance. The structural complexity of these Pro-PROTACs poses significant challenges for optimization and manufacturing. Additionally, Pro-PROTACs with larger molecular weights than parent degraders may have poor cell membrane permeability. The increased molecular size, rotatable bonds, and inherent instability of these molecules may negatively impact their pharmacokinetic characteristics. Resolving the rigidity issues of Pro-PROTACs, especially in their linker regions, may lead to better in vivo performance.

DACs have emerged as a new approach to reduce the toxicity associated with traditional PROTAC targeting. For instance, GNE-987, an effective PROTAC degrader targeting BET proteins, exhibited poor in vivo pharmacokinetic (PK) characteristics. To overcome this limitation, researchers designed a DAC named CLL1-1, connecting GNE-987 to the CLL1-targeting antibody using a disulfide-derived carbonate linker. CLL1-1 demonstrated biological activity, unlike the parent PROTAC GNE-987, showcasing the potential of converting suboptimal PROTAC candidates into DAC prodrugs.

  • Folate-Mediated PROTACs

Folate receptor alpha (FRα) is a widely recognized therapeutic target in cancer drug delivery. Pro-PROTACs designed with folate dependence aim to alleviate off-target effects caused by PROTACs. Folate-dependent Pro-PROTACs can target proteins like BRD, MEK, and ALK. It enters tumor cells via FRα-mediated internalization and is subsequently activated by intracellular hydrolases.

Nucleic acid aptamers are synthetic single-stranded oligonucleotides that bind specifically to certain target proteins. Aptamer-PROTAC conjugates enhance the tumor-targeting ability and anticancer efficacy of parent PROTACs. Researchers constructed an Aptamer-PROTAC conjugate (APC 13) by linking AS1411 with MZ1 using an ester-disulfide linker. APC 13 demonstrated effective BRD4 protein degradation in nuclear protein-positive MCF-7 cells, showing strong antitumor efficacy in MCF-7 xenograft models without significant side effects.

Light-Activated Pro-PROTACs

Light serves as a unique external control agent with high spatial and temporal resolution, providing new possibilities for targeted protein degradation. Light-activated Pro-PROTACs can be broadly categorized into photocaged and photoswitchable PROTACs.

For light-activated Pro-PROTACs, the required irradiation wavelengths may cause DNA damage, and the limited tissue penetration of light restricts their application to skin cancers and blood cancers. Further research can explore new photosensitive compounds activated by near-infrared light to broaden potential clinical applications.

Light-activated Pro-PROTACs. Fig. 2 Light-activated Pro-PROTACs. (Verma, 2020)

  • Photocaged PROTACs

Photocaged PROTACs typically involve incorporating a photolabile group into the parent PROTAC, preventing binding affinity to POI or E3 in the absence of light. Active PROTAC is rapidly released upon light exposure, inducing POI degradation.

Researchers introduced a 4,5-dimethoxy-2-nitrobenzyl (DMNB) group into the bromodomain (BRD) binding region of dBET1 (an effective BET protein degrader) to design BRD4-targeting photocaged PROTAC. The DMNB moiety in PROTAC hinders its binding affinity to BRD4 in the absence of light (a prerequisite for photocaged PROTACs and inherent stability). However, upon exposure to 365nm ultraviolet light, binding affinity is restored, leading to BRD4 degradation in Ramos cells.

  • Photoswitchable PROTACs

In contrast to most traditional PROTAC precursors, photoswitchable PROTACs (photoPROTACs) use azobenzene-based linkers instead of alkyl or ether linkers to connect the POI head and E3 ligase binding site. Therefore, photoPROTACs can effectively transition between "cis" and "trans" configurations under specific wavelength light. This transition results in significant changes in the topological distance and conformation between the POI head and E3 ligase binding site, leading to distinct biological activities. Unlike the light-cleavage mode of photocaged PROTACs, photoswitchable PROTACs utilize the change in spatial configuration of the photosensitive compound under light conditions to achieve compound activation.

Other Modes of Pro-PROTACs

  • Nitroreductase (NTR)-Responsive PROTACs

Hypoxia is a significant characteristic of many solid tumors associated with resistance and poor prognosis. Under normoxic conditions, nitroreductase (NTR) is overexpressed in hypoxic tumors, providing good selectivity for NTR-activatable prodrugs between tumor and normal cells. Evofosfamide, containing a 2-nitroimidazole moiety, is an NTR-activatable prodrug showing good safety and antitumor activity in clinical trials. Therefore, utilizing hypoxia-responsive NTR in PROTACs holds promise in expanding the therapeutic window compared to traditional PROTACs.

  • Radotherapy-Triggered PROTACs (RT-PROTACs)

Radiotherapy, utilizing high-energy X-rays to destroy cancer cells, is a frontline cancer treatment. The synergistic action of X-rays and active drugs may achieve superior antitumor effects.

  • Reactive Oxygen Species (ROS)-Responsive PROTACs

ROS, including hydrogen peroxide (H2O2), superoxide radicals, and hydroxyl radicals, are oxidative agents that are often elevated in tumors compared to normal cells. Therefore, prodrugs activated by ROS can selectively target tumor cells with lower toxicity to healthy tissues.


  1. Chen, C., et al., Recent Advances in Pro-PROTAC Development to Address On-Target Off-Tumor Toxicity, J. Med. Chem., 2023, 66, 13, 8428-8440.
  2. Verma, S., et al., Controlling PROTACs with Light, ChemMedChem, 2020, 15, 1258-1261.
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