Pro-PROTAC Development for Cancer Therapy
PROTAC's advantages and limitations
Proteolytic targeting chimera (PROTAC) was first proposed by Crews at Yale University in 2001. Given its unique mechanism of action (MOA), PROTAC exhibits a number of significant advantages. Traditional small molecule inhibitors (SMI) and monoclonal antibodies (Mabs) require high concentrations to obtain sufficient occupation levels at the active site of POI and impede its biological function. In contrast, PROTAC can perform ubiquitization and subsequent proteasome degradation of targeted proteins in a catalytic manner. Notably, PROTAC does not require a high affinity of the ligand to POI. In addition, PROTAC can overcome the problem of drug resistance arising from SMI.
Despite PROTAC's many advantages, the technology still has some drawbacks. A major problem is that PROTAC does not comply with Lipinski's five-drug rule (Ro5), resulting in poor drug permeability, solubility, and oral bioavailability. Another issue is the emergence of acquired resistance to PROTAC due to insufficient expression or mutation of the recruited E3 ligase, which can negatively impact drug efficacy. PROTACS-degrading drugs rely on E3 ligase, and some tumor cells can develop resistance by impacting E3 ligase function. It has been confirmed that tumor cells acquire resistance to VHL-recruited and CRBN-recruited PROTACs after prolonged treatment. Finally, while PROTAC can effectively inhibit protein activity, targeted toxicity is a major concern when the target POI is critical in normal tissue.
PROTAC related products and services at BOC Sciences
BOC Sciences has kept abreast of the cutting edge of PROTAC technology and is able to provide customized services for the development of prodrug-based PROTACs (pro-PROTAC).
PROTAC prodrug development for cancer therapy
Studies have shown that an effective strategy to expand the therapeutic window of conventional PROTAC is to utilize E3 ubiquitin ligases that are restricted in expression in cells or tissues associated with targeted toxicity. A similar approach utilizes E3 ligase that is specifically expressed in tumor tissue or cells. However, both approaches face significant challenges, as of the more than 600 E3 ligases that have been reported, only a very small fraction (less than 3%) have available ligands suitable for PROTAC design. More recently, the development of pro-PROTAC has emerged as a potential improved strategy to precisely release active PROTACs into tumors and minimize targeted extra-tissue toxicity.
PROTAC based on senescence-associated β-galactosidase (SA-β-Gal)
Professor Wei Wang and his team from the Department of Chemistry and Biochemistry at the University of Arizona have recently developed an innovative strategy to selectively eliminate aging cancer cells. This strategy utilizes galactose-modified PROTAC, a pro-PROTAC molecule, to specifically target specific protein degradation in senescent cancer cells.
Schematic diagram of the action mechanism of galactose-modified PROTAC. (Chang, M., 2024)
In the study, the scientists designed two prodrugs, Gal-ARV-771 and Gal-MS99. These two prodrugs contain SA-β-Gal sensitive galactose fragments that are linked via carbonates to VHL-based bromine domain (BRD) degraders such as ARV-771. In normal cells, the degradation activity of the prodrug is blocked, but in senescent cells, due to the presence of SA-β-Gal, the prodrug is activated, releasing PROTAC molecules with degrading activity.
The results showed that Gal-ARV-771 specifically induced the degradation of BRD4 protein in aged human lung A549 cells, but not in normal cells. Similarly, Gal-MS99 specifically degrades NPM-ALK protein in senescent Karpas 299 cells. In vivo studies using A549 xenografted mouse models found that the combination of etoposide and Gal-ARV-771 significantly inhibited tumor growth, and no significant toxicity was observed. These findings suggest that the selective elimination of senescent cancer cells by galactose-modified PROTACs may provide a novel and effective approach to cancer treatment, reducing the risk of drug resistance and leading to better treatment outcomes for patients. The preclinical findings of this strategy provide strong evidence for the therapeutic potential of Gal-ARV-771 as a pro-PROTAC.
Radiotherapy-activated pro-PROTAC in tumors
Radiation therapy, which destroys cancer cells using high-energy X-rays, is the first-line treatment for cancer. X-ray radiation has excellent precision and deep tissue penetration, enabling the local release of X-ray-activated prodrugs in tumor tissue. In addition, the synergistic effect of X-rays and active drugs may achieve excellent anti-tumor effects.
Li's team at Tsinghua University developed a radiation-triggered PROTAC (RT-PROTAC). Under X-ray radiation, the phenylazide group on RT-PROTAC is reduced to aniline, triggering a subsequent 1, 6-elimination and decarboxylation process that releases the active PROTAC molecule. The formation of active PROTAC not only leads to proteasomal degradation of BRD4 and BRD2 in vivo, but also exhibits synergistic effects with X-rays in MCF-7 xenograft models. This work provides a new way for the spatio-temporal release of pro-PROTACs at the tumor site, thereby improving the anti-tumor efficacy of conventional PROTACs and reducing adverse systemic toxicity.
Structure of a radiation-triggered PROTAC. (Yang, C., 2022)
Pro-PROTAC integrated nanosensitizer for potentiating cancer radiation therapy
Zhang et al. propose a new strategy to enhance the effects of RT for head and neck squamous cell carcinoma (HNSCC). In view of the issues of decreased resistance and sensitivity in RT, the research team developed a PROTAC-based nanosensitizer designed to increase the sensitivity of tumor cells to RT while reducing side effects.
Studies have revealed that RT can promote the DNA damage repair of tumor cells through BRD4-RAD51AP1 axis, forming a self-protection mechanism against RT. To break this mechanism, the researchers synthesized a H2O2-responsive BRD4 PROTAC prodrug BPA771, which can activate and degrade BRD4 under the action of hydrogen peroxide generated by radiation therapy, and then inhibit downstream RAD51AP1, making tumor cells more vulnerable to RT damage.
In addition, HfO2 nanoparticles were used as metal radiosensitizer and loaded into CrGDK-targeted RPB7H nanoparticles together with BPA771 predrugs. The CRGDK peptide recognizes the NRP-1 receptor on the surface of tumor cells, ensuring the specific delivery of HfO2 nanoparticles and BPA771 to the tumor site. HfO2 nanoparticles deposited X-rays inside tumor cells, enhancing DNA damage while generating ROS and activating BPA771, thereby enhancing RT effects.
In vivo and in vitro experiments, RPB7H nanoparticles have been confirmed to effectively accumulate in tumor areas, penetrate deeply into tumor tissues, synergistically interact with RT, significantly improve the sensitivity of tumor cells to RT, and prolong the survival of treated mice. This achievement provides a potential breakthrough strategy for the radiotherapy of HNSCC, especially by targeting the BRD4-RAD51AP1 axis to enhance the anti-tumor effect of RT, and is expected to become an innovation in the field of HNSCC therapy.
Pro-PROTAC used in microneedle-assisted cancer therapy
Professor Ping Yuan's team at the School of Pharmacy, Zhejiang University published a study in the Journal of Controlled Release, introducing a novel PROTAC prodrug MZ1-O, which achieves precise tumor therapy through bioorthogonal chemical activation. The prodrug remains inert in the absence of the tetraazine derivative (Tz), but when confronted with Tz, it is activated by the inverse electron demand Diels-Alder (iEDDA) reaction, which degrades the BRD4 protein and induces tumor cell apoptosis. To improve targeting, MZ1-O was encapsulated in polylactic acid-glycolic acid (PLGA) nanoparticles and modified with the targeting peptide RGD to form MZ1-O@NP, which can be administered throughout the body and actively search for subcutaneous tumors. In this study, Tz was encapsulated in dissoluble micronicles (MN-Tz), inserted into tumor sites in mice, and subsequently injected systemic into MZ1-O@NP to achieve precise intratumoral drug activation, effectively inhibiting the growth of subcutaneous tumors of B16-F10 and A549 with reduced potential side effects on healthy tissues. This study provides an example of a highly specific bioorthogonal chemically-activated PROTAC prodrug, demonstrating its potential for use in the treatment of cancer in vivo, particularly through microneedle-assisted local activation strategies that enhance therapeutic efficacy and safety.
The future of pro-PROTAC
Pro-PROTAC is a track full of potential for targeted delivery of potent degraders to specific tissues or cells, thereby reducing drug exposure to healthy organs and expanding the therapeutic window of traditional PROTAC.
However, there are challenges as well as opportunities. Due to the complexity of the structure, how to optimize and produce these pro-PROTAC faces a huge challenge. In addition, Pro-protacs with larger molecular weights can also lead to poor PK properties compared to pro-descending agents. Overall, the discovery of pro-PROTAC is expected to mitigate potential targeted toxicity, and advances in the field could further advance the clinical application of PROTAC technology.
References:
- Chen, C., et al. Recent advances in pro-PROTAC development to address on-target off-tumor toxicity. Journal of Medicinal Chemistry. 2023, 66(13): 8428-8440.
- Chang, M., et al. Selective elimination of senescent cancer cells by galacto-modified PROTACs. Journal of Medicinal Chemistry. 2024, 67(9): 7301-7311.
- Yang, C., et al. Radiotherapy-triggered proteolysis targeting chimera prodrug activation in tumors. Journal of the American Chemical Society. 2022, 145(1): 385-391.
- Zhang, S., et al. PROTAC prodrug‐integrated nanosensitizer for potentiating radiation therapy of cancer. Advanced Materials. 2024: 2314132.
