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A Universal Study to Verify PROTAC Animal in Vivo Test (II)

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Study details

  1. PROTAC design of degrading FKBP12

In this study, FKBP12 was selected as the target protein for the following reasons:

First, FKBP12 protein is widely expressed in mammals. By binding to the Ca2 + -release channel (RyR receptor), FKBP12 regulates Ca2 + signaling to travel important functions, especially in the heart.

Second, because FKBP12 is a highly conserved protein, this chemical knockdown strategy is expected to be used to generate large animal models with targeted protein knockdown (for example, pigs and non-human primates).

Third, the global knockout of FKBP12 by common methods can cause embryonic death from severe developmental heart defects (eg, hypertension, tight ventricles, and ventricular septal defects). The role of FKBP12 in adult heart function and disease development remains elusive.

Rapamycin has been shown to be a potent and specific ligand for FKBP12, which regulates mTOR signaling with high affinity (kd = 0.2nm). Therefore, researchers designed a PROTAC that degrades FKBP12 by linking rapamycin (a FKBP12-specific ligand) and pomalidamine (a specific ligand that binds to CRBN-containing E3 protein ligase) via polyethylene glycol. This heterobifunctional molecule ubiquitinates FKBP12 via CR3B-containing E3 ligase, and degrades FKBP12 via the proteasome pathway. Therefore, researchers synthesized a series of PROTACs and measured their potential to degrade FKBP12 in Jurkat cells.

Researchers named the chimeric molecule with rapamycin and pomalidomide as RC32, which showed the most effective FKBP12 degradation ability. After treating the cells for 12 hours, it caused 50% protein degradation, DC50 = 0.3 nM .

To determine whether RC32-induced ubiquitination degradation was caused by FKBP12 degradation via the ubiquitin-proteasome pathway. Researchers treated the cells with the proteasome inhibitor bortezomib or carfilzomib before adding RC32. In fact, inhibition of the proteasome completely blocked RC32-induced FKBP12 degradation, suggesting that this degradation is via the ubiquitin-proteasome pathway. The addition of the FKBP12 inhibitor rapamycin or the CRBN inhibitor pomalidomide effectively blocked the degradation of FKBP12 by RC32, further confirming that this degradation requires the combination of RC32 with FKBP12 and CRBN. It is worth noting that when degrading FKBP12.6, RC32 did not induce significant degradation of FKBP51 and FKBP11 in Jurkat cells. The use of a certain amount of RC32 can control the degradation of FKBP25. However, RC32 has no effect on the phosphorylation of S6K and S6, which may be beneficial for dividing the independent function of mTOR in FKBP12. When RC32 was washed away, FKBP12 protein levels completely recovered within 96 hours. To further evaluate the efficiency of RC32, tests were performed using different cell lines from different species and primary cells. FKBP12 is effectively degraded by RC32 in a highly consistent pattern in cells from humans, rats, mice and chickens. Importantly, RC32 showed high degradation efficiency in primary cardiomyocytes, which indicates its potential to degrade FKBP12 in vivo.

  1. Rapid and effective degradation of FKBP12 in mice and rats by RC32

After confirming the high degradation potency in cell lines and primary cells, researchers used RC32 to induce protein knockdown in mouse, rat, pig, and non-human primate models. These models are valuable tools for studying human diseases. Although mice and rats have been widely used in scientific research, it is much more difficult to construct protein knockdown models in pigs and non-human primates. Pig xenotransplantation is particularly attractive in biomedical research. Although non-human primates are more relevant to studying human diseases and developing treatment strategies, genetic modification of monkeys is very expensive, time-consuming, and technically challenging. These limitations in large animal models have severely hindered their application in biomedical research. Therefore, researchers tried to use PROTAC as a chemical method to construct a representative protein knockdown animal model and study the function of target proteins.

Researchers first investigated the effects of RC32 on mice. Surprisingly, after only 1 day of treatment with RC32 (intraperitoneal injection, 30 mg / kg, twice daily), FKBP12 protein was not detected in most organs of the treated mice, and the brain was removed. Interestingly, significant degradation of FKBP12 also occurred in the eyes. In contrast, RC32 has no effect on the degradation of FKBP12 in brain tissue, which may be due to the inability of RC32 to cross the blood-brain barrier. Interestingly, after 1 day of treatment (30 mg / kg, twice daily), RC32 was able to degrade FKBP12 for about 1 week. In addition, FKBP12 was recovered in different organs / tissues after RC32 was discontinued. Interestingly, the recovery rate of FKBP12 in the heart was the slowest in 13 days after RC32 was discontinued. After discontinuing PROTAC, FKBP12 mRNA levels showed an acute and compensatory increase, then quickly recovered and remained at near-normal levels. To degrade the FKBP12 protein in the brain, researchers use intraventricular (i.c.v.) administration. As expected, FKBP12 in tissues from the i.c.v. treated brain was degraded, while levels in other organs / tissues were not affected. FKBP12 is widely expressed in the nervous system and is known to regulate the localization and processing of amyloid precursor proteins. The study results suggest that local protein knockdown in the brain may open up a new avenue for treating Alzheimer's disease. When RC32 was delivered orally, FKBP12 was significantly degraded in mice, which highlights the clinical potential of oral PROTAC, which is more convenient than previously reported dependent injections. In addition, only two intraperitoneal injections every 20 hours (20 mg / kg; Figure 3e, f), high degradation efficiency was found in Sprague-Dawley rats.

To be continued in Part III…


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