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

Posted by beauty33 on February 20th, 2020

  1. Application of FKBP12 knockdown mice generated by PROTAC

FKBP12 is necessary for cardiac development, but its physiological importance in cardiac homeostasis remains unclear. Therefore, researchers used this new method in mice to further explore its function in the adult heart. When FKBP12 is depleted, the ryanodine receptor remains open, resulting in the loss of Ca2 +. Researchers examined the characteristics of Ca2 + sparks in response to RC32 dosing and found that their duration was significantly extended in the RC32 treatment group, as shown by confocal microscope line scan images of mouse cardiomyocytes treated with vehicle or RC32. Compared to the control, the properties of Ca2 + significantly altered in RC32-treated mouse cardiomyocytes. The full width at half maximum of the spark increased from 1.80 ± 0.22 to 1.97 ± 0.26 μm. The rise time was extended from 14.3 ± 4.67 to 17.2 ± 5.85ms, and the half-life decayed from 34.5 ± 12.7 to 44.5 ± 13.5ms (Figure 4d). These results indicate that RC32 induces Ca2 + loss in mouse cells. Continuous Ca 2+ loss is a cause of cardiac dysfunction and is a major cause of cardiac hypertrophy. Next, researchers used echocardiography to examine heart morphology and function in control and RC32 treated mice. Compared with the control heart, the RC32 treatment group had a lower ejection fraction (EF; 14.1%) and a shorter shortening fraction (FS; 18.0%), indicating that ventricular systolic activity was reduced. On day 11, a heart wall test treated with RC32 showed a 14.9% reduction in EF and a 18.0% reduction in FS in mice. In addition, left ventricular (LV) mass (19.9%), LV posterior wall thickness, diastolic blood pressure (LVPW; d) (12.5%) and ventricular diastolic septal width (IVS; d) during RV32 handling increased significantly. RC32 treated mice developed cardiac hypertrophy on day 30. However, LV quality, LVPW; d and IVS; d did not change in the vector group. Due to the development of myocardial hypertrophy, the RC32 treatment group had higher EF and FS at day 30 than at day 11. Echocardiographic data suggest that FKBP12 plays a key role in the adult heart, and its degradation leads to heart disease. Taken together, these results indicate that mice with FKBP12 knockout using PROTAC are valuable models for studying FKBP12 function, especially in the heart.

  1. Effective degradation of FKBP12 in Bama pigs

In view of the success of the mice and rats mentioned above, researchers established a protein knockdown pig model in subsequent experiments. When Bama pigs (20 kg) were given RC32 (8 mg / kg twice daily) for 2 days, FKBP12 protein was effectively degraded in most of the organs examined. After RC32 administration, only residual levels were detected in the heart, liver and kidneys. Consistent with the study findings in mice, peritoneal injection of RC32 had no effect on FKBP12 degradation in pig brains. Nevertheless, researchers have made significant progress in using PROTAC chemistry strategies in large animals and building protein knockdown models in such a short period of time. Therefore, researchers believe that chemical methods inspired by the success of RC32 may produce valuable specific protein knockdown pigs for use in human disease and xenograft research.

  1. Validate the degradation and function of FKBP12 in rhesus monkeys

Encouraged by the results of the pig model, researchers extended the study to rhesus monkeys. After 3 days of administration (8 mg / kg, intraperitoneal injection, twice daily), FKBP12 in the heart, liver, kidney, spleen, lung and stomach of monkeys was effectively degraded by RC32. Little or no FKBP12 can be detected in adipose tissue, bladder, and intestine. RC32 failed to degrade FKBP12 in the brain, consistent with the results in mice and pigs. In addition, researchers performed functional heart studies 3 and 15 days after RC32 administration, and 7 and 21 days after RC32 was discontinued. Similar to the effect of FKBP12 knockdown on mice, RC32 treatment reduced EF (6.7% on day 15) and FS (13.4% on Day 15) and systolic pressure line. In addition, heart rate decreases with treatment. After RC32 was discontinued, heart function gradually recovered and returned to a near-normal state after 22 days, accompanied by recovery of FKBP12 protein levels. These results indicate that the chemical protein knockdown method can be used for self-controlled studies. Because cardiovascular disease (CVD) is the leading cause of death worldwide, using this chemical method to knock out CVD-related proteins provides a new strategy for studying the causative factors and potential therapies for CVD. More importantly, the PROTAC study of degradation of FKBP12 provided proof of principle for this strategy for creating efficient and reversible protein knockdowns in monkey models.

  1. Expanding the applicability of chemical knockdown strategies in BTK

Finally, to broaden the applicability of this chemical knockdown method, based on the previous research, Bruton tyrosine kinase (BTK) -knockout mice were generated using a further optimized BTK degradation agent. After 11 days of treatment with P13IS (intraperitoneal injection, 33 mg / kg, three times a day), BTK in fat, thymus, abdominal lymph nodes, and axillary lymph nodes was effectively degraded. In addition, significant degradation of BTK was observed in bone marrow, lung, and intestinal lymph nodes. Similar to PROTAC that degrades FKBP12, BTK levels are restored 7 days after discontinuation of P13IS.


In summary, these in vivo protein degradation results indicate that the PROTAC method can be used to establish animal knockdown models at the protein level. This method requires only a few days of administration to achieve protein knockdown. This technology greatly reduces the time and cost of establishing preclinical animal models, especially for larger non-human mammalian models.

Researchers have developed a chemical method for general knockdown of target proteins using PROTAC. It is a novel, fast, and effective method for producing animal models of protein depletion, such as mice, rats, pigs, and rhesus monkeys. In addition, this strategy can also achieve target protein knockdown in the brain by i.c.v. administration. This method is also very effective when PROTAC is administered orally. After stopping PROTAC, FKBP12 protein levels recovered within a certain period of time. This model is suitable for self-controlled biomedical research. FKBP12 knockdown mice and rhesus monkeys exhibited Ca2 + loss and Ca2 + increased sparks through chemical strategies, leading to cardiac dysfunction. Therefore, these models can be developed for cardiac drug screening. In addition, the method can also be applied to other targets, such as BTK. For target proteins that do not have a well-documented specific conjugate, developing PROTAC can be a challenge. However, since ligands need only have a low affinity for the target protein to be sufficient for proteolysis using PROTAC, the PROTACs strategy will provide more ways to achieve effective protein knockdown. 


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