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

Brief introduction to PROTAC

PROteolysis TArgeting Chimeria (PROTAC) is derived from the 2004 Nobel Prize in Chemistry-Israeli scientists Aaron Ciechanover, Avram Hershko and American scientist Irwin Rose, who have jointly discovered how cells destroy harmful proteins by ubiquitin (Ub) regulating the process of protein degradation. Utilizing the mechanism of protein degradation by ubiquitin, Raymond J. Deshaies (Academician of the United States) and Craig M. Crews (Founder of Arvinas) and other scientists first proposed the concept of PROTAC in 2001 and successfully designed and synthesized the first batch of PROTAC bifunctional molecules. Those molecules were used to degrade methionyl aminopeptidase 2 (MetAP-2). Coincidentally, viruses and cancer cells also use this mechanism to degrade proteins that are resistant to them, such as SAMHD1, APOBEC3G, p53, and so on.

Advantage and disadvantage of PROTAC

The advantage of PROTAC is that it can effectively inhibit the target protein and can quickly degrade and clear it. In theory, it only needs a small amount of drugs to degrade almost all proteins (including membrane proteins) in the cell, so it has high safety, drug resistance and broad applications.

The disadvantage of PROTAC is that its high molecular weight results in poor water solubility, oral absorption and membrane permeability. PK is a major obstacle and the cost of chemical synthesis is high. Its off-target toxicity is worthy of attention, because ubiquitination labeling is not only related to protein degradation, but also related to methylation, acetylation, phosphorylation and other processes; not only protein but also DNA. Just like thalidomide’s teratogenicity to the fetus, its off-target effect is not easy to detect and track in preclinical toxicity screening, and its long-term and reproductive toxicity is a serious problem.

The background of study design

An animal model of protein knockdown is a powerful strategy for studying the consequences of functional loss of target genes. Traditionally, studying the consequences of loss of gene functionality has been mainly through genetic modification, such as RNA interference, transcription activator-like effector nucleases, recombinant-based gene knockouts, the CRISPR-Cas9 system, and other genome editing strategies. However, applying these strategies to large animals remains to be a challenge, especially in non-human primates. These animals have always played an important role in basic research and new drug discovery. However, these methods cannot control the acute and reversible changes in gene function to some extent. Furthermore, potential genetic compensation can produce complications of spontaneous mutations in gene knockout models, which may mislead results. In addition, the deletion of many genes also leads to the death of animal embryos, which has hindered related scientific research.

Protein degradation targeting chimeras (PROTACs) use a Linker with one end linked to the inhibitor of the target protein and the other end linked to the E3 ubiquitin ligase. PROTAC represents a chemical knock-down strategy. The PROTAC molecule pulls the target protein and E3 ligase, so that E3 can ubiquitinate the target degradation protein, and then degrade the target protein through the proteasome. The Crews research group first reported the development of peptide-based PROTAC. Subsequently, more and more researchers are committed to successfully degrading the protein of interest through PROTAC technology. Currently, PROTAC is mainly used to discover new anticancer agents because they have unique advantages over classic inhibitors. To our knowledge, this new strategy has not yet been used in large animal models to achieve global protein knockdown. The closely related model species of the rhesus monkey is a unique model that is often used to study various diseases caused by its humanoid genome, controllability of environmental factors, and real-time monitoring of metabolic phenotypes. However, it is unclear whether PROTAC works in non-human primates.

The study demonstrate that PROTAC technology is a convenient, fast and reversible chemical method that can globally and quickly (24–72 hours) degrade proteins in living animals, especially pigs and rhesus monkeys. After discontinuing PROTAC, protein levels recovered, suggesting that the method is cost-effective and time-efficient in self-regulated animals. In addition, the study used PROTACs to knock down FKBP12 in mice and rhesus monkeys, and studied the important role of FKBP12 in maintaining cardiac function.

To be continued in Part II…



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