The Workflow of PROTAC System (Part Two)

Posted October 6, 2020 by Bonnibelle

The nature of small molecules. Their small molecule properties make PROTACs as easy to use in experiments as inhibitors.

Pros and cons of PROTAC


Catalytic mechanism. Since PROTAC operates through an event-driven mechanism rather than an occupant-driven mechanism, they act as a catalyst for selective protein degradation because one molecule of PROTAC induces the ubiquitination of multiple molecules of target protein (Bondeson et al., 2015). Indeed, recent studies have shown that despite the covalent modification of E3, an E3 ligase occupancy rate as low as 10% can still effectively induce target degradation (Zhang et al., 2019b), thereby creating a permanently reprogrammed E3 Ligase. By using low-affinity ligands, the limited occupancy of the targeted ligand side has also been shown to be sufficient (Crew et al., 2018; Smith et al., 2019) or co-treatment with competitive agonists (Salami et al., 2018). This catalytic property of PROTAC is similar to RNAi.

The nature of small molecules. Their small molecule properties make PROTACs as easy to use in experiments as inhibitors. The application of PROTAC does not require special transfection reagents, culture conditions or viruses. In fact, in addition to tag-based systems, the use of PROTAC does not require genetic modification of the model system, so that it can query a completely endogenous system. In this way, the same method can be used to directly compare degradation and inhibition, thereby enhancing the biological understanding of protein functions, and eliminating the interference of transfection reagents that may induce their own effects or mask subtle effects on complex biological systems.

Another advantage caused by the small molecule nature of PROTAC is that the concentration of the compound can be controlled, so that the protein level can be quantitatively controlled. PROTAC can use an "analog" method to adjust protein levels between 0% and 100% without having to use a "digital" on/off switch as observed through gene editing. This may be particularly effective for dealing with proteins that are overexpressed in disease states but are essential in normal or healthy states. After performing a dose response experiment, it is possible to treat with a dose that induces sub-maximal degradation, so that it is possible to restore the overexpressed protein level to a normal level.

Time control. PROTAC allows precise time control of protein levels (degradation can be observed in as little as 1 hour), making it possible to study acute protein loss in ways that are not possible with many other technologies. This can prevent biological compensation after key proteins are missing in the selection process, just like various RNAi technologies and CRISPR/Cas9. A common CRISPR technology uses homology-directed repair (HDR) to insert GFP or resistance genes at the disturbed locus (Leonetti et al., 2016), allowing selection of cells that have been successfully edited. Although undoubtedly powerful, this multi-day option can select cell populations with alternative pathways that can drive the proliferation or bypass of problems caused by the loss of specific proteins. PROTAC can quickly deplete the protein in the entire cell population, thereby providing a more accurate method to query the function of the protein in the natural environment.

In addition, PROTAC withdrawal can quickly restore the target protein level at the speed allowed by protein re-synthesis. This provides reversibility and allows for elegant experiments, such as those described above for the WASH complex. Although some PROTAC may survive in the cell and continue to degrade after the compound is washed out, the simultaneous addition of excess E3 ligase ligand can competitively prevent any other degradation (Burslem et al., 2018b).

Portability. Another advantage of the PROTAC method is its portability. Generally, if PROTAC induces protein ubiquitination and degradation in a system, as long as the new system has the required mechanism (E3 ligase, etc.), it can be used more widely. This allows rapid screening of protein roles in different cell types without modifying their genes. In addition, it enables the study of proteins that are not suitable for other technologies, such as patient cells that cannot be cultured; for example, the CML stem cells discussed above (Burslem et al., 2019).

Another advantage of portability lies in the transition to in vivo experiments and potential clinical translation. The application of PROTAC in vivo does not require genetic manipulation of animals, so that the experimental process can be carried out faster and the system can be detected without interference. Although PROTACs may require optimization of their physicochemical properties to work in vivo, there are many examples of PROTACs functioning in the body, including mammals (Bondeson et al., 2015, Burslem et al., 2018C, Jain et al., 2019, Nabet Et al., 2018, Winter et al., 2015) and non-human primates (Sun et al., 2019). Although PROTAC presents any potential pharmacokinetic challenges, their small molecule nature and portability make them easy to translate in ways that other technologies cannot directly translate. It is important to note that PROTAC often exhibits better pharmacokinetic properties than expected by its molecular weight. In fact, it is possible to develop PROTAC with human oral bioavailability. The recent phase 1 clinical trial report of ARV-110 and ARV-471 confirms this. One oral administration can reach the effective range observed in preclinical studies every day.


Discovery stage. Although all the advantages of PROTAC are outlined above, they are not without challenges. A major disadvantage is the lead time for PROTAC development, which can be relatively long compared to designing and ordering siRNA or guide RNA. PROTACs not only need to be a ligand for the target protein, but they are also converted to PROTAC. This can be a time-consuming process involving a lot of synthetic chemistry and medicinal chemistry. Tag-based systems (HaloPROTAC and dTAG) bypass this discovery stage, but also abolish some of the advantages of the PROTAC system in terms of portability and lack of genetic manipulation.

Off-target effect. Like other technologies, PROTAC may have off-target effects. After PROTAC treatment, the use of proteomics to quantify proteins (including low-level expressed proteins) is a powerful tool to assess the off-target effects of PROTAC (Savitski et al., 2018). Even knowing the selectivity of recruiting elements, proteomics can reveal surprising new PROTAC substrates, which is probably due to the additive effect of the protein-protein interaction between the protein in question and the E3 ligase. To (Bondeson et al., 2018). Theoretically, the binding of ligands to E3 ligase may interfere with the binding of endogenous substrates. Fortunately, the ligand concentration is usually much higher than that of the PROTAC recruited by VHL (Frost et al., 2016) to stabilize its endogenous target HIF-1α (Burslem et al., 2017); however, off-target effects may affect other E3 Ligases cause greater problems, and these E3 ligases currently have poor or unknown characterization of natural substrates. The off-target effect of PROTAC recruiting CRBN must be evaluated very carefully, because the IMiD component of PROTAC alone or incorporated into PROTAC can induce degradation of the new zinc finger CRBN substrate (Fischer et al., 2014; Ishoey et al., 2018, Krönke et al., 2014, Matyskiela Et al., 2016).

Hook effect. PROTAC and other bifunctional molecules initially exhibit curiosity, but are completely rational phenomena, so higher concentrations do not always produce greater effects (Douglass et al., 2013). This so-called "hook effect" is caused by the formation of non-productive dimers at high PROTAC concentrations instead of degradation of the desired productive trimer complex. This raises concerns about the pharmacokinetic/pharmacodynamic (PK/PD) relationship and dosing regimen. However, it should be noted that the favorable protein-protein interaction between E3 and the target protein seems to increase the maximum concentration that can be used before the hook effect is observed (Buckley et al., 2015; Tovell et al., 2019).

Not all proteins or subcellular locations are suitable. Finally, because PROTAC is still an emerging technology, they have not been proven to work against every protein class or in every subcellular compartment. Cytoplasmic and nuclear proteins can be degraded routinely (Bondeson et al., 2015); in fact, some E3 ligases allow selective nuclear degradation (Zhang et al., 2019b). Several examples of receptor tyrosine kinase degradation have been reported (Burslem et al., 2018b, Burslem et al., 2018c), but no examples of single-pass transmembrane proteins have been reported. HaloPROTAC has been used to degrade proteins located in endosomal membranes (Tovell et al., 2019). Although hydrophobic labeling experiments have verified the unfolded protein response in the Golgi apparatus and endoplasmic reticulum (ER) (Hellerschmied et al., 2019; Raina et al., 2014; Serebrenik et al., 2018), protein degradation by PROTAC has not been Realization needs to be reported. Customized ligands for ligases located in these organelles may require PROTAC-mediated degradation at these subcellular locations (Smith et al., 2011).
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Last Updated October 6, 2020