Targeted Proteolytic Chimera (PROTAC) As A Therapeutic Tool

Posted October 4, 2020 by Bonnibelle

Given that patient studies with focal adhesion kinase (FAK) inhibitors failed to fulfill their preclinical promises, Cromm et al.

The role of FAK scaffolding is essential for cell migration, but essential for proliferation
Given that patient studies with focal adhesion kinase (FAK) inhibitors failed to fulfill their preclinical promises, Cromm et al. (2018) FAK inhibitor defactinib was incorporated into PROTAC to evaluate the effect of FAK degradation on inhibition (Cromm et al., 2018). Cell culture studies have shown that although FAK kinase inhibition cannot prevent cell migration in wound healing tests and prevent invasion in cross-hole tests, FAK degradation is effective, emphasizing the kinase-independent function of FAK. Interestingly, this work and a parallel study by Boehringer Ingelheim (Popow et al., 2019) failed to replicate the anti-FAK depletion mediated by short hairpin RNA (shRNA) (McDonald et al., 2017).

Tag-based system
The development of customized PROTACs for potential targets may be beyond the capabilities of many academic laboratories; therefore, tag-based methods have been developed that combine genetic modification with the power of PROTAC technology. The two most commonly used PROTAC systems are discussed below.

The HaloPROTAC system utilizes HaloTag protein, an engineered bacterial dehalogenase, which can orthogonally conjugate chloroalkane-labeled molecules with target fusion proteins (Los et al., 2008). It has been reported that both VHL recruitment (Buckley et al., 2015, Tovell et al., 2019) and HaloPROTAC recruited by cIAP (Tomoshige et al., 2015, Tomoshige et al., 2016) induced the degradation of various HaloTag fusion substrates, including cytoplasmic proteins (ERK, MEK and GFP), endosomal proteins (VPS34 and SGK3) and nuclear localization protein (CREB1). The HaloPROTAC system also provides biological insights into multiple systems, as described below.

The role of PNPLA3 in fatty liver disease
BasuRay et al. (2019) uses HaloPROTAC3 to degrade HaloTag fused with patatin-like phospholipase domain protein 3 (PNPLA3) in vivo. The I148M mutation in PNPLA3 is associated with non-alcoholic fatty liver disease, which causes steatosis through the accumulation of triglycerides into lipid droplets (Smagris et al., 2015). The I148M mutation results in a decrease of about 80% in triglyceride hydrolase activity, but surprisingly, the development of hepatic steatosis requires the presence of reduced or non-catalytic PNPLA3 protein. PROTAC-mediated degradation of I148M PNPLA3 in vivo restored normal triglycerides in mice, providing other evidence that the accumulation of mutant PNPLA3 is the cause of liver steatosis.

Kinetics of WASH complex formation
The HaloPROTAC system has also been used to confirm that heat shock factor binding protein 1 (HSBP1) is the assembly factor of the Wiskott-Aldrich syndrome protein and the SCAR homolog (WASH) complex, which plays a key role in endosome sorting. The degradation of the HaloTag: WASH fusion protein during PROTAC elution and subsequent siRNA knockdown of HSBP1 indicate that HSBP1 is an assembly factor required to remodel CCDC53 homotrimerization into a WASH complex (Visweshwaran et al., 2018). This method emphasizes the use of PROTAC to achieve temporary control of protein levels, thus realizing the pulse tracking experiment, otherwise the experiment may be challenging.

The dTAG system works similarly to HaloPROTAC, but fused the F36V FKBP mutant protein (Clackson et al., 1998) with the target protein instead of labeling the protein with HaloTag protein. The dTAG system uses F36V selective "bump" ligands, tethered with immunomodulatory imide drugs (IMiD) derivatives to recruit CRBN and subsequently degrade FKBP fusion proteins (Nabet et al., 2018). The system has been shown to be suitable for in vivo experiments via intraperitoneal injection of 25 mg/kg. The proof-of-principle research shows that dTAG is suitable for a variety of proteins, including HDAC1, MYC, EZH2, PLK1 and KRAS G12V, and the system has been used to solve biological problems, as shown below.

Basal-like breast cancer cells do not depend on MELK
Previous studies using shRNA silencing have shown that the survival of basal-like breast cancer (BBC) cells depends on the expression of maternal embryonic leucine zipper kinase (MELK) (Hebbard et al., 2010; Touré et al., 2016). However, an in-depth study using selective MELK inhibitors, CRISPR and PROTAC-mediated degradation found that BBC cells have nothing to do with MELK levels (Huang et al., 2017). Huang et al. (2017) used CRISPR gene editing technology to knock out MELK and observed no effect on proliferation. Similarly, MELK inhibitors have no anti-proliferative activity. Considering that compensatory signals appeared during the selection and/or cloning process to explain their observations, they also introduced a dTAG version of MELK before knocking out the endogenous MELK, so that MELK can be expressed continuously, thereby avoiding the occurrence any motivation to compensate. Despite acute MELK loss, the rapid and selective induction of dTAG-MELK degradation using dTAG PROTAC has no effect on proliferation, which proves that BBC cells are not dependent on MELK.

Cytoplasmic mutant nucleoprotein is critical to the development of leukemia
Brunetti et al. (2018) used CRISPR/Cas9 gene editing to prove that the relocation of mutant nucleophosphoprotein (NPM1) through the destruction of nuclear export sequence can inhibit cell proliferation and induce hematopoietic stem cell differentiation. This occurs by disrupting the HOX/MEIS1 transcription program, which is consistent with the hypothesis that the HOX/MEIS1 gene is the main regulator of the hematopoietic lineage (Argiropoulos et al., 2007). To further confirm that the lack of cytoplasmic NPM1 induced this phenotype, Brunetti et al., J. Med. Chem., 2000, 53, 1959. (2018) Use dTAG system to quickly deplete NPM1 (loss> 85% in 4 hours). This led to the same phenotype, confirming the direct correlation between the down-regulation of HOX/MEIS1 expression and the lack of cytoplasmic NPM1.

After the dTAG system was identified in the genome-wide CRISPR screen, it has also been used to confirm the addictive effect of acute myeloid leukemia cells on ENL (YEATS) domain-containing protein 1 (Erb et al., 2017) and prove that the degradation of SNF5 allows cMyc and staining qualitatively re-associated (Weissmiller et al., 2019). It has also been used to prove that YY1 has no direct role in the regulation of replication time (Sima et al., 2019), and that OCT4 is essential for the localization of coagulum mediated by hyperenhancers in embryonic stem cells (Boija et al. Et al. 2018).

Both HaloPROTAC and dTAG are powerful methods, and can be considered to be used at the same time due to their orthogonality. However, there are subtle differences between the two systems, and it is possible to determine which system is best for each application. For example, compared to HaloTag (33 kDa), dTAG requires a smaller tag (FKBP12 F36V, 12 kDa) to be incorporated into the target protein, which may be beneficial for the use of dTAG in crowded systems where the tags may interfere Protein-protein interaction. However, the commercial availability and other uses of the HaloTag fusion (England et al., 2015) are a perfect choice, for example, when one wants to see the subcellular localization of a protein and induce its degradation. In addition, the HaloPROTAC system did not show the “hooking effect” observed with dTAG. In addition, the use of VHL ligands in the HaloPROTAC method allows the use of diastereoisomeric controls and avoids the problems associated with the use of IMiD-based PROTAC.
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Last Updated October 4, 2020