Proteolysis-Targeting Chimeras

By Mark Comer

Cells tightly control the levels of proteins using intracellular machinery. The ubiquitin proteasome system (UPS) helps remove damaged, faulty, or surplus proteins by tagging them with ubiquitin to facilitate proteolysis by the proteasome. Defective UPS is implicated in diseases such as cancer, Alzheimer’s, and several inflammatory conditions (Subhankar et al,2008). A proteolysis targeting chimera (PROTAC) exploits this mechanism of protein degradation to overcome the limitations of small-molecule drugs and monoclonal antibodies (mAbs). A PROTAC will induce selective degradation of a specific protein of interest (POI) by bringing together the protein of interest and an E3 ligase, leading to the transfer of ubiquitin to the protein. Comparatively, most contemporary pharmaceutical agents derive their effects from occupancy of receptors/active sites or competition with endogenous molecules and are ineffective against a large proportion of the proteome. Potential targets, such as transcription factors and scaffold proteins, are considered ‘undruggable’ due to the absence of a suitable active site. Some protein-protein interactions can also be disrupted by monoclonal antibodies, but their effectiveness is limited. PROTAC development has largely focused on applications in oncology but applications in auto-immune disorders and viral infections are also being explored. By manipulating endogenous protein degradation pathways, PROTACs represent a potential improvement over current treatment options.

Only approximately 20-25% of the proteome is subject to ongoing drug development research (Sun et al, 2019). Proteins that have been successfully targeted are largely limited to membrane receptors, kinases, ion channels, nuclear receptors, or enzymes with suitable active sites. Other proteins like mutant K-Ras, MYC, and multiple oncogene products are attractive therapeutic targets but, as they lack obvious binding sites, they are very difficult to disrupt with a conventional approach. Although success has been achieved using small molecule drugs, acquired resistance often arises from mutations in the active site. Mutations in ‘gatekeeper’ residues in proteins like tyrosine kinases, BCR-ABL, and EGFR results in steric hindrance by disrupting hydrophobic interactions necessary for inhibitor binding (Chell et al, 2013). Likewise, cancer cells can acquire signalling redundancy and employ alternative pathways to survive treatment. Acquired resistance to kinase inhibitors is responsible for a significant proportion of treatment failures (Bhullar et al, 2018). However, it is undeniable that development of these inhibitors has boosted patient survival significantly (Wu, Nielsen, & Clausen, 2015). Small molecule drugs are entirely ineffective against pathogenic proteins such as Tau or alpha-synuclein/Lewy bodies common in neurodegenerative diseases. 

Antibody therapies are another strategy to target proteins. For example, antibodies targeting immunoregulatory CTLA-4 and PD-1 membrane proteins are the subject of ongoing research as a cancer immunotherapy. Nevertheless, antibody treatments are limited by their inability to bind intracellular targets and poor antibody uptake in the tissues- exposure to target tissues in murine models did not rise above 20% (Beckman, Weiner, & Davis, 2007). Heterogeneity in tumours also means that antibody design is difficult, largely held back by difficulties in identifying appropriate antigens. Resistance to mAb therapies also occurs, the mechanisms behind resistance are less well characterised, with mutations in the selected antigen being the most likely reason. This is seen with mutations in CD20 correlating with resistance to CD20 antibodies (Reslan, Dalle, & Dumontet, 2009). Additionally, hyperactivation of anti-apoptotic/pro-survival signalling pathways is another potential mechanism for resistance to treatment whilst mutant KRAS was another strong predictor of resistance (Reslan,Dalle, & Dumontet, 2009).

Treatments utilising small molecules or mAbs aimed at specific proteins and their interactions have boosted patient survival but have significant limitations. A PROTAC circumvents the issues faced by mAbs and small molecule drugs. A PROTAC is a “heterobifunctional molecule” (Sakomoto et al, 2001) composed of a ligand for the POI (a ‘warhead’) and a ligand for an E3 ligase, with a linker joining the two together. A PROTAC will complex with the POI and the chosen E3 ligase, an E2 ubiquitin conjugating enzyme will then ubiquitinate the POI leading to degradation by the proteasome. The PROTAC acts catalytically and remains unchanged, this means that PROTACs are effective in vitro at nanomolar concentrations (Pei et al, 20019). The primary benefit of such potency is a potential reduction in side effects that are common with other treatments. Moreover, unlike contemporary inhibitors, binding affinity to the POI is the only major concern in the design, as PROTACs do not require any inhibitory activity whatsoever. This may also help overcome problems arising from resistance as mutations are unlikely to significantly reduce the ability of a PROTAC to bind its POI. The first successful PROTAC constructed by Crews et al in 2001 utilised a phosphopeptide which was recognised by  β-TRCP (an E3 ligase) and a small molecule drug: ovalicin was used to bind METAP2 (methionine aminopeptidase), the POI. The experiment proved that the manipulation of the UPS to selectively degrade proteins is a viable strategy. 

Other constructs have since been developed, including PROTACs that utilise MDM2’s E3 ligase activity. These have successfully degraded the androgen receptor (Schneekloth et al,2008) and the oestrogen receptor in vitro (Okuhira et al, 2013). The repertoire of PROTACs was further boosted by development of cereblon PROTACs. A cereblon-based PROTAC was shown to degrade BRD4, an important driver of cancer. BRD4 is thought to drive expression of critical oncogenes such as c-myc, and bcl-xL, it is normally targeted using BRD4 inhibitors. A PROTAC, known as ARV-825, was found to be superior to the inhibitors as the PROTAC successfully suppressed c-myc and reduced the levels of BRD4 protein. ARV-825 was also more effective at suppressing cell proliferation and boosting caspase activity (Lu et al, 2015), clearly demonstrating the benefits of a PROTAC over small molecule drugs and antibodies. Further success has been achieved using PROTACs employing VHL (Von Hippel-Lindau) protein as its E3 ligase, these constructs degraded a significant amount of the target protein with up to 86% degradation of the oestrogen-related receptor in vitro and up to 40% degradation in vivo with xenograft mice models (Bondeson et al, 2015). 

PROTACs are likely to drive increasing accessibility to the proteome. As a developing technology, the potential applications against ‘undruggable’ targets has not yet been explored in much depth, nonetheless current research indicates that a PROTAC-based approach could represent a solution to the problems of small molecule drugs and antibody therapies. The ability to degrade specific proteins of interest and thus alter the phenotype of a cell on the protein level is a potentially powerful tool in academia and could help overcome serious challenges in drug development. In essence, a PROTAC combines the precision of small molecule inhibitors alongside a potent knockdown effect comparable to CRISPR or RNAi techniques (Bondeson et al, 2015). Whilst in vitro studies have indicated powerful therapeutic effects, increasing the repertoire of PROTACs alongside optimisation of delivery and construction will be necessary for any successful clinical application.


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