The accurate control of protein synthesis and degradation is key for cell homeostasis. Dysfunction of these processes can be damaging to the body and can result in life-threatening diseases, like abnormal cell proliferation in cancer.
For some conditions, the targeted inhibition of abnormal proteins and their pathways has been a successful therapy, but a novel group of small molecules has shown an alternative method that can be used to remove these proteins from the body.
Proteolysis Targeting Chimeras (PROTACs) are small heterobifunctional molecules that are able to target chosen proteins of interest (POI) and, by hijacking the body’s own natural disposal system, are able to degrade them.
PROTACs, seen in Fig 1, was first uncovered around 20 years ago and have been explored as an additional modality to the drug discovery toolbox since then.
Figure 1. Mechanism of action. Image Credit: SP Scientific Products
Studies have indicated that, compared to current drugs, smaller concentrations of PROTACs are required to be administered to achieve the therapeutic effect. This enables fewer adverse effects and reduces the possibility of drug resistance.
When treating cancer, many PROTACs targets have been developed to stop the protein which is responsible for abnormal or uncontrolled growth, like BCR-ABL, which can be found in human epidermal growth factor 2, which is associated with breast cancer or specific types of leukemia.
The first PROTAC, ARV110 (Arvinas, USA), has only recently entered phase I clinical trials for metastatic castration-resistant prostate cancer. Similarly, there has been a huge growth of scientific publications, some of which provide the potential to target the ‘undruggable’ proteome, which makes up around 85 % of human proteins.
Biopharma Group and SP Scientific Products recently held a webinar from three key opinion leaders outlining the design of PROTACs and the evaporation challenges relating to their synthesis, which is detailed in this article.
Molecular mechanism of PROTACs
PROTACs are made up of three components: a ligand for the POI, an E3 ligase ligand, and a linker that joins them together. The POI ligand and E3 ligand bind to their respective targets to form a ternary complex.
The POI and E3 are in close proximity once bound, which prompts the E3 to transfer numerous ubiquitin molecules to the POI. This is then recognized by the proteasome as part of the ubiquitin-proteasome system (UPS), which results in the degradation of the POI.
This is different from the mechanism of the majority of the inhibition drugs currently on the market, which bind to the POI and inhibit it but do not eradicate it from the body.
Understanding the ternary complex: On the road to better degraders
The design of PROTACs is mostly empirical and typically consists of the screening of small, sizable molecule libraries. A rational design method could relieve the burden of screening big libraries and lead to a selection of more specific small molecules.
Dr. Emelyne Diers from Prof Ciulli’s group at the University of Dundee, UK, talked about her work with Boehringer Ingelheim in the rational design of a PROTAC and the importance of the ternary complex.
Their group used a multidisciplinary method to design PROTACs. The process begins by examining the chemistry of the three components which make up a PROTAC – the POI ligand, the E3 ligase ligand (e.g., Von Hippel Lindau (VHL)), and the linker that binds them together to produce an optimal ternary complex.
For a novel method, there are fewer rules or standards of development, so a selection of candidates is selected for subsequent studies. Biophysical assays establish the stability and cooperativity of the chosen molecules and crystal structures visualize the interactions that are possible between the components in them.
Then, to determine how the ternary complex influences the efficacy of the degrader, the chosen structures can be tested in cell lines. Not all ternary complexes exhibit degradation in biological assays, though all PROTACs form ternary complexes. Crystal structures have proved to be advantageous for the design of better PROTACs.
MZ1 was the first reported ternary crystal structure; it is a bromodomain-containing protein 4 (BRD4) selective degrader. MZ1 brings the transcription factor, BRD4, and VHL (the E3 ligase ligand) in a highly cooperative complex that is long-lived, stable, and exhibits degradation of BRD4 at a quick rate.
Due to its part in heightening the expression of factors that are crucial for the pathogenesis of cancer, BRD4 has been the target of many inhibition drug candidates. More efficient anti-cancer activities have been demonstrated by some of the current BRD4 degraders than inhibition alone, which indicates that MZ1 may be a good candidate for cancer therapy.
The importance of examining the crystal structure when designing a PROTAC is demonstrated by another example, that of ACBI1, a small molecule-based on VHL and SMARCA binders. SMARCA is part of the BAF chromatin remodeling complex, which is at least 20% of cancers, is mutated.
Cancer cell growth is thought to depend on SMARCA4 in leukemia but has been considered undruggable and has proven to be difficult to treat with inhibition drugs. A small-molecule PROTAC (PROTAC1) was identified in a recent study from library screening and the degradation activity improved further by high-resolution ternary complex crystal structure analysis.
ACBI1 was discovered to be a potent degrader of PBRM1, SMARCA2, and SMARCA4 and induced apoptosis in cancer cell lines. It is clear from this study that an in-depth knowledge of the ternary complex is vital.
These experiments show that a structure-based design method used for ternary complex formation is also advantageous for producing optimal PROTACs that can even target previously undruggable targets.
Expanding the horizon of targeted protein degradation
Where and when and a protein is degraded, PROTACs have the potential to regulate protein levels in space and time controlling. For instance, a user could wish to degrade the protein in a specific part of the body or at a specific time in the cell cycle but not in other organs.
There could be dramatic consequences for a drug’s safety profile with the ability to turn a drug on and off. Mr. Cyrille Kounde describes the work that he and his colleagues in Prof Ed Tate’s group carried out at Imperial College London, the UK creating PROTACs with conditionally targeted degradation mechanisms.
Proteolysis is induced by light, an external stimulus that is quick and non-invasive fast in one instance. Another method involves delivering the PROTAC via an engineered antibody-conjugate.
Utilizing light as a precision tool has been shown in medicine as photodynamic therapy (PDT) for conditions like skin disorders and neck and head cancer. The intensity and duration of light can be easily controlled so that the drug is delivered precisely and quickly without invasion into the body.
An inactive complex must be designed when applying this to the delivery of PROTACs. This must only form an active ternary complex which induces degradation when exposed to light. No protein modification or UPS engineering is needed, which makes this an attractive choice for a conditional PROTAC molecule.
A light-activated PROTAC targeting the BRD4 transcription factor was designed by Mr. Kounde and his colleagues. The inactive form includes a caging group attached to the E3 ligase ligand, which is eradicated by exposure to 365 nm UV light for 1 minute.
This conditional PROTAC3 behaved with comparable kinetics and in a similar dose-dependent manner when activated by light when compared with an active non-conditional PROTAC2 in HeLa cells. After irradiation, PROTAC3 also exhibited inhibition of cell proliferation over time.
Antibody targeted degradation
Targeting the POI using a monoclonal antibody (mAb) system is an alternative strategy to treat cancer without affecting other cells. There are 79 mAbs approved by the United States Food and Drug Administration (FDA); 30 of these have been approved for cancer.
So it would be wise to design a PROTAC antibody-conjugate based on one of this approved mAbs. The HER2 mAb-PROTAC3 caged conjugate was created by Mr. Kounde and colleagues in order to target and degrade BRD44.
Utilizing cell lines that expressed BRD4 and either expressed or lacked expression of HER2, the HER2 mAb-PROTAC3 was only cleaved in the HER2+ cell lines, which resulted in HER2-dependent BRD4 degradation.
A fluorescent lysosome-targeting antibody was used to confirm evidence of the internalization of the PROTAC. Both multifunctional PROTACs degrade proteins with higher precision than a non-conditional PROTAC, which indicates a future in this targeted method for cancer therapy.
Eliminating bottlenecks in PROTAC synthesis – Focus on evaporation
A multidisciplinary approach between biology and chemistry is needed for small molecule or peptide-based PROTAC synthesis, as previously discussed. It has been demonstrated that understanding the ternary PROTAC complex will help to rationally design the small molecule libraries, which will ultimately decrease time and money spent.
In the first stages of this technology, however, the process is still time-consuming with multiple evaporation steps that cannot be eliminated. This leads to bottlenecks in the production of PROTACs and a slower development time for drug discovery.
The third presentation by Induka Abeysena, Portfolio Manager at SP Genevac, UK, outlined how to create a high-quality final product by overcoming the evaporation limitations in PROTAC synthesis.
Evaporation steps that happen throughout the small molecule synthesis include:
- Initial PROTAC complex synthesis, as cleavage or deprotection groups are removed
- Pre- and post-purification when concentrating the unprocessed mixture or the combined high-performance liquid chromatography (HPLC) fractions
- Post-reformatting, where the finished PROTAC molecule is transformed into the desired format for transportation.
These processes are crude and together with a range of different solvents, they involve vacuums, heat, and centrifugation, making it challenging to keep the integrity and quality of the final product. In some instances, the heat applied to a sample to speed up the evaporation can damage the compound if it is not controlled well.
It is also possible that compounds dry at different rates, which leads to heterogeneity in the quality of evaporation. The risk of contamination between samples when drying mixed solvent with different physical properties and boiling points is a further challenge in the evaporation process.
These samples are usually near to each other and can shower the neighboring samples with another sample or unwanted solvents. Lastly, each drying step takes time and this is dependent on volume size – the larger the volume to be evaporated, the slower the evaporation rates. More time also leads to increased labor costs.
The development of technology in commercial-scale evaporators has enhanced a number of these limitations. In order to fit various different requirements, SP Scientific Products manufacture centrifugal evaporators for parallel sample evaporation.
As they can be preprogrammed to dry many samples at once, keeping the consistency in all samples and allowing unattended operation, the SP Genevac HT Series 3i and EZ-2 personal solvent evaporators are perfect for medium to high-throughput sample processing.
They are compatible with a number of different solvents in multiple vessel types to assist the synthesis of a large range of small molecule and peptide-based PROTAC compounds. These systems are also able to freeze dry samples, particularly those which are challenging to dry.
The DriPure® and SampleGuard proprietary technologies allow these features by controlling the sample temperature and eliminating sample bumping to avoid overheating, compound sublimation, sample damage, and sample contamination.
The EXALT technology has been designed for the purposes of crystal production, to enable a large scope of solvents to be evaporated simultaneously at slow rates for structure characterization and polymorph screening.
Although these evaporators are mainly utilized on the benchtop, they can also be used for potent compounds, like Antibody Drug Conjugates (ADCs), which require safe handling in confined conditions such as installing the drying system in a glove box.
In addition to the parallel sample evaporator line, a more recent addition to the SP Genevac evaporator portfolio includes the Ecodyst® series. These are evolutionary single-sample evaporators with a built-in intelligent condenser.
Due to the lack of maintenance required and fast evaporation rates, these evaporators can help the medicinal chemist working with individual samples to work more efficiently. The requirement for consumables, like antifreeze and dry ice, is also removed.
Interest in PROTACs has grown at a fast rate in the last three years, with over 150 scientific publications on PROTAC research found on PubMed in 2020, compared to 30 in 2017. The majority of these are targeting cancer, but neurodegenerative diseases and others are also targeted.
ARV-110 for prostate cancer and ARV-471 for breast cancer (Arvinas, USA) are the furthest in clinical trials. The capability to target the POI without affecting other proteins is the holy grail, as with all cancer drugs.
Conditional PROTACs, which can be switched on at a specific time and place, could be a step towards this ideal. A multidisciplinary method is needed to design and create the optimal PROTAC studying the chemical interactions but also the manufacturing process and biological activity in vivo.
The presentations in this article give an outline of some of the areas that are vital to consider when going through this process, including optimal equipment to overcome operational bottlenecks.
Although there is potential for PROTACs to be powerful standalone therapeutic drugs, targeted protein degradation could be an advantageous additional modality to the drug discovery toolbox to work together with drugs on the market.
References and Further Reading
 Gadd, M.S. et al., Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 2017 13:514
 Farnaby, W. et al., BAF complex vulnerabilities in cancer demonstrated via structure based PROTAC design. Nat Chem Biol 2019, 15:672
 Kounde, C.S. et al., A caged E3 ligase ligand for PROTAC-mediated protein degradation with light. Chem Comm, 2020, 56(41): 5532
 Maneiro, M.; Forte, N.; Shchepinova, M. M.; Kounde, C. S.; Chudasama, V.; Baker, J. R.; Tate, Ed. W. ACS Chem. Bio. 2020 15 (6), 1306-1312
Produced from materials originally authored by Dr. Emelyne Diers from the University of Dundee, Cyrille S Kounde from Imperial College London and Dr. Induka Abeysena from SP Genevac.
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