An evolving class of therapeutics has the potential to allow the design of new drugs targeting “undruggable” proteins which make up to 85% of the proteome. They are called PROteolysis TArgeting Chimeras (PROTACs).
Image credit: Giovanni Cancemi | Shutterstock
Their innovative mode of action permits them to takeover a cells natural function and initiate degradation of the specific protein for a certain disease. The pharmaceutical and biotechnology industries have gained great interest due to this development.
Yet, synthesis of PROTACs needs several evaporation steps, leading to bottlenecks in their production and a reduction in the speed of the drug development process. Many have researched PROTACs and the benefits they can offer to the drug discovery field. Further discussions have investigated the issues with their synthesis, and how this can be overcome.
Conventionally, pharmaceutical drugs are small molecules that inhibit the active site of an enzyme target in a competitive way. The drug development process has depended on these types of agents in order to discover established methods. Therapeutics that have this occupancy-driven mode of action (MOA) make up most of the drugs on the market today.
However, this MOA has been known to have two noteworthy drawbacks. Firstly, high drug doses are usually needed to achieve the therapeutic effect, and secondly, most of the pharmacological protein targets do not have enzymatic activity.
A New Class of Therapeutics
In 2001, the Crews and Deshaies laboratories conveyed information about a new technology called PROteolysis TArgeting Chimeras (PROTACs). It was described as heterobifunctional molecules that takeover the body’s own normal disposal system in order to initiate selective degradation of the protein of interest (POI).
This MOA gives way to the possibility of targeting the “undruggable” proteome, which comprises of approximately 85% of human proteins, with the inclusion of scaffolding proteins, regulatory proteins and transcription factors. Additionally, a lower level of drug concentrations needs to be administered to achieve the therapeutic effect. This promises less adverse effects and reduces the potential of drug resistance.
The first PROTAC induced the degradation of methionine aminopeptidase-2. This is one of the main enzymes involved in blood vessel development of solid tumor cancers. It was peptide-based, just like several other early PROTACs.
These compounds increase the difficulty for the body to absorb them, resulting in research moving in the direction of the development of small molecule-based PROTACs. The first all small molecule-based PROTAC was noted in 2008. This degraded the androgen receptor which is a transcription factor with the ability to regulate the development and growth of the prostate.
Later, in 2013, evidence of PROTACs function inhibiting tumor growth was demonstrated in vivo for the first time. Since then, the development of further PROTACs has been reported, targeting a variety of POIs. These are inclusive of BCR-ABL which is found in types of cancer like leukemia, Tau which is linked with Alzheimer’s diseases and Parkinson’s disease, and human epidermal growth factor 2 which is indicative of breast cancer. Of these potential therapeutics, two PROTACs have currently progressed to phase I clinical trials, with the promise of further developments in the future.
The Event-Driven Mode of Action
In the human body, proteins are repeatedly degraded and replaced by newly synthesized molecules. Depending on the function of the molecule, the rate of protein degradation differs and can be from minutes to days.
For instance, regulatory proteins like transcription factors have a fast turnover to permit a rapid response to external stimuli. Additionally, this process of recycling allows proteins that are damaged or faulty to be removed in order to eradicate any problems in the future.
Most intracellular proteins undergo degradation due to the ubiquitin-proteasome system (UPS). This process involves a group of enzymes: E1 which is the ubiquitin-activating enzyme, E2 known as the ubiquitin-carrier and E3, ubiquitin-protein ligase.
These enzymes recognize the proteins for degradation and label them by attaching several ubiquitin molecules to their surface. Then, the polyubiquitinated proteins are identified by the proteasome which is a huge complex that subsequently degrades the polyubiquitinated protein into small peptides.
PROTACs are made up of three components most commonly known as an E3 ligand, a ligand for the POI, and a linker which connects the E3 ligand and the POI ligand together.
A ternary complex is formed when the E3 ligand and POI ligand bind to their respective targets. After they are bound, an E3 and POI are in close proximity, which triggers the E3 to transfer several ubiquitin molecules to the POI. Then, this polyubiquitinated POI is recognized and degraded by the proteasome as part of the UPS. After the POI degradation, the drug is released to continue its degradation task.
Figure 1. PROTAC mechanism of action. The heterobifunctional molecule comprises of an E3 ligase ligand (red square), a chemical linker (blue bar) and ligand for the protein of interest (black triangle). Image Credit: SP Scientific
Unlike the normal occupancy-driven drugs that function on a stoichiometric basis, this event-driven MOA leads to PROTAX molecules cycling through several rounds of activity, which removes super-stoichiometric quantities of the POI.
Additionally, taking over the cells natural protein degradation mechanism does not activate overexpression of the target protein which occurs to compensate for protein function loss. This action can be observed in drugs that inhibit a proteins active site. Due to these reasons, lower concentrations of PROTAC can be provided in order to create a therapeutic response. Low doses can be administered to patients, possibly lessening adverse effects and reducing the potential of drug resistance.
Synthesis of PROTAC
University science labs first discovered these heterobifunctional molecules, but now all major pharmaceutical companies like GSK, AZ, Pfizer and Bayer are investing in this emerging technology due to the potential to target the “undruggable” proteome, in combination with the promising pre-clinical results.
However, the challenge arises when developing the synthesis in the university in small batches, progresses to an industrialized, robust process that can yield high-quality products that meet the requirements for human administration. Additionally, a multidisciplinary approach between biology and chemistry is needed for synthesis because PROTAC molecules can be small molecule-based or peptide-based.
Evaporation Bottlenecks in PROTAC Synthesis
A number of evaporation steps are required in the production of both small molecule-based and peptide-based PROTACs. Firstly, during synthesis, for removal of cleavage or deprotection groups, and then, pre- and post-purification, for the crude mixture concentration or the combined high-performance liquid chromatography (HPLC) factions. Lastly, during post-reformatting, in transforming the completed compound into the chosen format for transportation.
During the drug discovery process, a large number of possible compounds are initially synthesized for the first non-clinical in vitro testing stages. This occurs before being narrowed down to a successful few that continue onto the clinical testing stage. With the many evaporation steps required for PROTAC synthesis, these drying processes have been highlighted as a major bottleneck with the many evaporation stops needed for PROTAC synthesis.
Additionally, there is a risk of the integrity and quality of the final product being negatively affected if these evaporation stages are performed badly. During evaporation, there are a variety of ways in which the compounds can be affected.
First of all, several evaporators use heat lamps to increase the speed of the evaporation process. During this method, the sample is directly heated, however, it is done in an uncontrolled way which can result in damage to the compounds.
Secondly, within the evaporators, different samples may dry at varying rates which can result in overheating of some compounds, while others are still wet. If some samples have dried, but still continue to be heated because others are wet, these dried samples may sublime and end up being lost.
Lastly, samples that bump can lead to solute and solvent showering the adjacent samples and causes contamination or loss of other samples.
SP-Genevac Evaporators can Eliminate Bottleneck Issues
Currently, drug development is a long and expensive process without the issues like time-consuming evaporation steps, cross-contamination and sample loss. Consequently, it is important that these issues can be solved to make PROTACs a viable option for biotechnology and pharmaceutical businesses to invest in.
Within the centrifugal evaporation and concentration processes sector, SP-Genevac is the industry leader and has developed two systems created to eradicate evaporation problems. These two systems are called the HT Series 3i Evaporator (HTS3i) and the EZ-2 Personal Solvent Evaporator (EZ-2).
The HTS3i and the EZ-2 both have the capabilities to dry a number of samples at one time, so they are suitable for high throughput. These evaporators can also accomodate a large range of sample formats, including vials, flasks, tubes and plates. The processes of drying, concentration and lyophilization can be completed in parallel.
Additionally, the systems are able to be pre-programmed and can distinguish when samples are dried, or can be set to a timed run so there is potential for unattended operation. These features help decrease the time-consuming issues that PROTAC synthesis currently involves.
These evaporation systems use SampleGuard technology - this can monitor and directly control sample temperature to prevent the potential of overheating, sample damage and compound sublimation. The DriPure, anti-bumping technology removes bumping and thus the potential sample loss and cross-contamination, ensuring the integrity and purity of the final product.
In addition, the HTS3i and the EZ-2 are both compatible with all common organic solvents, inclusive of corrosive acids like hydrochloric acid (HCl) and trifluoracetic acid, explosive solvents like diethyl ether, and high boiling point solvents like dimethyl sulfoxide (DMSO) and N-Methyl-2-pyrrolidone. This compatibility allows the synthesis of a wide variety of small molecule and peptide based PROTAC compounds using the same equipment.
Figure 2. Left image: HTS3i., Right image: EZ-2. Image Credit: SP Scientific
SP-Genevac has additional components for the HTS3i and the EZ-2 evaporators on offer, like the SampleGenie™. This equipment can allow large-volume samples to be dried or lyophilized directly into a small vial. Four steps are typically required for this process. Firstly, drying the large volume, secondly, redissolving the sample in a small volume of strong solvent (for example, DMSO), next, pipetting the sample into the end vial, and finally, drying the sample in the end vial.
The SampleGenie™ cuts down this multi-step process into a single step. This further reduces bottlenecks in PROTAC synthesis and is predominantly beneficial when combining HPLC fractions after sample purification.
Another supplementary component that can be used with HT series evaporators is EXALT. This option facilitates the ability for many solvents to be evaporated simultaneously and at slow rates for polymorph screening to produce crystals. This feature is extremely advantageous because it identifies the stable and meta stable forms of a small molecule at an early stage with just a few milligrams and the process can be automated so there is potential for unattended operation.
It is compatible with a large range of solvents that have a boiling range from 40 °C to 165 °C and if required, the evaporation time can be programmed to take 6 to 72 hours, or more. The EXALT operates with baffles to control the solvent evaporation rate and modular toolkit lets researchers create their own baffle configurations to attain their chosen evaporation profiles.
PROTAC Therapeutics in the Future
Over the last few decades, the development of PROTAC technology has grown considerably, with several types of heterobifunctional molecules created to target a large range of POIs in cancer, neurodegenerative diseases and beyond.
Arvinas are at the forefront in this field and two of their PROTACS; ARV-110 for prostate cancer and ARV-471 for breast cancer, are now undergoing phase I clinical trials. It is expected that these trials will be completed by mid-2020 and if they successfully reach the clinic, there is great potential that they will be the very first PROTAC drugs on the market.
Yet, there are problems that remain to be unsolved and need to be addressed before the compounds reach the market, particularly, the evaporation steps during PROTAC synthesis. To enable these issues to be eliminated, centrifugal evaporators with the potential for running multiple samples, and technology to overcome sample loss and cross-contamination, such as the HTS3i and EZ-2, need to be provided by SP Genevac.
References and Further Reading
- Cooper G.M. (2000). The Cell: A Molecular Approach. 2nd Edition. Sinauer Associates. https://www.ncbi.nlm.nih.gov/books/NBK9957/
- Lai A.C. et al. (2017). Induced Protein Degradation: An Emerging Drug Discovery Paradigm. Nature Reviews Drug Discovery. doi:10.1038/nrd.2016.211.
- Pettersson M. et al. (2019). PROteolysis Targeting Chimeras (PROTACs) – Past, Present and Future. Drug Discovery Today: Technologies. doi.org/10.1016/j.ddtec.2019.01.002
- Ottis P. et al. (2017). Proteolysis-Targeting Chimeras: Induced Protein Degradation as a Therapeutic Strategy. ACS Chemical Biology. doi.org/10.1021/acschembio.6b01068
- Wang P. et al. (2018). Proteolysis Targeting Chimera (PROTAC): A Paradigm-Shifting Approach in Small Molecule Drug Discovery. Current Topics in Medicinal Chemistry. doi:10.2174/1568026618666181010101922
- Darrington R. (2002). Evaporation Issues in Compound Supply. Genevac Ltd.
- HT Series 3i. SP Scientific. (2019). https://www.spscientific.com/HT3/
- EZ-2 Series. SP Scientific. (2019). https://www.spscientific.com/Products/Centrifugal_Evaporators___Sample_Concentrators/Genevac/EZ-2_Series/EZ-2_Series/
- SampleGenie. SP Scientific. (2019). https://www.spscientific.com/ContentBlock.aspx?id=3505
- EXALT Controlled Crystallisation. SP Scientific. (2019). https://www.spscientific.com/ContentBlock.aspx?id=3762
- Ciulli et.al. (2019) in their research into iterative design and optimization of initially inactive PROTACs used the Genevac EZ-2 to dry fractions
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