The ubiquitin proteasome system (UPS) is the body’s built-in garbage recycling system. Its function is to degrade unwanted, used, or misfolded proteins back into amino acids, and scientists have recently found a way to use this to fight against cancer.
The newly popularized small molecule proteolysis-targeting chimeras (PROTACs) acts on the UPS by tagging cancerous proteins with identification molecules that can be recognized by the body’s recycling plant, the proteasome.
According to Roots Analysis, the protein degradation technologies market is anticipated to be worth over USD 3.3 billion by 2030, driven by strong growth in drug discovery interest and a healthy pipeline of novel therapeutics in pre-clinical phases.
In the Asia Pacific, PROTACs research have received widespread adoption since 2017, with organizations such as Merck Singapore, University of Tokyo, Yonsei University, and the Development Center for Biotechnology Taiwan contributing efforts into conducting characterization research and identifying lead compounds.
Figure 1. Results per 100,000 citations in PubMed for “PROTACs” have spiked since 2017. Data retrieved from PubMed by Year
The PROTAC is a bifunctional small molecule consisting of an E3 ligand and a ligand designed for a specific protein of interest (POI), called the warhead. The two ligands are joined together by a chemical linker, which aims to bring an E3 ubiquitin ligase within proximity to the POI, forming a ternary complex of E3 ubiquitin ligase – PROTAC – POI. This results in the recruitment of ubiquitin onto the protein of interest in a process called polyubiquitination. This process will signal the proteasome to degrade the POI, leading to targeted protein knockdown.
Figure 2. Mechanism of action for PROTACs. A PROTAC molecule links an E3 ubiquitin ligase with a target protein of interest (POI), resulting in polyubiquitination and protein degradation. Retrieved from: Generating a chemical toolbox to support PROTAC
Drugs designed using the PROTAC mechanism have shown a high level of selectivity, potency, and oral bioavailability. They are differentiated pharmacology unlike traditional enzyme inhibitor drugs.
The benefits of PROTACs over traditional small molecule drugs include:
The characterization of protein-protein interactions and protein-ligand interactions are particularly important for PROTACs due to the ternary complexes that are formed. Bifunctional molecules can experience a “hook effect,” which is when the binary interaction (for example, E3 ubiquitin ligase - PROTAC), outcompetes the desired ternary complex. This reduces the range of concentration of ligands present where the activity of PROTACs is desirable.
The cooperativity is a value measured by the binding affinity KD of the binary complex divided by the KD of the ternary complex. It is shown that positive cooperativity increases the stability of the ternary complex, and negative cooperativity can reduce activity through competition.
Figure 3. ITC-guided improvements of PROTAC drug design by fine-tuning cooperativity and Gibbs free energy values. Assays and technologies for developing proteolysis targeting chimera degraders
The isothermal calorimetry (ITC) technique is excellent in evaluating the cooperativity of PROTACs. Gadd, M. et al. demonstrates a method using the ITC to quantify the KD of ternary complexes and binary complexes separately through a reverse titration reference to give a more accurate measurement.
The method is also useful for characterizing the thermodynamics of the system. A negative Gibbs free energy, coupled with positive cooperativity, results in the most stable PROTAC ternary systems.
PROTACs work by bringing together an E3 ligase and a warhead to proximity. The ability to function is therefore related to the lifetime of the ternary complex. Surface Plasmon Resonance (SPR) is the only label-free technique to measure the dissociation lifetime and the choice for many researchers working on the characterization of PROTACs.
By immobilizing the E3 ligase onto an SPR sensor chip and running experiments to determine the dissociation half-life of the system, Roy MJ et al. suggests that there is a mechanistic link between the relative half-life of a given ternary complex and the initial rates of target degradation. Additionally, just a single amino acid difference can make large changes to the dissociation.
It is useful to measure using the SPR and ITC techniques for the following:
SPR and ITC are two complementary protein-ligand binding assay techniques that address challenging new drug designs. SPR is the gold standard label-free kinetics tool and ITC is the gold standard label-free affinity tool. Read more about these tools below:
Other biophysical characterization techniques available at DKSH include:
James Hsu joined DKSH in 2019 and is part of the Business Development, Business Unit Technology team in Taiwan. In this role, he is responsible for growing the life sciences and scientific instrumentations business. His previous experience was accumulated in the bustling Asian genomics and proteomics sector, where he worked on bringing a digital PCR startup to market. James graduated from the University of California, San Diego.