A PROTAC bridging two protein targets, showing how these bifunctional molecules recruit E3 ligases to facilitate selective protein ubiquitination and degradation. [WuXi AppTec]
This article is written to shed light on the intricacies of preclinical development for PROTACs. Our goal is to provide insights into the key obstacles that researchers and developers must overcome and the strategies and solutions available to navigate these hurdles successfully. As industry professionals working closely with emerging therapies, we recognize the importance of bridging the gap between discovery and clinical application. A solid understanding of the preclinical complexities of PROTACs guides strategic decision-making and lays the groundwork for their success in human trials.
Additionally, the evolving landscape of drug discovery emphasizes the need for collaboration with specialized testing partners who possess the expertise to address the unique challenges of PROTAC development. By leveraging external knowledge and resources, developers can accelerate drug development, streamline regulatory approval, and ultimately bring innovative treatments to patients more efficiently. In this article, we focus not only on the challenges but also on how partnering with experienced preclinical testing providers can mitigate risks and enhance the success of PROTAC development.
Understanding PROTACs: What sets them apart?
PROTACs are bifunctional molecules designed to induce targeted protein degradation. Each PROTAC consists of two distinct components: one that binds to the target protein and another that recruits an E3 ubiquitin ligase (Figure 1). By bringing these two molecules together, PROTACs promote the ubiquitination of the target protein, which marks it for degradation by the proteasome. This mechanism is distinct from traditional small-molecule inhibitors, which merely block protein function. Instead, PROTACs eliminate the target protein entirely, offering a more permanent therapeutic effect.
Figure 1: Schematic of typical PROTAC structure and an example (ARV-110).
This novel mode of action holds tremendous promise, particularly for targeting proteins that have been historically difficult to drug. Many diseases, including cancer, neurodegenerative disorders, and autoimmune conditions, involve proteins that play a key role in disease progression but are not easily inhibited by conventional therapies. PROTACs offer a potential solution by degrading these pathogenic proteins altogether rather than just inhibiting their function.
However, while the mechanism of action of PROTACs is revolutionary, it also introduces a range of new challenges in preclinical development. Their bifunctional structure, complexity, and reliance on intracellular machinery create unique hurdles that must be addressed before advancing to human trials.
Key challenges in preclinical development
The preclinical development of PROTACs is not without its hurdles. From understanding their pharmacokinetic properties to predicting off-target effects, PROTACs present a series of obstacles that differ from those of traditional small molecules. Below, we outline some of the most significant challenges.
- Pharmacokinetics and biodistribution
Given their large and complex structure, PROTACs often face challenges with PK and biodistribution. Many PROTACs suffer from poor bioavailability, limited cellular uptake, and rapid clearance from the body, which can hinder their therapeutic efficacy. Their bifunctional design also complicates absorption, distribution, metabolism, and excretion (ADME) profiles. For instance, the E3 ligase-recruiting ligand or the linker between the two ligands can significantly affect a PROTAC’s stability and distribution in tissues. Optimizing these properties requires an iterative process of molecular design, formulation, and PK modeling. - Cell permeability and target engagement
For PROTACs to work effectively, they must enter cells, bind to the target protein, and recruit the E3 ubiquitin ligase. However, cell permeability can be a significant barrier, particularly in tissues with limited penetration, such as the brain. Some PROTACs may struggle to cross the plasma membrane due to their size or polarity. Furthermore, once inside the cell, the extent and kinetics of target engagement can vary depending on tissue-specific factors, making it challenging to predict therapeutic outcomes. Early preclinical studies must, therefore, focus on optimizing cell permeability and ensuring robust target engagement. - Off-target effects and toxicity
While PROTACs’ ability to degrade specific proteins is a major advantage, unintended degradation of off-target proteins can lead to significant toxicity. Off-target degradation could result from non-specific binding to other proteins or E3 ligases, leading to unintended ubiquitination. Preclinical toxicology studies are critical in identifying these off-target effects and ensuring that PROTACs are specific and safe. Additionally, the potential for immunogenicity, especially in degrading proteins involved in immune regulation, must be carefully evaluated. - Species differences in E3 ligase expression
The efficacy of PROTACs hinges on the presence of appropriate E3 ligases in target tissues. However, E3 ligase expression can vary widely between species, complicating the use of animal models for preclinical evaluation. For example, ligases that are highly expressed in rodents may not be as prevalent in human tissues, and vice versa. This complicates the translation of preclinical findings to human applications, particularly when predicting efficacy and safety. Careful consideration of species-specific differences in E3 ligase expression is crucial when designing preclinical studies. - Drug-drug interactions (DDIs)
The degradation of target proteins by PROTACs can lead to complex drug-drug interactions, especially when co-administered with other therapies that rely on similar cellular pathways. For example, the degradation of proteins involved in drug metabolism could affect the pharmacokinetics of other drugs, leading to altered efficacy or toxicity. Early identification of potential DDIs during preclinical testing is vital to avoid complications in later stages of development.
How to overcome the challenges
Overcoming challenges in the preclinical development of PROTACs requires a combination of scientific rigor, advanced technological tools, and strategic partnerships. Below are key strategies to address the complexities associated with PROTACs.
- Comprehensive PK/PD modeling
Given PROTACs’ unique PK/PD profiles, advanced modeling techniques are essential. In vitro and in vivo models should be leveraged to predict how PROTACs behave in different biological systems. By integrating these models with bioanalytical data, researchers can gain valuable insights into how PROTACs are absorbed, distributed, and eliminated, allowing for better optimization of drug properties. Additionally, PK/PD modeling can inform dose selection and scheduling to maximize target degradation while minimizing off-target effects. - Target engagement assays
Robust assays that assess target engagement and protein degradation in relevant tissues are critical for determining the efficacy of PROTACs. These assays can help identify the concentration of PROTACs needed to achieve therapeutic effects while minimizing off-target activity. Techniques such as Western blotting, mass spectrometry, and proteomics are valuable tools for measuring protein levels in cells and tissues, providing direct evidence of target degradation. - Species-appropriate models
To address the challenge of species differences in E3 ligase expression, preclinical studies should employ models that are as close to human biology as possible. Humanized mouse models, in which human E3 ligases are expressed, can provide more accurate predictions of how PROTACs will perform in human tissues. In vitro models using human cell lines can also provide valuable data on the efficacy and specificity of PROTAC-induced degradation. By selecting species-appropriate models, researchers can improve the translatability of preclinical findings to human clinical trials. - Early DDI screening
To mitigate the risk of drug-drug interactions (DDIs), early screening for potential interactions is essential. In vitro assays that assess the effects of PROTACs on key drug-metabolizing enzymes, such as cytochrome P450, can help identify possible interactions with other drugs. Early identification of DDIs ensures these risks are managed before clinical trials, reducing the likelihood of adverse reactions. - Partnering with an experienced testing partner
One of the most effective strategies for navigating the challenges of PROTAC development is to partner with a qualified and experienced preclinical testing laboratory. A specialized service provider can offer technical expertise, state-of-the-art technologies, and a deep understanding of the regulatory landscape, which are crucial for overcoming the unique obstacles posed by PROTACs.
Experienced testing partners can also help design and execute preclinical studies that address the specific challenges of PROTAC development. From optimizing PK/PD studies to conducting bioanalytical testing, these partners provide valuable insights that streamline the development process and reduce the risk of failure in clinical trials. Additionally, a knowledgeable testing partner can assist with regulatory submissions, ensuring that studies comply with global standards.
A final word
PROTACs represent a groundbreaking approach to drug development, offering the potential to degrade disease-causing proteins that have long been considered undruggable. However, the preclinical development of PROTACs is complex and requires careful attention to pharmacokinetics, cell permeability, target engagement, off-target effects, species differences, and drug-drug interactions. Addressing these challenges early in development is critical to ensuring the success of PROTACs in clinical trials.
By employing advanced modeling techniques, robust target engagement assays, species-appropriate models, and early DDI screening, developers can overcome the hurdles associated with PROTAC development. Moreover, partnering with an experienced preclinical testing organization provides the expertise and resources to navigate these complexities, streamline development, and mitigate risks. With the right strategies and collaborations, PROTACs’ potential to transform the treatment landscape is within reach, bringing innovative therapies to patients who need them most.
Author bios
Dr. Liping Ma holds a Ph.D. in Pharmaceutical Analysis and is an expert in pharmacokinetic evaluation. As a Senior Study Director at WuXi AppTec, she has over 10 years of experience in preclinical and clinical pharmacokinetics, successfully supporting more than 20 global IND applications for new drugs.
Xiaoying Jin is the Senior Director of the Suzhou Bioanalysis Department at WuXi AppTec, where she leads the establishment and management of bioanalysis laboratories (LC-MS/MS and immunology) to support non-clinical safety evaluations.
Dr. Xiaoxia Li serves as Executive Technical Director at WuXi AppTec, bringing over 17 years of expertise in preclinical drug development and providing clients with fast, efficient scientific and technical support.
Filed Under: Biologics, clinical trials, Drug Discovery, Regulatory affairs
