In a significant advancement, cancer researchers have found a long-sought way to overcome a critical design barrier in the generation of small-molecule chemical tools and drugs that work by targeted protein degradation.They demonstrated the usefulness of their approach by improving the targeted depletion of a protein, Aurora A, that is involved in cancer cell proliferation and associated with poor clinical outcomes.
By taking a novel approach to the design of proteolysis targeting chimeras (PROTACs) – molecules that selectively degrade disease‑driving proteins – the scientists successfully engineered a second‑generation degrader that more effectively eliminates Aurora A. They achieved this primarily by overcoming the problematic “hook effect”, a counterproductive phenomenon that often limits PROTAC effectiveness.
The team’s findings are not only valuable in creating a best-in-class research tool for studying the effects of degrading Aurora A in cells in the lab, but they may also ultimately influence the broader development of PROTAC tools and drugs designed to eliminate key target proteins in cancer cells – which could lead to significantly better outcomes for people living with cancer.
Scientists at The Institute of Cancer Research, London, led the study, which was supported primarily by Cancer Research UK funding awarded to the CRUK Children’s Brain Tumour Centre of Excellence (CBTCE) and involved an international collaboration with researchers at the University of Kiel, Germany. The findings were published in the Journal of Medicinal Chemistry.
Targeting Aurora A: a long‑standing challenge
Aurora A is a kinase enzyme that is essential for several aspects of cell division, including mitotic spindle formation, chromosome alignment and centrosome maturation. Its activity is tightly controlled in healthy cells but frequently dysregulated in a wide range of cancers.
Although researchers have long recognised Aurora A as a promising therapeutic target, its multifunctional role has hindered the attempts to inhibit it. Aurora A is not only a kinase but also a scaffold protein responsible for assembling and regulating multiple protein partners, including oncoproteins, which are involved in a range of cellular processes. Traditional kinase inhibitors have been unable to disrupt all of these activities. As a result, they can leave cancer cells with enough functional support to continue proliferating.
PROTACs offer a tantalising solution, as they work by marking an entire protein for degradation rather than merely switching off a single function. Thus, in the case of Aurora A, targeted degradation eliminates both the kinase and the scaffolding activities from the cell. But despite their promise, PROTACs commonly exhibit a paradoxical flaw in the form of the hook effect, whereby higher PROTAC concentrations display reduced effectiveness, thereby limiting the extent of the targeted degradation.
This counterintuitive drop‑off stems from imbalanced interactions between PROTAC molecules, their target protein and the enzyme – an E3 ubiquitin ligase – responsible for tagging proteins for destruction. At higher concentrations, PROTACs bind separately to either the target protein or the E3 ligase, preventing the formation of the three‑component complex needed for degradation.
Removing the hook effect, increasing stability and selectivity
As Aurora A is important for cancer cell replication, many conventional kinase inhibitory drug candidates have been developed and entered the clinic, but none of them has been approved so far. More recently, several Aurora A PROTAC degraders have been published for experimental lab use, but the limitations of the hook effect, together with instability and lack of selectivity, have hindered their usefulness.
The researchers behind the current study wanted to find a way to develop a PROTAC tool that would not only eliminate Aurora A’s kinase activity but also destroy its scaffolding properties and interactions with protein partners. Their underlying aim was to overcome the limitations of previous Aurora A PROTACs and offer a more versatile and effective Aurora A degrader reagent for lab experiments.
The researchers approached this challenge through rational molecular redesign, using the investigational, conventional Aurora A kinase inhibitor alisertib as a basis from which to develop a second‑generation PROTAC known as CCT400028.
To overcome the hook effect, the researchers engineered the part of the PROTAC responsible for recruiting the E3 ligase to decrease its affinity for this enzyme – thereby reducing the production of an unproductive binary complex and enhancing the formation of the ternary complex that leads to degradation. This rationally-based optimisation resulted in the successful removal of the hook effect and enabled a greater depth of Aurora A degradation. Importantly, laboratory tests in paediatric cancer cells demonstrated that the optimised PROTAC maintains potent activity across a wider and higher concentration range without loss of performance.
In addition to successfully removing the hook effect, the researchers modified the PROTAC to achieve an increase in stability – and hence more prolonged activity in cell culture experiments. They were able to achieve a half‑life exceeding 120 hours – far surpassing the 25 hours of its predecessor – meaning that the new PROTAC tool can be used in cell culture experiments over a longer period while still sustaining very effective Aurora A depletion.
The researchers demonstrated the improved and sustained effectiveness of the new CCT400028 PROTAC tool in depleting Aurora A using paediatric cancer cell lines representing leukaemia, neuroblastoma and glioma.
Moreover, CT400028 was shown to exhibit a third important and advantageous feature – greatly improved selectivity towards Aurora A. Profiling of CCT400028 demonstrated high specificity in terms of degrading Aurora A but not other proteins in the cancer cells. It also did not show binding to other kinases – thus minimising the risk of unintended off‑target effects seen with previous PROTACs.
A chemical probe built for scientific exploration
In addition to reporting an optimised degrader, the researchers introduced a carefully matched inactive control analogue. Such a companion molecule is a standard requirement for chemical probes, allowing scientists to distinguish effects caused by targeted degradation from unrelated cellular responses.
Going further, the researchers demonstrated that CCT400028 meets all of the best-practice criteria for a high‑quality degrader probe and represents a best-in-class Aurora A PROTAC probe for use in cell culture. It is already recommended as a top-quality Aurora A PROTAC on the Chemical Probes Portal. This versatile tool could help laboratories around the world to understand the consequences of selectively depleting Aurora A in their own cell culture models. The control that PROTACs provide over the concentration- and time-dependent degradation makes them complementary to genetic approaches.
Implications for future discovery of chemical probes and drugs
Aurora A inhibitors have long been explored as potential anticancer agents, but they have failed due to toxicity, limited efficacy, minimal therapeutic window and potentially the inability to disrupt all of the protein’s functions, including scaffolding roles. The selective degradation of Aurora A could open new therapeutic avenues, including in paediatric cancers where the scaffolding roles of Aurora A have been implicated in the disease mechanism.
The broader significance of the work lies in the methodological insights provided into the future design of PROTAC tools and potential drugs.
The researchers hope that their achievement in overcoming the hook effect in Aurora A PROTACs – a widely observed PROTAC flaw – may be generalisable to PROTAC probes and potential drugs acting on other protein targets. The work marks a promising sign that degraders can be tuned, controlled and optimised with increasing precision.
A new chapter in targeted degradation
Co-senior author Dr Lindsay Evans, Staff Scientist in the Division of Cancer Therapeutics at The Institute of Cancer Research (ICR), said:
“This work is the culmination of great team science, and it represents a steep learning curve. It took an intense iterative design process to untangle the hook effect’s biochemical roots, but our findings demonstrate that even apparently counterintuitive design choices – like weakening a key protein interaction – can strengthen a molecule’s real‑world performance. The next phase of the work is to further optimise the structure of CCT400028 for use in mouse models of human cancer.
“Our work also potentially provides a more general blueprint for PROTAC design. Other PROTAC programs targeting different disease‑related proteins may be able to adopt similar strategies.”
Co-senior author Dr Gary Newton, Group Leader of the Medicinal Chemistry 3 Group at the ICR, said:
“By eliminating the hook effect and stabilising the PROTAC molecule, we’re finally able to study Aurora A biology in depth – without the noise that distorted earlier generations of degraders. As our research community continues to refine degrader technology, tools like CCT400028 illustrate how sophisticated molecular engineering can overcome fundamental obstacles.
“Ultimately, the insights gained from this work may help bring targeted degradation closer to clinical reality. I’m hopeful that we’ve opened the door to more reliable and effective degraders across the biomedical landscape, offering hope for patients with cancers where traditional inhibitors have fallen short.”
Professor Paul Workman, Co-Director of the CBTCE, Harrap Professor of Pharmacology and Therapeutics at ICR and an author on the paper, said:
“The research in this publication exemplifies one of the important roles of the CBTCE, which is to use our chemical biology and drug discovery expertise at the ICR to validate new treatment approaches and develop chemical tools and prototype drugs to aid therapeutic approaches in paediatric cancers, in partnership with other affiliated collaborators.
“Although CCT400028 is not a therapeutic candidate – it is intended strictly as a laboratory probe – it represents a meaningful advance in the science of targeted protein degradation. By providing a stable, selective and hook‑free degrader, the research offers new clarity for biologists studying Aurora A and new design opportunities for chemists engineering the next generation of PROTACs.”
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