Unlocking New Avenues in Cancer Therapy by Targeting Ubiquitin-Specific Proteases
Dr. Oon Chern Ein, DPhil (University of Oxford, UK), Associate Professor in Molecular Oncology Institute for Research in Molecular Medicine Universiti Sains Malaysia.
Ubiquitin-specific proteases (USPs) are crucial in cancer biology, influencing protein stability, cell signaling, and DNA repair. While targeting USPs holds promise for cancer therapy, developing effective inhibitors remains a challenge. Induced proximity strategies show potential but are hindered by issues of selectivity, efficacy, and resistance. This article discusses the advancements and obstacles in targeting USPs for cancer treatment and explores innovative approaches for achieving more effective and less toxic therapies.
Introduction
The ubiquitin-proteasome system (UPS) is a crucial mechanism that helps maintain balance within our cells by breaking down damaged or excess proteins. Central to this process is ubiquitination, where a small protein called ubiquitin (made up of 76 amino acids) attaches to target proteins, thus influencing the protein's stability, location within the cell, and its ability to interact with other molecules. In essence, it acts as a signal for certain proteins to either form complexes with others or kickstart specific cellular processes. On the other hand, deubiquitinating enzymes (DUBs) reverse this process. Among them, ubiquitin-specific proteases (USPs) are the largest group and play key roles in the regulation of cellular processes, including the stabilisation and degradation of key proteins. Their dysregulation has been increasingly linked to various types of cancers. USPs regulate key pathways that drive tumor growth and resistance to treatment, such as Wnt/β-Catenin, EGFR, and ERK/MAPK signaling. These enzymes also influence the tumor microenvironment (TME) by affecting processes like immune suppression, cancer spread, and blood vessel formation. Additionally, USPs are involved in DNA repair, which is crucial for maintaining genome stability and allowing tumors to survive treatments. While chemotherapy remains the cornerstone of cancer therapy, the development of resistance and severe side effects limits its effectiveness. Targeting USPs offers a promising avenue for overcoming these barriers, as their ability to modulate key cancer-related signaling pathways presents potential for more targeted and less toxic therapeutic strategies in the fight against cancer.
In the past decade, scientists have developed small-molecule inhibitors (SMIs) that block USP activity, aiming to trigger the death of cancer cells and slow down tumor growth. Despite this progress, no USP inhibitor has been approved for clinical use so far. The drugs currently being developed have limited efficacy or unwanted side effects, underlining the complexity of targeting this family of proteins. This article provides insights into the hurdles in developing drugs to target USPs and emerging modalities to address these challenges.
Small molecules: from inhibitors to modulators of protein-protein interaction
In traditional drug development aimed at targeting ubiquitin-specific proteases (USPs), the primary focus has been on small molecules (SMs) that bind to either the active or allosteric sites of these enzymes. By inhibiting USPs' ability to remove ubiquitin from substrates, these compounds promote the degradation of proteins that drive cancer progression. However, recent developments have shifted towards modulating protein-protein interactions (PPIs), which aim to disrupt the interactions between USPs and their substrates or other cellular partners.
For example, UAT-B, a novel compound derived from Streptomyces conglobatus, can interfere with the interaction between USP25 and Tankyrase (TNKS), effectively inhibiting Wnt signaling in colorectal cancer. Similarly, the synthetic peptide PSH-4 disrupts the USP25-shieldin complex, impairing DNA repair processes like non-homologous end joining (NHEJ) and promoting cancer cell death [2]. Despite these advancements, developing effective PPI modulators remains a challenge, as large, flat binding surfaces complicate the achievement of selectivity and potency. Future strategies could involve structure-based drug design, stabilised peptides, and fragment-based approaches, while exploring allosteric sites or using artificial intelligence-driven design may help address these issues.
Although non-selective inhibitors have demonstrated promising anti-cancer activity, their lack of specificity raises concerns regarding toxicity in cancer therapy. This emphasizes the need for more precise inhibitors or novel strategies that balance selectivity with efficacy. However, selectivity alone has not ensured clinical success. For example, inhibitors like ML323 and FT671, which selectively target USP1 and USP7, have shown strong preclinical results but face challenges in advancing to clinical trials. ML323, the first selective USP1/UAF1 inhibitor and FT671, a selective USP7 inhibitor; both have potent IC50 values of under 100 nM [3] [4]. Despite their promising selectivity in biochemical assays, both inhibitors encounter off-target interactions and toxicity in more complex in vivo settings.
These challenges highlight the importance of continued research into the design of selective SMIs that can target USPs effectively while minimising off-target effects and undesirable pharmacokinetics. A deeper understanding of the conserved catalytic domains of USPs and the key residues involved in substrate recognition and catalysis will be critical in developing more refined and effective inhibitors.
Current landscape of USP-targeted drugs in the pharmaceutical industry
The development of therapies targeting USPs has mainly focused on USP1 and USP7 because of their importance in cancer biology and therapeutic potential. Drugs targeting USP1 aim to leverage the concept of synthetic lethality, especially in cancers with DNA repair defects or BRCA1/2 mutations. In certain cancer types, blocking the Poly (ADP-ribose) polymerase (PARP) DNA repair protein can kill cancer cells, but these cells can develop resistance. Combining USP1 inhibitors with PARP inhibitors have shown promising results in ovarian and breast cancers [5], This is further supported by the clinical development of KSQ-4279 (Roche) [5], XL-309 (Exelixis) [6] and TNG348 (Tango Therapeutics) [7], which are currently in Phase I/II clinical trials as monotherapies or in combination with PARPi (olaparib) for patients with solid tumours. However, TNG348, had to be discontinued due to severe liver damage in patients, highlighting the need for more research into the safety of these treatments.
USP7 is another important target, but developing drugs for it has been more challenging. Several different approaches have been tried, including indirect methods, covalent binding, and non-covalent binding, but many of these drugs have struggled with side effects, lack of target specificity, and limited effectiveness in the body. Despite these challenges, USP7 inhibitors are still being tested, particularly in combination with other therapies. Other USPs have also been explored, but many of these efforts have been terminated due to issues with targeting, safety, and clinical application. Overall, while there is progress, turning early-stage discoveries into effective cancer treatments remains a tough challenge.
Modalities of induced proximity as emerging strategies
New strategies in cancer treatment are focusing on "induced proximity" techniques, which aim to specifically target and degrade cancer-causing proteins. PROTACs (Proteolysis Targeting Chimeras) are bifunctional small molecules designed to target specific proteins for degradation. Unlike traditional small molecules that simply inhibit a protein's function, PROTACs work by recruiting the body's natural protein degradation machinery to break down the target protein. A PROTAC consists of two functional parts: one that binds to the target protein and another that binds to an E3 ubiquitin ligase, an enzyme responsible for tagging proteins for destruction. By bringing these two components together, the PROTAC forms a ternary complex that signals the targeted protein for degradation through the ubiquitin-proteasome pathway. This mechanism offers a more effective way to eliminate "undruggable" proteins that have previously been challenging for traditional SMIs. However, a challenge remains in overcoming resistance to these treatments, and PROTACs can unintentionally degrade other important proteins, causing unwanted side effects [8]. Researchers are now looking at ways to improve these treatments by making them more specific and reducing off-target effects.
Another approach, known as molecular glue degraders (MGDs), is being explored for its potential to degrade proteins that were previously challenging to target. Imagine a sticky substance that can bring two things together – in this case, the "glue" helps connect a specific protein to the cell's waste disposal system. This system normally gets rid of damaged or unnecessary proteins. By sticking the protein to this system, the drug ensures the protein is broken down and removed from the cell. In the context of cancer treatment, molecular glue degraders can target and degrade proteins that help cancer cells survive, essentially "turning off" those survival signals and helping to stop cancer growth. MGDs present multiple benefits compared to PROTACs, such as a lower molecular weight, better oral bioavailability. Additionally, MGDs like lenalidomide have already shown clinical efficacy and therapeutic advantages in cancer treatment [9]. However, MGDs can face problems because they may unintentionally stabilize proteins that should be degraded, leading to resistance. Researchers are trying to better understand these mechanisms and improve the design of MGDs to make them more effective and specific.
A new development is the use of molecular glue stabilizers, which work by increasing the stability of tumor-suppressing proteins. One example is bromocriptine (BC), which has been shown to stabilize the p53 protein, a key tumor suppressor [10]. While the drug shows promise in early studies, further research is needed to make sure it only targets the desired proteins and does not cause unwanted side effects.
Another strategy involves Deubiquitinase Targeting Chimeras (DUBTACs), which aim to prevent the degradation of important proteins by targeting deubiquitinases (DUBs), enzymes that help regulate protein levels. While DUBTACs have potential, their development is slower due to challenges with their design and the need to improve their selectivity [11]. The clinical translation of DUBTACs may face obstacles due to pharmacokinetic and structural issues. If these challenges can be overcome, DUBTACs may become an important tool in cancer therapy. Overall, these innovative approaches offer new ways to treat cancer, but researchers still face challenges in making them safe, effective, and specific to the right targets.
Concluding remarks:
USPs play a critical role in various aspects of cancer biology, including protein stability, cell signaling, and DNA repair. This broad involvement makes them an attractive target for cancer therapies, offering the potential for a more comprehensive treatment approach compared to single-target therapies. Despite promising preclinical progress, translating USP-targeted strategies into effective clinical treatments remains challenging. One significant obstacle is the multifunctionality of USPs. In addition to their catalytic deubiquitinating activity, many USPs also act as scaffolds, influencing key cellular processes independently of their enzymatic function. This complexity complicates drug design, as traditional SMIs typically target the enzymatic activity, while targeted protein degraders (TPDs) like PROTACs and MGDs aim to eliminate the entire protein, affecting both catalytic and non-catalytic roles. Furthermore, USPs regulate extensive networks of substrates, meaning that disrupting a single USP can trigger widespread effects across multiple signaling pathways. Additionally, substrates are often regulated redundantly by other USPs, deubiquitinases, or E3 ligases, creating compensatory mechanisms that may drive therapeutic resistance. Combining the targeting of multiple USPs or pairing USP inhibition with complementary pathways may offer a solution to bypass resistance and improve treatment effectiveness.
The development of selective USP inhibitors remains a significant challenge due to the highly conserved catalytic sites across USP family members. The reactive cysteine residue in these sites increases the risk of off-target interactions with other cysteine-dependent enzymes, such as DUBs and cysteine proteases. To address this, researchers are now focusing on targeting allosteric sites and non-catalytic domains, which exhibit more structural variability among USPs and offer the potential for enhanced selectivity. Emerging strategies like PROTACs, molecular glues, and DUBTACs are reshaping the therapeutic landscape by enabling the selective degradation or stabilisation of USPs. However, these approaches introduce new challenges around linker design, pharmacokinetics, and stable ternary complex formation. Future research will be essential to refine ligand design, improve drug selectivity, and anticipate resistance mechanisms to fully harness the therapeutic potential of these novel strategies. Despite these hurdles, these advancements offer promising possibilities to transform cancer treatment and provide new hope for patients, marking a significant advancement in precision oncology.
Acknowledgment:
This article has been adapted from the original scientific publication authored by Dr. Oon Chern Ein and colleagues (Ref 1). It has been simplified for a general audience while maintaining the core findings and insights of the original work.
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Associate Professor Dr. Oon Chern Ein is dedicated to advancing innovative cancer therapies. She earned her BSc (First Class Hons) in Biotechnology from Universiti Kebangsaan Malaysia and pursued her DPhil in Medical Oncology at the University of Oxford, United Kingdom. Following this, she trained as a postdoctoral fellow at Karolinska Institutet, Sweden. She has served as a lecturer at Universiti Sains Malaysia since 2013. Chern’s work in molecular therapeutics has earned her numerous prestigious awards, including the L’Oreal-UNESCO For Women in Science National Fellowship (2015), the Women of the Future Awards (South East Asia), and the Malaysian National Young Scientist Award (2018). She was further recognized with the Promising Academician Award and Excellent Service Award by the Malaysian Ministry of Higher Education (2019), alongside the British Council Study UK Alumni Award (Professional Achievement Category) and MINDS Women Scientist Award in 2020. Most recently, she received the Singapore Academies-ASEAN Fellowship from the Singapore National Research Foundation to train in drug discovery at A*STAR Singapore to strengthen her industrial insights. Her multidisciplinary research encompasses drug discovery, cancer-targeted therapies, and bioengineering solutions through collaborations with industry partners. She holds two granted patents in Malaysia (MY-203445-A, MY-199524-A) for small molecule inhibitors targeting anti-cancer and anti-angiogenesis pathways. As a member of the Global Young Academy, Chern actively champions female empowerment and education for all. Chern’s research continues to bridge academia and industry, striving to transform cancer therapy through novel therapeutic strategies.
