The ubiquitin-proteasome system serves as one of the crucial pathways for the "digestion" of intracellular proteins, regulating numerous vital biological processes such as cell proliferation, differentiation, apoptosis, and DNA repair. In recent years, it has become increasingly recognized that abnormalities in the ubiquitin-proteasome system are intimately linked to the onset of various diseases, including neurodegenerative disorders like Alzheimer's disease and Huntington's disease, as well as cancer, cardiovascular, and respiratory diseases. In neurodegenerative diseases, external or internal factors can trigger misfolding of proteins both inside and outside nerve cells, thereby disrupting the degradation function of the ubiquitin-proteasome system and leading to the abnormal accumulation of a large number of proteins, and even cell death. On the other hand, the development of tumors is associated with excessive cell proliferation and enhanced anti-apoptotic capacity. The ubiquitin-proteasome system affects the survival of tumor cells by promoting the degradation of tumor suppressor proteins like P53 or blocking the degradation of oncogenic proteins. Over the past few decades, as a vast protein machinery, the ubiquitin-proteasome system has emerged as a significant target for scientists in developing potentially effective drugs against various diseases.

Mechanism of the Ubiquitin-Proteasome Pathway

The ubiquitin-proteasome pathway necessitates the participation of several key components, including ubiquitin (Ub), ubiquitin-activating enzyme E1 (ubiquitin-activating enzyme, E1), ubiquitin-conjugating enzymes (also known as ubiquitin-carrier proteins, E2s), ubiquitin ligases (ubiquitin-ligating enzymes, E3s), 26S proteasomes, and deubiquitinating enzymes (DUBs).

The ubiquitin-proteasome pathway comprises two main processes: ubiquitination of the substrate protein and degradation of the substrate through the proteasome. Ubiquitin is a highly conserved polypeptide chain consisting of 76 amino acids. Ubiquitin exists in two primary forms: one is in a free state, and the other is covalently bound to receptor proteins. The process of ubiquitin covalently binding to the ε-amino group of the lysine side chain of a receptor protein or the α-amino group of the N-terminus of a receptor protein through its C-terminus is referred to as ubiquitination. Ubiquitination is a multi-enzyme cascade reaction mediated by ubiquitin-activating enzyme E1, ubiquitin-conjugating enzymes E2, and ubiquitin ligases E3.

Ubiquitin-activating enzyme E1 is highly conserved during evolution and is an ATP-dependent enzyme that activates ubiquitin by forming a high-energy thioester bond with the C-terminus of ubiquitin. Unlike E1, multiple types of E2 exist within cells. E2 binds to activated ubiquitin to form an E2-ubiquitin complex, which assists specific E3 in transferring the activated ubiquitin to the substrate. Ubiquitin ligase E3 is the most abundant, structurally diverse, and complexly regulated member of the ubiquitination reaction system. It can directly interact with the substrate or through adaptor proteins to determine the selectivity of ubiquitin-mediated substrate protein degradation. There are three major classes of E3 in mammalian cells: those containing the HECT (homologous to E6-AP C-terminal) domain, those containing the RING finger domain, and those containing the U-box. The role of E3 in the ubiquitination process mainly encompasses three stages: mutual recognition and interaction between E3 and E2, mutual recognition and interaction between E3 and the substrate protein, and E3-catalyzed linkage of ubiquitin to the substrate protein as well as between ubiquitin molecules. Ultimately, ubiquitin covalently binds to the ε-amino group of the lysine side chain of the receptor protein through its C-terminus, and the lysine residues at positions 48 and 63 on these ubiquitin molecules can serve as modification sites. In this way, polyubiquitin chains can form on the substrate protein. Generally, substrate proteins modified with four or more ubiquitins linked through the 48th lysine residue can be recognized and degraded by the 26S proteasome, while ubiquitin chains linked through the 63rd lysine residue do not participate in protein degradation but are primarily associated with intracellular signal transduction processes and DNA repair.

Before the ubiquitinated substrate protein is degraded by the 26S proteasome, ubiquitin can be hydrolytically removed by deubiquitinating enzymes (DUBs), thereby preventing its degradation along with the substrate protein. The dissociated free form of ubiquitin can then be recycled for reuse. The proteasome is a massive, multi-subunit complex (approximately 2000 kD) with a quaternary structure and possesses a variety of proteolytic activities, and its protein degradation function is primarily dependent on the ubiquitination process. The 26S proteasome consists of a 20S core particle (CP) and a 19S regulatory particle (RP, also known as PA700). The 20S CP has a hollow barrel-like structure formed by four coaxial rings of seven subunits stacked on top of each other. The outer rings on both sides are composed of α1-α7 subunits, while the two inner rings are composed of β1-β7 subunits. Three constitutively expressed β-subunits (β1, β2, β5) possess threonine protease active sites (specifically, "caspase-like," "trypsin-like," and "chymotrypsin-like" active sites, respectively), which are enclosed inside the degradation chamber, effectively preventing normally folded proteins from entering the chamber. The 19S RP recognizes ubiquitinated substrates, unfolds them, releases free ubiquitin, opens the degradation channel on the α-rings, and feeds the unfolded substrates into the degradation chamber. Thus, the rapid and irreversible specific protein degradation mediated by the ubiquitin-proteasome system is a highly complex and tightly regulated biological process.

Ubiquitin-Proteasome System and Drug Development

Multiple human diseases have been reported to be associated with abnormalities in the proteasome pathway. Approved proteasome inhibitors have already been successfully used in the treatment of cancers such as multiple myeloma and mantle cell lymphoma. Furthermore, numerous drugs targeting different components of the ubiquitin-proteasome pathway, including deubiquitinating enzyme inhibitors (preclinical drugs), ubiquitin ligase modulators, and new proteasome inhibitors (clinical trial drugs), are currently under development and have shown promising therapeutic effects.

1. Proteasome Inhibitors

There are currently over ten drugs reported to have significant anti-tumor effects, with most of the clinically used drugs being proteasome inhibitors. Bortezomib (PS-341) is the first proteasome inhibitor drug targeting the ubiquitin-proteasome pathway to be approved by the FDA. Bortezomib is a boronic acid dipeptide that specifically binds to the threonine active site of the β5 subunit of the 20S proteasome, effectively inhibiting chymotrypsin-like activity. Additionally, it can also bind to the threonine active site of the β1 subunit, inhibiting caspase-like activity. Bortezomib is a highly selective reversible inhibitor and an approved drug for the treatment of multiple myeloma. Exposure to Bortezomib stabilizes proteins such as p21, p27, p53, and the apoptotic proteins Bid and Bax, blocking the activation of the NF-κB pathway within cells, ultimately leading to tumor cell death.

Carfizomib, as a second-generation proteasome inhibitor, was approved by the FDA in 2012 for the treatment of multiple myeloma patients who have already received Bortezomib therapy. Unlike Bortezomib, Carfizomib belongs to the epoxyketone class of compounds, featuring a unique chemical structure and selectivity. Its epoxyketone moiety interacts with the hydroxyl group and free α-amino group of threonine, forming a morpholine adduct in an irreversible manner. Carfizomib exhibits weaker binding affinity to other β-subunits of the proteasome, such as β1 and β2, compared to Bortezomib, thus reducing its inhibitory effect on trypsin-like and caspase-like activities.

Ixazomib is the first approved oral boronic acid ester proteasome inhibitor. Data indicates that Ixazomib can be used as an "orphan drug" or in combination with other proteasome inhibitors for the treatment of multiple myeloma, systemic light-chain amyloidosis, and other malignancies. Additionally, specific subunit inhibitors of the proteasome (β1i, β2i, β5i) can occupy the binding sites for protein degradation by reversibly or irreversibly binding to the subunits, inhibiting degradation and blocking the cell cycle, thereby effectively suppressing tumor recurrence.

2.Deubiquitinating Enzyme Inhibitors

Deubiquitinating enzyme inhibitors target cysteine proteases and metalloproteases. Currently, several deubiquitinating enzymes associated with the 19S proteasome have been identified, including UCHL5 (or UCH37), USP14, USP7, and PSMD14 (POH1). P5091, a thiophene-based small molecule inhibitor, primarily acts on USP7 along with P22077. Studies have found that administering P5091 to multiple myeloma patients who have developed resistance to Bortezomib can induce tumor cell death. WP-1130 and b-AP15 are both capable of inhibiting multiple deubiquitinating enzymes. The accumulation of polyubiquitinated proteins disrupts the cell cycle, effectively inhibiting the development of tumors such as colon cancer. WP-1130 exhibits synergistic tumor-suppressing effects when combined with Bortezomib, while b-AP15 is more commonly used to treat tumors that are resistant to Bortezomib. PSMD14, a member of the metalloprotease family, negatively regulates the proliferation of multiple myeloma cells and patient survival rates at the cellular level. Consequently, Cleave Biosciences has designed and synthesized specific inhibitors targeting PSMD14, which have entered the preclinical stage of clinical trials.

3.Ubiquitin Enzyme Modulators

E1 is the first enzyme in the ubiquitination system. Two types of E1 enzymes, Uba1 and Uba6, regulate downstream ubiquitination reactions. Therefore, modulating E1 activity can regulate the ubiquitination of certain tumor-related proteins downstream. Currently, various E1 inhibitors have been reported, such as PYR-41 and PYZD-4409, which target Uba1. However, only the E1 activating enzyme inhibitor of the ubiquitin-like molecule NEDD8, MLN4924, has entered clinical trials. Inhibition of E1 leads to the accumulation of ubiquitination substrates, but the poor specificity and druggability of E1 have hindered the development of E1 inhibitors.

E2 inhibitors, on the other hand, exhibit strong selectivity, offering greater potential for tumor therapy. For instance, inhibitor CC0651 blocks the Ub cascade reaction by binding to CDC34 and inducing a conformational change. There are approximately 700 E3 enzymes, which specifically recognize protein substrates, making them attractive therapeutic targets for various diseases. By modulating the E3 of substrate proteins, it is possible to regulate the target protein and enhance therapeutic efficacy in a targeted manner. For example, MDM2 is the E3 for the tumor suppressor gene p53. Blocking the interaction between MDM2 and p53 can stabilize p53, leading to the arrest of the tumor cell cycle and promotion of apoptosis. Nutlin-3 is the first E3 inhibitor targeting MDM2. It mimics and occupies the binding site between p53 and MDM2, preventing p53 from being ubiquitinated and degraded. RITA, another small molecule drug that stabilizes p53, inhibits the interaction between p53 and MDM2 by binding to the N-terminus of p53.

Drugs that have been approved for the treatment of multiple myeloma (MM), mantle cell lymphoma (MCL), and myelodysplastic syndromes (MDS) include those that target similar pathways or mechanisms, but specific examples directly related to these modulators may vary depending on their exact mechanisms of action and approved indications.

References

[1] Chen Yushan, Jiang Tianxia, Zhou Luming, Feng Rentian, Qiu Xiaobo. The Ubiquitin-Proteasome Pathway and Drug Development [J]. Chinese Journal of Biochemical Pharmaceutics, 2016, 36(12): 1-6.

[2] Liu Yang. The Ubiquitin-Proteasome System and Its Applications in Drug Development [J]. University Chemistry, 2019, 34(07): 60-66.

About the Author

Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.