R&D Progress of Ubiquitin-proteasome Pathway and Its Drug

Ubiquitin-proteasome is one of the important ways to "digest" protein in cells, and it can regulate many important life activities, such as cell proliferation, differentiation, apoptosis, DNA repair and so on. In recent years, people gradually realize that the abnormality in the ubiquitin-proteasome system is closely related to many different diseases, including neurodegenerative disease Alzheimer's disease, Huntington's disease, cancer, cardiovascular disease, respiratory disease and so on. In neurodegenerative disease, exogenous or endogenous factors can cause the misfolding of protein inside and outside nerve cells, affecting the degradation function of ubiquitin-proteasome and leading to abnormal accumulation of many proteins and even cell death. The occurrence of tumor is related to the excessive proliferation of cells and the enhancement of anti-apoptosis ability. Ubiquitin-proteasome affects the survival of tumor cells through promoting the degradation of cancer suppressor proteins such as P53 or blocking the degradation of oncogenic proteins. As a great protein machine, ubiquitin-proteasome has become an important target for scientists to develop potentially effective drugs for diseases in recent decades of research.

Mechanism of action of ubiquitin-proteasome pathway 

Ubiquitin-proteasome pathway needs the participation of ubiquitin (Ub), ubiquitin-activating enzyme (E1), ubiquitin polymerase (ubiquitin-carrier protein) (E2), ubiquitin-ligating enzyme (E3), 26S proteasomes, deubiquitinating enzymes (DUBs) and other related substances.

Ubiquitin-proteasome pathway is made of 2 processes: substrate protein ubiquitination and substrate degradation by proteasome. Ubiquitin is a highly conserved polypeptide chain consisting of 76 amino acids. It has two forms, one is free ubiquitin, and the other is ubiquitin covalently binding to the receptor protein. Ubiquitin is covalently bound to ε-amino group of lysine side chain or α-amino group of N-terminal of receptor protein through its C-terminal, which is called ubiquitination. Ubiquitination is a multi-enzyme cascade reaction mediated by ubiquitin-activating enzyme E1, ubiquitin polymerase E2, ubiquitin-ligating enzyme E3, etc. 

Ub: Ubiquitin; E1: Ubiquitin-activating enzyme; E2: Ubiquitin-carrier protein; E3: Ubiquitin-ligating enzyme; DUB: Deubiquitinating enzyme

Ubiquitin-proteasome and drug R&D

At present, many reported human diseases are related to the abnormality of proteasome pathway. The approved proteasome inhibitors have been used to treat multiple myeloma, mantle cell lymphoma and other cancers successfully, and some drugs under research, which target different components of ubiquitin-proteasome pathway, including preclinical trial drugs such as deubiquitinating enzyme inhibitor and clinical trial drugs such as ubiquitinating enzyme regulator and new proteasome inhibitor, have shown a more anticipated curative effect. 

1. Proteasome inhibitor 

At present, there are more than 10 kinds of drugs reported to have a good anti-tumor effect, and proteasome inhibitors are most commonly used in clinical treatment. Bortezomib (PS-341) is the first proteasome inhibitor drug approved by FDA which targets ubiquitin-proteasome pathway. Bortezomib, a kind of boracic acid dipeptide, can effectively inhibit chymotrypsin activity by specifically binding to the active site of threonine at β5 subunit of 20S proteasome. It can also inhibit Caspase activity by binding to the active site of threonine at β1 subunit. Bortezomib is a reversible inhibitor with a high selectivity and a drug approved for treating multiple myeloma. Exposure to Bortezomib can stabilize p21, p27, p53 proteins, promote apoptosis of Bid and Bax proteins, block the activation of NF-κB pathway in cells, and lead to the death of tumor cells.

As the second generation proteasome inhibitor, Carfizomib was approved by FDA for treating multiple myeloma patients who have been treated with Bortezomib in 2012. Different with Bortezomib, Carfizomib is an epoxy ketone compound, with an unique chemical structure and selectivity. Its epoxy ketone part interacts with hydroxyl and free α-amino of threonine to form morpholine compound in an irreversible way. The binding of Carfizomib to other β subunits of proteasome, such as β1 and β2, is weaker than that of Bortezomib, which reduces the activity inhibition of trypsin and caspase. Ixazomib is the first approved oral borate proteasome inhibitor. Data shows that Ixazomib can treat multiple myeloma, systemic light chain amyloidosis and other malignant tumors in the form of "orphan drug" or combined with other proteasome inhibitors. What's more, the special subunit inhibitors of proteasome (β1i, β2i, β5i) can inhibit degradation and block cell cycle to inhibit tumor recurrence effectively by binding with subunits and occupying the binding site of protein degradation in a reversible or irreversible way. 

2. Deubiquitinating enzyme inhibitor

Deubiquitinating enzyme inhibitor acts on cysteine protease and metalloproteinase. The developed 19S proteasome-related deubiquitinating enzymes mainly include UCHL5 (or UCH37), USP14, USP7 and PSMD14 (POH1). P5091 is a thiophene small molecule inhibitor, which mainly acts on USP7 with P22077. The study found that it can induce the tumor cell mortality to use P5091 for treating patients with multiple myeloma who are Bortezomib-resistant. WP-1130 and b-AP15 can also act on multiple deubiquitinating enzymes. The accumulation of polyubiquitination modified proteins blocks the cell cycle, effectively inhibiting the development of tumors such as colon cancer. WP-1130 and Bortezomib can treat for tumor synergetically, while b-AP15 is more used to treat Bortezomib-resistant tumors. As a member of metalloproteinase family, deubiquitinating enzyme PSMD14 can negatively regulate the proliferation of multiple myeloma cells and the survival rate of patients at the intracellular level. Thus Cleave Biosciences has designed and synthesized a specific inhibitor for PSMD14, which has entered the early stage of clinical trials. 

3. Ubiquitinating enzyme regulator 

E1 is the first enzyme in ubiquitination system. Two kinds of E1 (Uba1 and Uba6) regulate the downstream ubiquitination reaction. Thus, the regulation of E1 activity can regulate the ubiquitination of some downstream proteins relating to tumor. At present, many E1 inhibitors have been reported. For example, inhibitors of Uba1, PYR-41 and PYZD-4409. However, only the E1 activating enzyme inhibitor MLN4924 of ubiquitin-like molecule NEDD8 has entered clinical trials. The inhibition of E1 will accumulate the ubiquitination substrate, and the poor specificity and drug-forming ability of E1 block the development of E1 inhibitors. E2 inhibitor has a good selectivity, providing a more space for tumor treatment. For example, inhibitor CC0651 blocks the cascade reaction of Ub by the conformational changes caused by binding to CDC34. There are about 700 E3, which is a therapeutic target for many related diseases by specifically recognizing protein substrate. The target protein can be regulated to improve the curative effect in a targeted way by regulating the E3 of substrate protein. For example, MDM2, as an E3 of tumor cancer suppressor gene p53, can block tumor cell cycle and promote apoptosis by blocking the interaction between MDM2 and p53 and stabilizing p53. Nutlin-3 is the first E3 inhibitor targeting MDM2. Nutlin-3 can simulate and occupy the binding site of p53 and MDM2 to avoid p53 degradation by ubiquitin labeling. RITA, as another small molecule drug for stabilizing p53, can inhibit the interaction between p53 and MDM2 by binding to the N-terminal of p53. Drugs approved for the treatment of multiple myeloma (MM), mantle cell lymphoma (MCL) and myelodysplastic syndromes

References

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

[2] Liu Yang. Ubiquitin-Proteasome Related Drug Applications [J]. University Chemistry, 2019, 34 (07): 60-66.

About the Author

Xiao Nisha, a food science and technology worker working in a large R&D company of drugs in China, engages in the R&D of nutritious food and functional food.