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Medicilon Pharmacokinetic Services for First-in-Class Ubiquitination Inhibitor Development

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Dysfunction of the ubiquitin-proteasome system (UPS) is closely related to the development of many diseases, including tumors, cardiovascular diseases, and neurodegenerative diseases. Therefore, UPS has become one of the popular targets for the treatment of related diseases. Inhibiting the ubiquitin-proteasome system and regulating the degradation of related substrate proteins to achieve disease therapy is an important drug development strategy.

The current research on small molecule inhibitors targeting the ubiquitin-proteasome system mainly includes the following:

Ubiquitin-activating enzyme E1 inhibitors.

Ubiquitin-binding enzyme E2 inhibitors.

Ubiquitin ligase E3 inhibitors.

Proteasome inhibitors.

TAK-243 is a First-in-Class, highly potent ubiquitin-activated enzyme E1 (UAE) inhibitor. TAK-243 was found to have significant antitumor activity. Among other things, researchers conducted in vivo efficacy experiments using the HCC-70 model through Medicilon. The results revealed a tumor growth inhibition (TGI) rate of 91%.

>> Learn more about "Medicilon's Xenograft Models"

In this article, we will share a case study of pharmacodynamic research on the development of ubiquitination inhibitors. It combined the literature and the experience of Medicilon's pharmacodynamic department in the hope that it will be helpful for all researchers who are concerned about ubiquitination research and tumor pharmacodynamics.


Ubiquitination is a process by which ubiquitin molecules break down proteins in cells under the action of a series of special enzymes, select target protein molecules from them, and make specific modifications to the target proteins. Ubiquitination plays a vital role in protein localization, metabolism, functional regulation, and degradation. It also regulates numerous life activities such as cell cycle, cell proliferation, apoptosis, cell differentiation, metastasis, gene expression, transcriptional regulation, signaling, damage repair, immune inflammation, etc.

Ubiquitin-proteasome system

The Ubiquitin-Proteasome System (UPS) is a multi-component system responsible for cell protein degradation. It mediates more than 80% of protein degradation in eukaryotes and is involved in cell growth and differentiation, DNA replication and repair, cell metabolism, immune response, etc. The ubiquitin-proteasome system (UPS) consists of ubiquitin (Ub), ubiquitin-activating enzyme E1, ubiquitin-binding enzyme E2, ubiquitin ligase E3, deubiquitylating enzymes (DUBs), and 26S proteasome.

The ubiquitin-proteasome system (UPS).jpg

The ubiquitin-proteasome system (UPS)[1]

Ubiquitin (Ub)

Ubiquitin is a highly conserved small molecule protein widely distributed in eukaryotic cells. A single ubiquitin molecule consists of 76 amino acid residues. The primary function is to label target proteins that need to be broken down to be degraded by the 26S proteasome. Ubiquitin can also label transmembrane proteins and participate in the transport of proteins across membranes.

Structure diagram of ubiquitin.jpg

Structure diagram of ubiquitin (Source: Wikipedia)

A ubiquitin-activating enzyme (E1)

The ubiquitin-activating enzyme, the E1 enzyme, is the first enzyme required for ubiquitin binding to substrate proteins. It contains a conserved cysteine residue in a fixed position in its structure. It activates ubiquitin by forming a high-energy thioester bond between the cysteine residue and the C-terminus of ubiquitin. Necessary for regulating ubiquitin homeostasis and downstream ubiquitination-dependent cellular processes, including protein hydrolysis and selective autophagy via the ubiquitin-proteasome system.

The ubiquitin-binding enzyme (E2)

The ubiquitin-binding enzyme E2 plays a vital role in the ubiquitination process and is an essential intermediate. The ubiquitin-binding enzymes E2 are a superfamily of many structurally and functionally diverse proteins. All ubiquitin-binding enzymes contain a conserved structural domain, the UBC domain. The ubiquitin-binding enzymes (E2) play a vital role in the translocation of ubiquitin from ubiquitin-activating enzymes (E1) to ubiquitin-ligases (E3) and substrates.

Ubiquitin ligase (E3)

The third enzyme in the ubiquitination cascade reaction catalyzing the delivery of ubiquitin bound to the ubiquitin-binding enzyme to the target protein, ubiquitin ligase E3 determines the specific recognition of the target protein. It has a vital role in the ubiquitination pathway. Ubiquitin ligase E3 is involved in various physiological processes in the cell by regulating the ubiquitination process of regulatory proteins. All E3s can link target proteins to specific E2s. The ubiquitin ligase E3 structural domains mainly include the HECT structural domain, the RING structural domain, and the U-box structural domain. Due to the specific recognition of these complex and variable E3 family members for different substrates, the ubiquitination pathway exhibits a high degree of selectivity for protein degradation.

Deubiquitinating enzymes (DUBs)

A family of deubiquitinating enzymes is responsible for the deubiquitination of ubiquitin molecules by hydrolyzing the ester, peptide, or isopeptide bonds at the carboxyl terminus of ubiquitin to specifically hydrolyze ubiquitin molecules from proteins or precursor proteins linked to ubiquitin, which acts as a deubiquitinating agent to regulate protein degradation inversely and thus affect the function of the protein.


The 26S proteasome is an ATP-dependent protein hydrolysis complex that degrades ubiquitinated substrates and consists of a 20S core particle, a 19S regulatory particle, and an 11S regulatory factor. The 26S proteasome is the central protease for ubiquitin-mediated protein degradation. It plays a vital role in cellular homeostasis by controlling the degradation of critical proteins involved in apoptosis, cell cycle, and signal transduction. The proteasome has multiple functions and is an important target for drug development, particularly in tumors and neurodegenerative diseases. Many selective proteasome inhibitors have been identified and approved for clinical use in the last two decades (e.g., Bortezomib, Carfilzomib, etc.).

26S proteasome structure.jpg

26S proteasome structure (Source: Wikipedia)

The process of ubiquitination

Activation of ubiquitin molecules by ubiquitin-activating enzyme E1 in the presence of energy supplied by ATP; formation of an E1-ubiquitin intermediate.

Ubiquitin-activating enzyme E1 passes the activated ubiquitin molecule to ubiquitin-conjugating enzyme E2; an E2-ubiquitin intermediate is formed.

Ubiquitin ligase E3 first recognizes the target protein to be degraded. Then it attaches the ubiquitin bound to E2 to the target protein, forming a complex containing the E2-ubiquitin intermediate, E3 enzyme, and the target protein.

E2 enzyme and E3 enzyme are released from the above complex, resulting in the formation of the ubiquitin-tagged target protein.

Repeat the above process until multiple ubiquitins attached to the protein form a ubiquitin chain.

The target protein, which is ubiquitinated, is recognized and degraded by the 26S proteasome.

Ubiquitin-proteasome pathway.jpg

Ubiquitin-proteasome pathway [2]

Inhibitors of ubiquitin-activated enzyme E1

Two key enzymes initiate ubiquitin coupling in mammals, the ubiquitin-activating enzymes UAE and UBA6, collectively referred to as E1 enzymes. UAE (encoded by the UBA1 gene) is responsible for over 99% of intracellular protein ubiquitination, whereas UBA6 is responsible for < 1% of ubiquitination. Classical E1 enzymes all have an adenylation structural domain (AD), a catalytic cysteine structural domain (CCD), and a ubiquitin folding structural domain (UFD). First, in the presence of ATP and Mg2+, the E1 enzyme catalyzes the formation of the Ub-AMP adduct with ATP. It binds to the AD structural domain of the E1 enzyme while releasing phosphate group PPi, the cysteine sulfhydryl nucleophile of the CCD structural domain, attacks the Ub-AMP bond to form a Ub-E1 thioester bond and releases AMP. The other Ub is non-covalently bound to the AAD structural domain to create an E1 enzyme carrying two Ub molecules. Then, the E1 enzyme, having two molecules of Ub, binds to the E2 enzyme and transfers Ub to the E2 enzyme via the UFD to form the Ub-E2 complex, which then activates the next Ub. Therefore, inhibition of E1 enzyme activity can block the activation of ubiquitin molecules and inhibit the ubiquitination process, which is a potential antitumor target.

First-in-Class UBA1 Inhibitor TAK-243

TAK-243 (MLN7243) is the first UBA1 inhibitor developed by Millennium to enter the clinic. It was discovered through screening of over 700 compounds and can form Ub-TAK-243 adducts with Ub, which in turn binds to the ATP binding site of UBA1 and inhibits UBA1 activity. It can induce ubiquitin-mediated accumulation of p53, c-Jun, c-Myc, XIAP, and other proteins in various cancer cells and exhibits significant antitumor activity. It is currently in phase I clinical setting for treating advanced malignant solid tumors.

TAK-243 is a first-in-class ubiquitin-activating enzyme (UAE) inhibitor. It effectively inhibits UAE by forming a TAK-243-ubiquitin adduct. Treatment of cells with TAK-243 in vitro leads to loss of cellular ubiquitin coupling, resulting in defective ubiquitin-dependent protein switching and signaling, impaired cell cycle progression and bad DNA repair, increased proteotoxic stress, and ultimately cancer cell death. In vivo treatment of tumor cells with TAK-243 resulted in a dramatic reduction in cellular polyubiquitination and induced significant antitumor activity in mice bearing human xenograft tumors.

TAK-243 was highly selective and inhibited UAE, UBA6, NAE, and SAE with IC50 values of 1, 7, 28, and 850 nM, respectively. Once formed, TAK-243-ubiquitin adducts bind tightly to UAE and block the catalytic activity of UAE. TAK-243 inhibits the transfer of ubiquitin molecules from UAE to the E2 enzyme.

A Chemical structure of TAK-243 B Structure of TAK-243-ubiquitin adduct.jpg

A: Chemical structure of TAK-243 B: Structure of TAK-243-ubiquitin adduct [3]

In vivo antitumor activity study of TAK-243

The antitumor activity of TAK-243 was verified using PDX and CDX tumor models, respectively. Lateral abdominal subcutaneous injection of tumor fragments or tumor cell suspensions in serum-free medium was performed in 8- to 12-week-old mice. Researchers have established subcutaneous tumors in mice using CDX models: WSU-DLCL2 (diffuse large B-cell lymphoma), HCT-116 (colon cancer), THP-1 (acute myeloid leukemia), CWR22 (prostate cancer), Calu-6 (non-small cell lung adenocarcinoma), HCC-70 (triple negative breast cancer) and MM1.S (multiple myeloma), and PDX models: PHTX-24c (colon cancer), PHTX-132Lu (primary NSCLC), PHTX-55B (triple-negative breast cancer), PHTX-235O (ovarian cancer) and HNM626 (cervical cancer).

>> Learn more about "Medicilon's PDX Models"

In this study, researchers conducted experiments using the HCC-70 model through Medici. The HCC-70 model was formed by transplanting 5.0×106 HCC-70 cells in the abdomen of each female nude mouse. When the average tumor volume reached approximately 200 mm3, the animals were randomly grouped into eight animals per group. TAK-243 (12.5 mg/kg) and control (20% HPβCD) were administered twice weekly over the next 21 days. Tumor growth and animal weight were monitored twice weekly. TGI was calculated within five days after the last dose.

Growth inhibition curves of TAK-243 against HCC-70.jpg

Growth inhibition curves of TAK-243 against HCC-70 [3]

It was found that TAK-243 induced significant antitumor activity responses in all models examined. Among them, the TGI reached more than 90% against CWR22, PHTX-235O, HCC-70, and PHTX-55B. Also, there was almost no effect on the body weight of the animals.

PHTX-235OPrimary ovarian97
PHTX-55BPrimary breast91
PHTX-24CPrimary colon87
HNM626Primary neck85
PHTX-132LuPrimary NSCLC84
MM1.SMultiple myeloma80

The antitumor activity of TAK-243 against different tumors in vivo [3]

Summary and outlook

The different roles of ubiquitin in regulating cellular proteostasis and signaling highlight the possibility of targeting UPS to control human diseases. Despite this potential, only a tiny fraction of the more than 500 enzymes involved in UPS are targetable by drugs that have entered clinical studies. The clinical success of the proteasome inhibitor Bortezomib has generated interest and enthusiasm to investigate other components of the UPS. TAK-243 is a first-of-its-kind, potent UAE inhibitor that inhibits cellular ubiquitination, leading to impaired ubiquitin-dependent protein hydrolysis, ER stress, and impaired cell cycle progression and DNA damage repair. In vitro and in vivo data suggest that TAK-243 exerts its effects through UAE inhibition. The discovery of TAK-243 provides a valuable tool to study protein ubiquitination and offers new opportunities to explore protein homeostasis and ubiquitin signaling for use in tumor research. The ubiquitin-proteasome system is an essential target for various diseases, especially tumors, and targeting this system will yield higher selectivity.


[1] Marie W Wooten, et al.  Signaling, polyubiquitination, trafficking, and inclusions: sequestosome 1/p62's role in neurodegenerative disease. J Biomed Biotechnol. 2006;2006(3):62079.doi: 10.1155/JBB/2006/62079.

[2] Ashok N Hegde, et al. The ubiquitin-proteasome pathway in health and disease of the nervous system. Trends Neurosci. 2007 Nov;30(11):587-95. doi: 10.1016/j.tins.2007.08.005. Epub 2007 Oct 24.

[3] Marc L Hyer, et al. A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment. Nat Med. 2018 Feb;24(2):186-193. doi: 10.1038/nm.4474. Epub 2018 Jan 15.

[4] Isabella A Lambert-Smith, et al. The pivotal role of ubiquitin-activating enzyme E1 (UBA1) in neuronal health and neurodegeneration. Int J Biochem Cell Biol . 2020 Jun;123:105746. doi: 10.1016/j.biocel.2020.105746. Epub 2020 Apr 18.

[5] H. Yang, X. Chen, K. Li, et al. Repurposing old drugs as new inhibitors of the ubiquitin-proteasome pathway for cancer treatment. Seminars in Cancer Biology. 2021, 68:105-122.

[6] L. Cappadocia, C. D. Lima. Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism. Chem Rev. 2018, 118(3): 889–918.

[7] J. A. Harrigan, X. Jacq, N. M. Martin, et al. Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat Rev Drug Discov. 2018, 17(1): 57–78.

Medicilon Pharmacology & Pharmacodynamic Services

In recent years, pharmacological and pharmacodynamic evaluation, animal models, and gene & cell therapies have been iteratively updated and rapidly developed. Animal models have played an essential role in new drug development.Establishing different disease animal models provides a powerful tool to study the mechanism of disease occurrence and development, screening, and evaluation of anti-disease drugs.

Medicilon has approximately 30,000 square meters of laboratories, is AAALAC accredited, and has an adequate supply of animals for animal model construction for mice, rats, rabbits, pigs, dogs, monkeys, and other animals.

Keeping pace with industrial development and market demand, Medicilon's Pharmacology and Pharmacodynamics Department has years of experience and established a complete animal model library based on verifications and practices for precise and efficient drug efficacy testing.The test subjects include non-human primates, dogs, rats/mice, rabbits, guinea pigs, and miniature pigs.

Medicilon boasts nearly 300 orthotopic evaluation models. At the same time, we are empowering innovative therapies to evaluate and study immuno-oncology comprehensively. We have completed model establishment and efficacy evaluation of immuno-therapies such as CAR-T, TCR-T, CAR-NK, oncolytic virus, antibody (monoclonal antibody, double antibody, polyclonal antibody, etc.), siRNA, and AAV.

Medicilon can provide various animal models (including renal failure model, anemia animal model, gastric acid secretion animal model, and gastric ulcer model) to test drug effectiveness according to client needs. Using rats as subjects, we can test specific digestive system diseases, including gastric acid secretions, ulcers, and renal failure.

In the field of metabolic diseases, we are proud of our portfolio of stable and effective animal models, especially those for Non-alcoholic Fatty Liver Disease (NAFLD). Though popular recently, they still lack effective drug treatment.

Our Preclinical Pharmacodynamics Department has been deeply involved in this field for years, developing reliable animal-based efficacy evaluation models aimed at different targets and pathways, thus facilitating the clinical transformation of new drugs.

Our Pharmacodynamics Department is proud of its multiple nervous system models based on anti-depressants, anti-Alzheimer's drugs, sedative-hypnotic and anti-anxiety medications, analgesics, anti-convulsants, anti-Parkinson's drugs, and anti-schizophrenia drugs. Those models can effectively evaluate Type-1 innovative medicines at the molecular and cellular level, ex vivo, and in vivo.

Medicilon offers various effective animal models based on clients' needs. Typical models include full-thickness skin trauma, pressure ulcers, and spontaneous hypertension, with rats/mice and SHR as subjects.

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