Development of Deubiquitinase-targeting Chimeras (DUBTACs)
Targeted chimeric proteolysis (PROTAC) technology represents a breakthrough development in the field of drug discovery, utilizing the ubiquitin proteasome system to specifically degrade disease-associated proteins, and multiple projects have entered clinical development. However, for CF and some cancers, targeted protein stabilizers TPS need to be developed to combat abnormal protein degradation.
PROTAC at BOC Sciences
Targeted protein stabilization (TPS) is an emerging technology in the biomedical field in recent years, which aims to stabilize proteins in cells by a specific method, thereby affecting their function and behavior. This technology has a wide range of potential applications, from basic research to the development of new drugs.
What is DUBTAC?
Deubiquitinase-targeting chimeras (DUBTACs) are a kind of targeted protein stabilization technology gradually developed in recent years. DUBTACs generally consist of target protein ligand, deubiquitinase ligand and linker. This structural design allows the DUBTAC molecule to bind to both the target protein and the deubiquitination enzyme at the same time, inducing the target protein and the deubiquitination enzyme to approach and remove the ubiquitin chain on the target protein, thus achieving a level of stabilization of the specific protein degraded in a ubiquitination dependent manner.
Although DUBTAC seems to be a very close concept to PROTAC, there are a number of key differences in implementation. One is the ligand binding site; The E3 targeted by PROTAC is mainly a skeleton protein, but the DUB of DUBTAC needs to retain its catalytic function. Second, the requirements for enzyme activity may be different. PROTAC may only need a low level of ubiquitination target to degrade the target protein, but DUBTAC may need to hydrolyze most of the ubiquitin to avoid the degradation of the target. Third, the requirements for ubiquitination are different. PROTAC does not require the ubiquitination level of the target protein, but for DUBTAC to work, the target protein must first be ubiquitinated.
DUBTAC platform. (Henning, N. J., 2022)
In this article, we summarize the development of DUBTAC technology in recent years, and discuss its application prospects and potential challenges in the field of biomedicine in detail.
Outb1-based DUBTACs
In early 2022, Professor Daniel K. Nomura and his team at the University of California, Berkeley used activity-based protein profiling (ABPP) to analyze the reactive cysteine of 65 deubiquitinating enzymes globally.
The results showed that the C23 site of ubiquitination enzyme OTUB1, which does not participate in the regulation of enzyme activity and has high nucleophilicity, is an ideal ligand binding site. Since the most abundant ubiquitin chain in cells that primarily mediates protein degradation is the K48 chain, OTUB1 is a deubiquitination enzyme that targets the removal of the K48 chain.
In theory, it is very effective to use OTUB1 to de-target ubiquitin chains on specific proteins and prevent specific proteins degraded in a ubiquitination-dependent manner from being degraded by the proteasome.
ΔF508-CFTR-DUBTACs
Based on the above findings, Professor Daniel K. Nomura's team selected a covalent ligand EN523 that binds to OUTB1 with high affinity. EN523 binds to OTUB1 through covalent interaction between the acrylamide part of EN523 and the allosteric cysteine residue Cys23 of OTUB1. They demonstrated in vitro that EN523 had no significant effect on the enzyme activity of OTUB1.
Structure of EN523, with cysteine-reactive acrylamide highlighted in red. (Henning, N. J., 2022)
The researchers then linked the screened EN523 with Lumacaftor, a regulator of ΔF508-CFTR, to the heterobifunctional small molecule NJH-2-057, which was used to target the stabilization of the CFTR mutant ΔF508-CFTR to improve cystic fibrosis.
The experimental results show that NJH-2-057 can effectively stabilize ΔF508-CFTR, while the results of Lumacaftor alone are not significantly different from the control group. This result provides a proof of concept for the development of DUBTAC technology.
WEE1-DUBTACs
Professor Daniel K. Nomura's team then applied DUBTAC technology to improve the stability of the tumor suppressor gene WEE1, a tumor suppressor kinase in non-malignant eukaryotic cells that phosphorylates the cyclin-dependent kinase (CDK1)-cyclin B1 complex. To inhibit the progression of the cell cycle in the S and G2 phases of mitosis, and its activity must be down-regulated for mitotic progression to occur. WEE1 is degraded in many tumors to promote cancer cell proliferation. Stabilizing levels of WEE1 kinase in cancer cells is expected to prevent tumor growth.
EN523 was combined with AZD1775, a clinical WEE1 inhibitor, to synthesize DUBTAC targeting the tumor suppressor kinase WEE1 via either an alkyl chain linker or a peg-based linker link. The study showed that DUBTAC significantly stabilized WEE1 in liver cancer cell lines, while EN523 or AZD1775 alone had no effect on WEE1 levels.
This further demonstrates the universality of the DUBTAC technique for TPS, and in addition, the research by Professor Daniel K. Nomura's team highlights the practicality of using a covalent ligand discovery platform supported by chemical proteomics.
TF-DUBTACs
In June 2022, Professor Wenyi Wei's team from Harvard Medical School in the United States, based on previous studies, used EN523 to recruit the degeneralizing enzyme OTUB1 to further develop the first TF-DUBTAC drug platform that can stabilize cancer-inhibiting transcription factor (TF). The aim is to realize the treatment of tumor by stabilizing TF.
Schematic diagram of TF-DUBTAC platform. (Liu, J., 2022)
The researchers connected the binding ligand of FOXO3A, p53 and IRF3 transcription factors, azide-modified DNA oligonucleotides, with the covalent ligand (DUBL-X-BCN 1-10) of the deubiquitination enzyme OUTB1 modified by BCN (bicyclononyne) via SPAAC reaction. A series of DUBTAC molecules targeting FOXO3A, p53 and IRF3 transcription factors were designed and synthesized. Then, the researchers determined the stabilizing effect of the compound on the target protein at the cellular level through in vitro experiments, and screened out effective TF-TUBTAC molecules. The corresponding TF-DUBTAC stabilized intracellular FOXO3A, p53, and IRF3 in an OUTB1-dependent manner.
This further demonstrates that TF-DUBTAC is a generable platform to achieve the selective stability of cancer-suppressing transcription factors and can be used as a therapeutic means to inhibit tumorigenesis. It also demonstrates the universality and effectiveness of DUBTAC technology in cancer treatment.
DUBTACs based on USP7
Since DUBTACs developed based on the covalent ligand of deubiquitinating enzyme OTUB1 may have disadvantages such as toxicity or hypersensitivity similar to other covalent inhibitors, the development of reversible recruitment ligands based on other deubiquitinating enzymes is an important development direction in the field of TPS.
In April 2024, Professor Wenyi Wei's team at Harvard Medical School in the United States developed the first DUBTAC based on the deubiquitination enzyme USP7, which uses non-covalent ligands of USP7 to selectively stabilize CFTR and AMPK proteins.
ΔF508-CFTR-DUBTACs
First, the researchers selected the previously reported USP7 ligand#1 as the ligand of the deubiquitination enzyme USP7. By analyzing the eutectic structure of the ligand and USP7 complex, the amino group located outside the binding pocket in the structure of the ligand molecule USP7i-#4a was selected as the linker linking site for molecular design.
We synthesized a series of CFTR-targeting DUBTAC molecules by linker linking USP7 ligand#1 and the ΔF508-CFTR modulator Lumacaftor, and measured the stabilizing effect of the compound on CFTR protein at the cellular level.
Usp7-based DUBTAC to stabilize the target protein. (Liu, J., 2024)
The findings suggest that the compound MS6869 promotes the stabilization of CFTR in a relatively specific manner, with effectiveness comparable to that of the previously reported OTUB1-based CFTR-DUBTAC.
AMPK-DUBTACs
Subsequently, the researchers designed and synthesized a series of AMPK-targeting DUBTAC molecules based on two different USP7 ligands, and tested these compounds for AMPK protein stabilization. The study showed that AMPK-DUBTAC derived from different USP7 ligands can selectively stabilize different subtypes of AMPK β. AMPK signal transduction is increased.
These results show the feasibility of DUBTAC technology development based on USP7.
Future prospect of DUBTAC
With the emergence of DUBTAC technology, many fields are expected to benefit from active ubiquitination and targeted deubiquitination of degraded proteins. Examples include stabilizing BAX levels in mitochondria to induce apoptosis, stabilizing STING for immunooncology applications or stabilizing glucokinase in pancreatic cells for adult-onset diabetes type Ⅱ. Other targets that benefit from DUBTAC include various tumor suppressors that are actively ubiquitinated and degraded in order to maintain cancer cell proliferation.
In addition to cystic fibrosis, there are many other genetic diseases that can also benefit from dubtac stabilization. These include glucocerebrosidase mutations in Gorscher's disease or Parkinson's disease and phenylalanine hydroxylase and fumarylacetoacetate hydroxylase mutations in phenylketonuria.
This article summarizes the research progress of DUBTAC technology in recent years, showing the significant advantages of DUBTAC technology in improving the efficiency of protein stabilization, and also looks forward to the development prospect of DUBTAC technology. It is expected that more researchers can join this innovative field and jointly promote the development of biomedical technology.
References:
- Henning, N. J., et al. Deubiquitinase-targeting chimeras for targeted protein stabilization. Nature Chemical Biology. 2022, 18(4): 412-421.
- Liu, J., et al. TF-DUBTACs stabilize tumor suppressor transcription factors. Journal of the American Chemical Society. 2022, 144(28): 12934-12941.
- Liu, J., et al. USP7-Based Deubiquitinase-Targeting Chimeras Stabilize AMPK. Journal of the American Chemical Society. 2024,146(16): 11507-11514.
