Deubiquitinase: A New Opportunity for Small Molecule Drug Discovery

preface

Deubiquitinase is widely involved in various physiological processes such as cell proliferation, autophagy, DNA damage repair and immune response, and is closely related
to the occurrence and development of tumors, Alzheimer's and other diseases.

Deubiquitinase can not only regulate druggable proteins, but also regulate the level of proteins that are insensitive or difficult to intervene with small molecules with traditional targeted therapies, and are a class of promising drug targets
.

At present, the small molecule inhibitor of deubiquitinase is still in the early stage of development, and two molecules are undergoing clinical phase I trials, and the difficulty of drug development lies in the development of accurate and effective biochemical analysis methods, screening technologies and improving the selectivity
of congener proteins.

01Deubiquitinase is numerous

Widely involved in the regulation of cell life activities

Ubiquitination is a post-translational modification that links the carboxyl group at the C-terminus of the ubiquitin molecule to the N-terminus of the substrate protein or the amino group of lysine through a cascade catalyzed by ubiquitin ligase, which can change its function, localization, stability, and protein-protein interaction
.

Deubiquitination is the opposite process of ubiquitination, in which ubiquitin molecules on the substrate protein are removed under the catalysis of deubiquitinases (DUBs), thus avoiding degradation by the proteasome
.
Inhibition of deubiquitinase can lead to selective degradation of proteins and may affect other "undruggable" targets
.
Therefore, DUBs are potential drug targets
.

Figure 1, Ubiquitination/Deubiquitination System

There are about 100 different deubiquitinating enzymes in the human body, which can be divided into ubiquitin-specific proteases (USPs), ubiquitin-specific proteases (USPs), ubiquitin carboxyl-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado-Joseph disease protein domain proteases (MJDs), JAMM/MPN region-associated metallopeptidases (JAMMs), zinc-containing ubiquitin peptidases 1 ( ZUP1) and 7 families
including the new DUB family (MINDYs) in which motif interacts with ubiquitins.
Among them, the structure and function are the most in-depth research, and the USP family
is the most reported small molecule inhibitor.
Figure 2, Classification of deubiquitinase

02 Mechanism of action of deubiquitinase (USBs).

When DUBs play a catalytic role, they first identify the hydrophobic Ile44 patch on ubiquitin and the His72 and His74 residues through the S1 pocket, thereby locating the peptide bond to be broken by ubiquitin to the catalytic site of the DUB, while the proximal S1' pocket determines the specificity of the substrate protein and can distinguish
different substrate proteins with the same ubiquitin 。 The catalytic pockets of DUBs are generally composed of cysteine, histidine and aspartic acid, in which the -SH of cysteine with sulfhydryl groups initiates a nucleophile attack on the peptide bond chain between ubiquitin and substrate protein, causing it to break; The catalytic cysteine-free JAMM family breaks peptide bonds by inducing zinc atoms
.

Through their catalytic action, DUBs can specifically dissociate ubiquitin from ubiquitin-tagged substrate proteins, maintaining free ubiquitin levels, while releasing substrate proteins from the ubiquitin degradation pathway to prevent degradation
.
Figure 3, Catalytic mechanism of deubiquitinase

03DUBs are associated with a variety of diseases

It is a potential drug development target

Studies have shown that DUBs play an important role in the occurrence and development of malignant tumors, neurodegenerative diseases and other immune diseases, in addition, some bacteria and viruses such as COVID-19 also inhibit the host's immune response by expressing pathogenic DUBs to increase the chance of survival and replication
.

DUBs are related to tumorigenesis and development at multiple levels, including but not limited to regulating key epigenetic changes, regulating oncogene/tumor suppressor protein activity, and regulating DNA damage repair
.
For example, USP7 can deubiquitinate the E3 ubiquitin ligase MDM2 to increase the level of MDM2 in cells, while MDM2 ubiquitinates the tumor suppressor p53 and inhibits p53-mediated cell cycle arrest and apoptosis, creating conditions
for tumor development and development.
In addition, USP7 can also deubiquitinate PTEN protein and transcription factor FOXP3, thereby promoting tumor growth and metastasis
.

In neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD), disease-related nerve fiber tangles, Lewy bodies and mitochondrial homeostasis, etc.
, are regulated by the ubiquitination and deubiquitination system, which promotes the ubiquitination of pathogenic proteins/damaged mitochondria, that is, inhibits the activity of related DUBs, which can play a therapeutic role
in the treatment of diseases 。 Studies have shown that the accumulation of damaged mitochondria will lead to defects in cellular metabolism, which is associated with the occurrence of PD, and the deubiquitinase USP30 is thought to prevent autophagy of damaged mitochondria, thereby causing damage mitochondrial accumulation, therefore, inhibiting the function of USP30 will benefit the treatment of PD and other mitochondrial diseases, a conjecture that has been verified
in animal models.
USP14 is thought to reduce the ubiquitination of the α-synuclein of PD and Tau protein in AD, and inhibiting the function of USP14 will also help in the treatment of
the disease.
Figure 4, Deubiquitinating enzymes that play an important role in a variety of diseases

04DUBs small molecule inhibitor development

It's an early stage where challenges and opportunities coexist

At present, there have been more reports of small molecule inhibitors of DUB, which can be roughly divided into three categories: ubiquitin analogues, catalytic inhibitors, and allosteric inhibitors, and the development method is generally through DUBs-based high-throughput screening, phenotypic screening and NMR-based fragment screening to obtain suitable hits, and then optimize the hit to improve its activity and druggability
.

Due to the fact that the biological mechanism of DUBs association with disease has not been fully resolved, there are many family members and the structure is conserved, the development of DUBs inhibitors is more challenging
than kinase, membrane receptor and other types of targets.
But at the same time, DUBs can also regulate the level of proteins that are not sensitive or directly inhibited by traditional targeted therapies, which may include "undruggable" targets, such as transcription factors, drug-resistant enzymes, and proteins
in protein interactions that are difficult to directly intervene with small molecules.

As of October 2022, 3 DUBs small molecule inhibitors have entered the clinical phase I trial, of which 1 has been discontinued from development and no drug has been approved
.
The remaining reported small molecule inhibitors are in preclinical research, and more than half of the small molecules are multi-target inhibitors, and companies involved in research and development include Novartis, Roche, Mission Therapeutics, KSQ Therapeutics and other companies
.

(1) VLX1570, developed by Vivolux, is the first DUBs inhibitor to enter clinical trials, which can simultaneously inhibit USP14 and UCHL15 for the treatment of multiple myeloma, but the clinical trial was terminated
due to severe dose-limiting toxicity causing patient death.

(2) KSQ-4279 was developed by KSQ Therapeutics as an allosteric inhibitor of USP1, with high selectivity for other USP members, and is currently in phase I clinical trials for advanced solid tumors
.

(3) MTX652 developed by Mission Therapeutics is a selective inhibitor of USP30, which can promote the degradation of damaged mitochondria for the treatment of chronic kidney disease, and has opened clinical phase I trials
in April this year.
In addition, more than 60 small molecules are in the preclinical research stage
.
Figure 5, some of the reported structures of DUBs inhibitors

summary

DUBs are widely involved in various physiological processes in cells, including transcription, apoptosis, autophagy, DNA repair, immune response, etc.
, and their dysfunction is closely related to Alzheimer's disease, tumor and other diseases, and are a class of drug targets with great market potential
.

DUBs small molecule inhibitors have shown good efficacy in preclinical studies, and it is necessary to further improve their selectivity to members of the same family in the future to accurately guide related disease pathways and improve the safety of
drugs.
Although no product has yet been approved, the research on the biological regulatory mechanism and structural function of DUBs is deepening step by step, and the eutectic structure of more than 10 DUBs and small molecules has been disclosed on PDB, providing a solid foundation
for the development of DUBs inhibitors.

We have reason to believe that in the near future, safe and effective DUB inhibitors will also be approved by regulatory agencies, providing more treatment options
for patients with multiple diseases.