The latest research progress and prospect of PROTAC for non-enzymatic function of target proteins

Studies have shown that the non-enzymatic function of target proteins plays a key role in the regulation of multiple cell signaling pathways and is closely related
to many human diseases.
However, traditional small molecule inhibitors typically target the catalytic function domain directly, acting by inhibiting the enzymatic function of the target protein without affecting non-enzymatic functions
.
The targeted proteolytic chimera PROTAC technology that has emerged in recent years has the advantage of regulating both the enzymatic and non-enzymatic functions of the target protein, thus providing a potential strategy
for improving the deficiency of small molecule inhibitors and exploring new treatment options.
This article summarizes the latest progress of PROTAC for non-enzymatic functions of target proteins and looks forward
to future development trends.

Classical enzymes typically consist
of enzyme domains and non-enzyme domains.
The functional domain of an enzyme refers to the region with a specific spatial structure in
which the enzyme performs its catalytic function.
Specifically, the enzyme active site contains a binding region and a catalytic zone that are involved in the binding of the substrate and the catalytic substrate undergoing specific chemical reactions
, respectively.
The non-enzymatic functional region mainly regulates substrate protein activity through protein-protein interaction (PPI), which is independent of catalytic function and mediates the interaction between different protein components in the substrate and signaling pathway, such as allosteric regulation, scaffold protein function, etc
.

Numerous studies have shown that non-enzymatic functional regions of various target proteins are involved in the regulation
of cell division, differentiation, RNA metabolism, DNA repair and genome stability.
In addition, these non-enzymatic functions are closely related to various human diseases, such as cancer, cardiovascular disease, and play a key role
in regulating cell signaling and determining cell fate.
However, traditional small molecule inhibitors typically target the catalytic domain of the enzyme directly and exert therapeutic effects
by inhibiting the enzymatic function of the target protein.
However, these inhibitors are often unable to block the non-enzymatic function of the target protein and face problems such as
low selectivity, poor specificity, limited clinical efficacy, and drug resistance.

Targeted protein degradation (TPD) is considered a promising and attractive therapeutic strategy
.
PROTAC typically consists of three parts: the target protein ligand, the E3 ubiquitin ligase ligand, and the linkage chain
.
PROTAC can recruit E3 ubiquitin ligase to promote ubiquitination and degradation
of target proteins through the ubiquitin-proteasome system (UPS).

Unlike traditional drug development strategies that directly inhibit target proteins, PROTAC works
by modulating the host protein degradation system.
In addition, PROTAC can induce degradation of the entire target protein, which can block both enzymatic and non-enzymatic functions of the target protein (Figure 1).

Therefore, blocking the non-enzymatic function of target proteins through PROTAC strategies may be a new and promising strategy
to solve the disease treatment problems faced by traditional small molecule inhibitors.

1 PROTAC targeting EZH2

Epigenetics is the study of the heritable modification
of a gene's nucleotide sequence without altering gene expression.
Abnormalities of epigenetic modification are widely present in the process of tumor occurrence and development, and are a research hotspot
in the development of antitumor drugs.

PRC2 is a member of the multi-combination histones, which is an important target for epigenetic cancer therapy and has histone methyltransferase activity
.
The PRC2 complex consists of
four main members: EZH2, EED, SUZ12, and RbAp46/48.
Overactivation of the PRC2 complex induces malignancy by silencing tumor suppressor genes, and EZH2 is a core and multifunctional catalytic subunit of PRC2 that is overexpressed in multiple cancer types, such as lung, bladder, and breast cancer
.
EZH2 inhibits the expression
of tumor suppressor genes by methylating the 27-position lysine of histone H3.

In recent years, significant progress
has been made in the development of inhibitors that directly or indirectly target EZH2.
There is growing evidence that EZH2's carcinogenic function is not entirely dependent on its enzyme activity
.
In addition to catalyzing H3K27me and mediating the silencing of genes associated with various cellular processes, EZH2 mediates the activation of genes in a variety of cancers that are not associated
with the enzymatic function of EZH2/PRC2.

However, the currently reported EZH2 inhibitors only downregulate H3K27me3 levels by targeting histoprotein methyltransferase activity
.
Due to insufficient inhibition of EZH2's carcinogenic activity, EZH2 inhibitors have limited clinical efficacy and are only effective
in some cancers.
Therefore, there is an urgent need to develop a new therapeutic strategy
targeting EZH2.

The targeted protein degradation strategy PROTAC provides a new opportunity
to completely block the carcinogenic activity of EZH2.

Recently, the Yu research group at Sichuan University designed and synthesized EZH2 PROTAC 1 by linking the EZH2-selective inhibitor EPZ6438 to the CRBN ligand thalidomide through a linkage chain (Figure 2).

The experimental results show that compound 1 can completely inhibit the carcinogenic activity
of EZH2.
Among them, compound 1 showed significant degradation efficiency for the PRC2 subunit, and the degradation was complete
at 72h.
The study found that compound 1 binds directly to the EZH2 protein instead of SUZ12, EED and RBAP48
.
Thus, compound 1 can recruit E3 ubiquitin ligase to the vicinity of the PRC2 complex, leading to ubiquitination and degradation
of EZH2.
EZH2-mediated indirect interactions induce degradation of other PRC2 subunits by proteasomes, including EED, SUZ12, and RBAP48
.
However, due to the poor selectivity of the PRC2 subunit, Compound 1 may have potential toxicity or side effects
.
Future research on compound 1 should focus on how to improve target selectivity
for protein degradation.

The Wen group linked the selective EZH2 inhibitor EPZ6438 and VHL ligands through a linkage chain to obtain the selective EZH2 degraders YM281 (3) and YM181 (4).

The results show that these two EZH2 PROTACs can target the entire EZH2 in lymphoma, thereby blocking the non-enzymatic function of EZH2 by degrading EZH2 (Figure 3).

In addition, EZH2 PROTAC significantly reduced levels of H3K27me3 and induced cell cycle arrest and apoptosis
.
At the same time, lymphoma cell lines resistant to the inhibitor EPZ6438 showed complete inhibition and good inhibition in in
vivo anti-tumor models.

Because EZH2-PRC2 and EZH2-TAD-cMyc-coactivator, two EZH2 complexes, are very important for EZH2-mediated pro-cancer function, the current limitations of EZH2 inhibitors in tumor treatment have been caused
.
cMyc is a carcinogen that is difficult to target by small molecules, has a direct PPI effect with EZH2, and is independent of the PRC2 complex
.

Therefore, the researchers designed the degrader MS177 (5) using PROTAC technology (Figure 4), which can target both the enzymatic and non-enzymatic functions
of EZH2.
The experimental results showed that compound 5 degraded the components of PRC2 complex (including EZH2, SUZ12, EED), downregulated the level of H3K27me3, and inhibited the traditional enzymatic function
dependent on PRC2.
In addition, it can effectively degrade cMyc and inhibit the non-enzymatic function of
EZH2.

Jian Jin's team developed MS8815 (7), a selective EZH2 degrader, to explore its role in
triple-negative breast cancer (TNBC) cells.
The experimental results showed that compound 7 achieved efficient degradation of EZH2 (DC50 = 140 nm)
in the breast cancer cell line MDA-MB-453 cells.
In addition, compound 7 has good antiproliferative activity
in multiple TNBC cell lines.
Thus, the development of compound 7 overcomes the limitation
that traditional catalytic site inhibitors target only the catalytic domain of EZH2 in TNBC cells.

2 PROTAC targeting HDAC6

Histone deacetylase 6 (HDAC6) is a microtubule-related member of the HDAC family, located primarily in the cytoplasm, involved in regulating the degradation, cell morphology, and migration
of misfolded proteins.
The abnormal regulation of HDAC6 is closely related
to cancer, neurodegenerative diseases, autoimmune diseases and other diseases.
Known HDAC6 selective inhibitors block the function
of enzymes by binding to the C-terminal catalytic domain of HDAC6.
However, due to the presence of multiple domains in HDAC6 (Figure 5), such as the C-terminal ubiquitin-binding domain (UBD), the N-terminal catalytic domain, and the zinc finger ubiquitin-binding domain (ZNF-UBP), as many as 45 HDAC6 inhibitors currently fail to target these functional domains
.
Therefore, there is an urgent need for a new R&D strategy
for HDAC6.

A variety of PROTACs targeting HDAC6 have been successfully reported (Figure 6).

Among them, compound 9 is PROTAC based on VHL ligand and compounds 12-14 are PROTAC based on CRBN ligand (compounds 13 and 14 are negative controls).

These PROTACs all exhibited significant HDAC6 degradation and inhibition of tumor cell growth
.
These degraders do this by inducing degradation of the entire target protein while blocking both enzymatic and non-enzymatic functions
of HDAC6.

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis, which plays a key role
in tumor metabolism and inflammation.
In addition to its function in the proliferation and differentiation of tumor cells, NAMPT affects the immune microenvironment
due to its cytokine-like effects.

NAMPT is called a "bilateral protein" and is divided into two types
: intracellular NAMPT (iNAMPT) and extracellular NAMPT (eNAMPT).
Studies have shown that NAMPT inhibitors only block their enzymatic functions and do not regulate non-enzymatic functions
through eAMPT.
Inhibition of the enzymatic function of NAMPT alone is not enough to completely inhibit the carcinogenic function
of AMPT.
Clinical trials of two NAMPT inhibitors (FK866 and CHS-828) were discontinued
due to limited antitumor efficacy and dose-dependent toxicity (e.
g.
, thrombocytopenia and gastrointestinal side effects).
Therefore, new strategies are urgently needed to interfere with the non-enzymatic function of
NAMPT.

Sheng Chunquan's research group reported that the first PROTAC 15 that can degrade NAMPT and reduce eNAMPT secretion (Figure 7), compound 15 directly degrades iNAMPT through the UPS pathway, thereby reducing the secretion of eNAMPT and promoting anti-tumor immunity, thereby blocking the enzymatic and non-enzymatic functions
of NAMPT.
In tumor mouse models, compound 15 also showed good inhibition
.

In addition, compound 15 can activate the immune response
with lower cytotoxicity and better pharmacokinetic properties.
The development of PROTAC 15 has led to a better understanding of the role of NAMPT's non-enzymatic functions in reconstructing the immunosuppressive tumor microenvironment, thereby facilitating the development of
NAMPT-targeted tumor immunotherapy methods.

1 PROTAC targeting the mitotic kinase AURORA-A

The mitotic kinase AURORA-A plays an important role in phosphorylation of various proteins during mitosis, and its catalytic activity is critical for the entire cell cycle, and AURORA-A is considered an important target for anti-cancer drug discovery
.
However, due to the low clinical response rate, the development of AURORA-A kinase inhibitors has been stagnant
.
Studies have shown that non-enzymatic functional regions of AURORA-A can bind to proto-oncogene proteins of the MYC family, so that N-MYC and C-MYC cannot be degraded
by proteasomes.
Also, AURORA-A kinase inhibitors are insufficient to eliminate the carcinogenic activity
of AURORA-A.

To explore the non-enzymatic functions of AURORA-A kinase, Wolf's group developed PROTAC JB170 by combining the AURORA-A clinical inhibitor alisertib17 with CRBN's E3 ligase ligand (Figure 8).

JB170 was found to induce rapid, effective and highly specific degradation
of AURORA-A.
The enzymatic activity of AURORA-A is thought to work primarily in the G2/M phase of the cell cycle, while its function in the S phase may be independent of
its enzymatic activity.
The authors found that compound 18 could induce strong S-phase blockade, but had no discernible effect
on cell aggregation in the G2/M phase.
In addition, the authors also experimentally demonstrated that the S phase of compound 18 block is mainly caused
by the non-enzymatic function of AURORA-A during DNA replication.

2 PROTAC targeting FAK

FAK is a cytoplasmic protein tyrosine kinase
that mediates the transduction of growth factor receptors and integrin-associated signals.
FAK consists of three main domains: the N-terminal FER domain, the central kinase domain, and the C-terminal local adhesion-targeting (FAT) domain (Figure 9).

While traditional FAK inhibitors only act on protein kinase domains to block enzyme function, non-enzymatic functions of FAK are also critical in cancer development and progression and cannot be blocked by reported FAK inhibitors
.
Therefore, inhibiting the kinase-dependent enzyme function and kinase-independent scaffold function of FAK at the same time is a new research direction
in the development of antitumor drugs.

Yale's Crews group has developed a targeting FAK PROTAC 19
.
Compound 19 is formed
by linking the FAK inhibitor defactinib (20) and the VHL ligand through the polyethylene glycol (PEG) of 1,2,3-triazole.
PROTAC 19 is selectively degraded at low nanomolar concentrations (DC50=3.
0nM, Dmax=99%)
.
In addition to affecting its kinase-dependent signaling activity, degradation of FAK also attenuates its kinase-dependent signaling
.
For example, since FAK-mediated cell motility is primarily controlled by kinase-independent pathways, removal of FAK significantly hinders the migration and invasion capacity
of TNBC cells.

Rao Liao's research group at Tsinghua University developed FAK PROTAC FC-11 (Figure 11), and further studied the non-enzymatic functions
of FAK on this basis.
Experimental results show that FC-11 can effectively degrade FAK
quickly.
At the same time, FC-11 can effectively degrade FAK in the reproductive system of mice and significantly downregulate the level
of phosphorylated FAK protein.

Law's research team designed a highly efficient and selective FAK degrader, GSK215
.
The researchers linked the VHL ligand and the clinical FAK inhibitor VS-471 through a rigid and short linkage chain
.
The compound GSK215 induced sustained degradation of FAK and prolonged PK/PD effect in mouse liver (Dmax was 85% within 18 hours, and FAK levels decreased by 60% after 96 hours of administration).

Notably, the researchers showed through structure-activity relationship (SAR) and X-ray crystallography that the compound's high degradation capacity stems from an unusually short and rigid linkage chain and produces a highly synergistic ternary
complex.

1 PROTAC targeting FKBP12

Pulmonary hypertension (PAH) is the most common and serious complication of congenital heart disease and a major problem in
the prevention and treatment of cardiovascular disease.
Recent studies have found that iron plays a crucial role
in the occurrence and development of PAHs.
Bone morphogenetic protein (BMP) is one of the key proteins in PAH and is involved in the expression
of hepcidin, a key protein that regulates the balance of iron metabolism in the human body.

FK506 binding protein 12 (FKBP12) binds to BMP type I receptors and subsequently inhibits hepcidin
.
This non-classical function of FKBP12 is similar to non-enzymatic function in that it regulates its interaction
with different components of the cell signaling pathway in a catalyst-independent manner.
The currently reported immunosuppressants rapamycin and FK506 block the binding of FKBP12 to BMP type I receptors, thereby increasing hepcidin
.
However, in clinical use, FKBP12 inhibitors exhibit immunosuppressive side effects
.

Rao's team reported PROTAC RC32 (Figure 13).

RC32 is formed
by linking rapamycin with pomalidomide.
In a mouse model, compound RC32 successfully upregulated
hepcidin gene expression by activating BMP signaling.
With rapamycin (24) or FK506 (25), the compound RC32 has no immunosuppressive activity
.
This work confirms the reliability
of PROTAC-mediated non-classical functional degradation of FKBP12 for the treatment of low hepcidin-related diseases.

2 PROTAC targeting USP7

As an important tumor suppressor factor, p53 is one of
the most frequently mutated genes in cancer.
The mutation rate of P53 in human cancer is greater than 50%.

The study found that the mutated p53 not only loses the regulation of normal biological functions of cells, but also inhibits the function of wild-type p53 proteins, resulting in cell cancer.

Therefore, the development of new drugs to treat p53-mutated cancers remains an urgent issue
.
However, mutations in p53 are relatively random, making it very difficult
to develop targeted drugs that directly target p53 mutants.

Ubiquitin-specific protease 7 (USP7) plays a key role
in regulating p53 content by deubiquitination and stabilization of MDM2.
Recently, Zhou Bing's research group at Shanghai Institute of Materia Medica, designed and synthesized the first-generation small molecule degrader U7D-1 (Fig.
14), which can effectively and selectively degrade USP7 (DC50=33 nM).

Compound U7D-1 exhibits inhibitory activity comparable to or stronger than USP7 inhibitors against the growth of p53 wild-type cancer cells, especially in p53 mutant cancer cells (Jeko-1 cells, IC), while USP7 inhibitor 27 shows weak activity
.
Further studies of the mechanism of action suggest that U7D-1 may induce USP7 degradation by regulating non-enzymatic functional regions of USP7 (apoptosis and E2F pathway), thereby exerting antitumor activity
against p53-mutant cancer cells.

In recent years, non-enzymatic functions of target proteins have attracted increasing attention
.
PROTAC has attracted attention because it can block both the enzymatic and non-enzymatic functions of the target protein and does not occupy the enzyme binding pocket for a long time to induce degradation of the whole protein
.

Current studies have shown that PROTAC shows good advantages in studying the non-enzymatic function of target proteins and exerts effective therapeutic effects
.
Compared with small molecule inhibitors, PROTAC has higher target selectivity, stronger efficacy, lower risk of drug resistance and prolonged effect
.

Therefore, PROTAC-mediated inhibition of enzymatic and non-enzymatic functions of drug targets can lead to a significant increase in drug activity, providing a new therapeutic strategy and providing a basis
for studying the non-enzymatic function of target proteins and the mechanisms of related diseases.
Although PROTAC's research on non-enzymatic functions is still in its infancy, more research is needed to discover more strategies to modulate non-enzymatic functions
of target proteins.
However, we still believe that using PROTAC to block the non-enzymatic function of proteins may become a new direction in drug development, and the future is bright
.

References

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       2、Proteolysis Targeting Chimeras (PROTACs) for Epigenetics Research.
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       3、Discovery of a Potent and Selective Degrader for USP7.
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       4、Targeting the Non-Catalytic Functions: a New Paradigm for Kinase Drug Discovery? J.
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(15) Wang, Y.
; Jiang, X.
; Feng,