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PROTACs and Targeted Protein Degradation
Curing malignant carcinomas is a grand ambition in the development of human
health. Over the past decades, targeted therapies have become one of the
most successful ways of achieving this. Of these approaches, small molecule
inhibitors and monoclonal antibodies are two major methods, however several
barriers to their development and clinical use still exist. PROTACs
(proteolysis targeting chimera) induced targeted protein degradation has
emerged as a novel therapeutic strategy in drug development and attracted
the favor of academic institutions, large pharmaceutical enterprises, and
biotechnology companies. PROTACs opened a new chapter for novel drug
development.

What is PROTAC?
Proteolysis targeting chimeras (PROTACs) are heterobifunctional small
molecules consisting of a ligand that binds to an E3 ligase, connected by a
linker to another ligand that binds to the protein of interest (POI). PROTAC is a
chemical knockdown strategy that degrades the target protein through the
ubiquitin-proteasome system. PROTACs' two covalently linked protein-binding
molecules can engage an E3 ubiquitin ligase and binds to a target protein
meant for degradation respectively. Recruitment of the E3 ligase to the target
protein results in ubiquitination and subsequent degradation of the target
protein by the proteasome.

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Image source: https://pubs.rsc.org/

Comparison of PROTAC and Other Therapeutics
The mechanism of action of PROTAC is different from traditional drugs. Most
small-molecule

inhibitors

and

antibody

drugs

are

competitive-

and

occupancy-driven process, that is, by binding to the active site of the target
protein, or by competing with the endogenous substrate to play the role of
inhibiting the function of the protein or enzyme. This has higher requirements
on the affinity between the drug and the target protein and the half-life of the
drug. Small-molecule interfering RNAs play their roles by gene silencing to
regulate genes. However, RNA has a short half-life and is easily degradable.
Although GalNAc-coupled RNA technology in recent years has successfully
achieved liver targeting and improved the escape rate of RNA, in general, the
delivery of RNA drugs is still a problem.
PROTAC is different from the above drugs. First of all, it does not necessarily
need to bind to the active site of the target protein to play a role, and it can
achieve the degradation of the target protein with moderate binding force to
the non-active site of the target protein. Studies have shown that when the

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binding force of PROTAC molecules to the target protein p38α reaches
Kd=11μM, degradation can be effectively achieved. Secondly, in the whole
process, PROTAC plays a role as a "matchmaker", matching between the E3
ligase and the target protein. After the target protein and E3 ligase are close to
each other and receive ubiquitination, the "matchmaker" will be released from
the two and continue to be the next pair of matchmaking. Therefore, PROTAC
degradation proteins have catalytic properties, and low doses can play a
role.

Intracellular target
Systemic administration
Tissue penetration
Target scaffold protein
Eliminate pathogenic proteins
Oral bioavailability
Catalytic properties
Route of administration

Traditional small
molecule drugs



×
×
×
PO/IV/SC

mAb

RNAi

PROTAC

×

weak

×
×
×
IV/SC



×

weak





×


IV/SC PO/IV/SC

The History of PROTACs
PROTACs technology was first proposed in 2001. The research group of
Crews and Raymond Deshaies synthesized the first batch of PROTACs
bifunctional molecules to induce the degradation of MetAP-2. The E3 ligase
recognition ligand is a decapeptide. This laid the foundation for the first
generation of PROTACs - PROTACs designed based on peptide fragments.
Following MetAP-2, Crews' research group has successively designed
PROTACs that target the degradation of MetAP2 and androgen receptors.
After microinjecting these molecules into cells, they found that they could
specifically bind to the target protein and degrade the protein. And they also
found that the target protein recognition ligand is not limited to inhibitors,
agonists can also play the role of degradation of the target protein. However, it
is not satisfactory that the first-generation PROTACs contain peptide
sequences, resulting in relatively high molecular weight, labile peptide bonds,
poor cell penetration, and low potency which was typically in the micromolar
range. These shortcomings make peptide-based PROTACs poor

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pharmaceutical candidates.
To avoid the weaknesses of peptide-based PROTACs, all
small-molecule-based PROTACs, in which E3 binding ligands are also small
molecules, were created. The first small-molecule PROTAC was reported in
2008. This PROTAC includes a non-steroidal androgen receptor ligand which
is a selective androgen receptor modulator (SARM), a MDM2 ligand known as
nutlin, and a PEG-based linker. The SARM-nutlin PROTAC triggered the
ubiquitination and degradation of androgen receptor. Cell-level experiments
show that the PROTAC molecule can effectively degrade androgen receptors
at a concentration of 10 μmol/L. Although the drug activity is not high,
compared with the first-generation PROTACs, the drug-forming property is
improved. In 2012, Crews' research group designed a series of hydroxyproline
derivatives based on HIF-α1. These molecules can bind E3 ligase VHL with
high affinity. The molecular weight of the optimized ligand was reduced to 400
Daltons, and the drugability was further improved. In 2013, the first discovery
of a PROTAC molecule that can efficiently degrade the target protein in the cell
at 1 nM. In 2015, Crews' research group and GSK developed a PROTAC
molecule that targets the degradation of serine/threonine protein kinases, and
uses a small molecule VHL as a recognition ligand for E3 ligase to degrade
target molecules at low nanomolar levels. In addition, the James Bradner team
found that thalidomide can bind to the E3 ligase CRBN, and they designed a
PROTAC molecule that targets the degradation of the BET family based on
this. These molecules have been shown to effectively degrade target proteins
at the level of humanized tumors in cells and mice. So far officially set off the
R&D boom of PROTACs.
Recently, light control PROTAC technology has also become a research
hotspot. Achieve the time-conditioning control of the PROTAC molecule by
manipulating the light. In 2019, an article on JACS successfully constructed
the light-regulated PROTAC molecule, which can effectively control the
degradation of Brd4 and BTK under 365nm light. In 2020, light-controlled
PROTAC molecules were designed in two back-to-back articles on Science
Advances. By adding a photosensitive group on the ligand, the degradation of
ALK fusion protein and BRD can be achieved under visible light/UV irradiation.

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Image source: Drug Discovery Today:Technologies

As of today, the research of PROTACs not only targets classic drug targets
(estrogen and androgen receptors, protein kinases, etc.) but also includes
transcription factors and protein skeletons. In the future, it is expected to play a
role in the treatment of various diseases, of which the treatment of cancer is
most worth looking forward to. In addition, PROTAC is also involved in virus
infection and immunity.

PROTAC Technology: Opportunities and Challenges

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PROTACs have opened a new chapter for the development of new drugs and
novel chemical knockdown tools and brought unprecedented
opportunities to the industry and academia, which are mainly reflected in the
following aspects:
1) Degrade the "undruggable" protein target.
At present, traditional small molecule drugs and antibody drugs can only work
on about 15% of protein targets, and the remaining 85% of protein targets
make scientists helpless and difficult to design effective drugs. The reason why
these targets are undruggable is mainly due to their structural limitations. Their
active sites and their nearby surfaces are smooth and flat, and they lack
pockets that are conducive to the binding of small molecules. Moreover, many
proteins are located inside the cell, and it is difficult for macromolecular drugs
to enter the cell. PROTAC is expected to break this deadlock thanks to its
unique mechanism of action. As mentioned above, the role of PROTAC is
different from that of traditional drugs that rely on the tight binding of molecules
and target proteins, which mainly depends on the ligand-induced proximity of
E3 ligase and target protein.
The currently recognized undruggable targets mainly focus on transcription
factors, protein backbones, and non-enzymatic proteins. For example, STAT3,
MYC, RAS, etc. STAT3 is a signal transduction and activation factor, lacking
binding pockets in the structure. In November 2019, Shaomeng Wang’s group
first reported a potent PROTAC targeting STAT3 with potent biological
activities in vitro and in vivo. This successful case confirms the key potential of
PROTAC technology, especially in the field of "undruggable" targets.
2) Overcoming drug resistance of cancer.
The emergence of targeted drugs prolongs the survival time of cancer patients
and improves the quality of life of patients, but these drugs can cause acquired
resistance problems, resulting in poor treatment of patients with the disease. A
part of the resistance comes from the mutations generated during the
treatment, such as EGFR produces T790 mutation, BTK produces C481S
mutation. Although drug developers are constantly designing a new generation
of targeted drugs for these resistance mutations, it is difficult for new
generation drugs to avoid the emergence of new drug resistance. The
characteristic of PROTACs degrading target protein provides a new idea for
solving the problem of drug resistance. Taking the example of overcoming
drug resistance caused by ibrutinib, 80% of patients with chronic lymphocytic
leukemia will develop C481S mutation after receiving ibrutinib treatment. In
2018, Crews' research group developed the PROTAC molecule MT-802,
which can effectively overcome the C481S resistance mutation produced by

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ibrutinib. In 2019, Rao Yi and other scholars designed a protein targeting BTK
based on CRBN ligands that can effectively degrade various clinical mutant
BTK proteins and overcome the resistance to ibrutinib caused by BTK
mutations.
3) Eliminating both the enzymatic and nonenzymatic functions of the
kinase.
PROTAC's ability to degrade proteins gives it the ability to clear target proteins,
which means it not only affects not only the enzymatic activity of the protein but
also nonenzymatic activity. For example, in addition to the kinase domain, the
FAK protein also has a scaffold region, which in addition to participating in the
exercise of FAK kinase activity, also affects the function of the plasma
membrane and plays an important role in the process of tumor invasion and
metastasis. In 2018, Crews's research group designed a highly efficient and
selective FAK degrading agent. Compared with devatinib, PROTAC molecules
can simultaneously clear the kinase activity and scaffold function of FAK,
which significantly reduces the ability of cancer cells to move.
Although PROTAC technology has a bright future in drug development, it also
has many challenges as follows:
1) Expansion of E3 ligase and its ligand.
The development of the second generation of PROTAC is based on the
discovery of small molecule E3 ligase ligands. But so far, the E3 ligase and E3
ligase ligands commonly used in PROTAC are few in number. The most
commonly used are CRBN and VHL, followed by MDM2 and IAP. But the
human genome encodes more than 600 kinds of E3 ubiquitin ligase, and there
is still a lot of room for the development of E3 ligase and its ligands.
2) Selection problem of PROTAC linkers.
The length of the linker and the site of attachment to the ligand will affect the
selectivity and degradability of PROTACs. An article published by Pfizer in
PNAS shows that BTK protein degradation can be achieved by regulating the
length of the linker. This may be due to the change in the linker resulting in the
change in the contact interface between the target protein and E3 ligase. In
addition, linker may affect PROTAC's cell permeability, tissue distribution,
metabolism/elimination. Therefore, it is very important to optimize the linker to
maximize the synergy with the ligand, but there is currently no theoretical basis
to guide the design. Based on the length and connection position of the
eutectic linker, it is possible to provide direction and basis for the linker design.

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The commonly used linkers in the development of PROTACs are PEGs,
Alkyl-Chain and Alkyl/ether. Biochempeg provides multi functionalized PEG
derivatives as PROTAC linkers.
3) Hook Effect
The formation of ternary complex is the key to PROTAC degradation of target
protein, but the Hook effect will occur when the concentration of PROTAC
molecule is high. PROTAC can interact with E3 ligase ligand and target protein
ligand to form a binary complex, resulting in a decrease in degradation activity.
Therefore, the drug concentration of PROTAC should be regulated.
4) Druggable problem of PROTAC.
For traditional small molecule drugs, the five principles of drugability can be
used to make a preliminary judgment, but for PROTAC, this rule is no longer
applicable. PROTAC is equivalent to a dual-target drug. The molecular weight
of the drug is larger than that of ordinary small molecule drugs, which can be
more than 700 Daltons, and some can even exceed 1000 Daltons, which may
affect the water solubility and permeability of the molecules. However, some
studies have shown that PROTAC has excellent solubility and can even cross
the blood-brain barrier.
In addition, traditional PK and PD properties may not be suitable for PROTAC.
The catalytic properties of PROTAC and the relatively complex triplet structure
may make the tissue distribution not follow the traditional pH distribution theory,
and there may be target-mediated drug disposal (TMDD) phenomenon that
leads to the complexity of PK/PD. However, there are few relevant in vivo tests,
and the PKPD model for PROTAC has not been established.
5) Safety
Off-target is the biggest concern about PROTAC's safety. The mechanism of
action of PROTAC is to use the ubiquitin-proteasome pathway to clear proteins.
If non-target proteins are unfortunately cleared, serious adverse reactions may
occur. And experiments have proved that the PROTAC molecule based on
CRBN can induce the degradation of zinc finger protein.
In addition, ubiquitination markers not only play a role in protein degradation,
but also participate in methylation, acetylation, and phosphorylation processes.
Whether the removal of the target protein will create new safety hazards, and
whether the cells can still perform normal physiological functions require our
attention.

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All in all, the PROTAC molecule looks modular, but it is not simple to design a
drugable PROTAC molecule. The three components need to be continuously
optimized to achieve the best combination. In addition, the adverse reactions
that occur in preclinical studies should be closely monitored. Basic research on
E3 ligase is expected to help PROTAC's drug development.
Reference:
PROTACs: great opportunities for academia and industry
PROTAC Technology: Opportunities and Challenges
The PROTAC technology in drug development


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