A new technology that combines covalent inhibitors and PROTACs has emerged—covalent PROTAC technology, including reversible covalent and irreversible covalent PROTACs.

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Covalent PROTACs, An Emerging Protein
Degradation Technology
Covalent inhibitors and PROTACs are two forms of small molecule drugs with
great development potential, which have been extensively and intensively
studied. PROTAC technology has the potential advantages of catalyzing
degradation, expanding the target range and overcoming drug resistance, and
is widely used in chemical biology and new drug development. However,
limited E3 ligase ligands and high affinity requirements for POIs limit the range
of POIs that PROTACs can target. In recent years, a new technology that
combines the two has emerged—covalent PROTACs technology, including
reversible covalent and irreversible covalent PROTAC. Covalent PROTAC
technology combines the dual theoretical advantages of covalent inhibitors
and PROTACs, and is expected to help overcome the above shortcomings of
PROTACs and further increase the upper limit of the application of PROTACs.
1. PROTAC, A Potential New Drug Development Technology
Proteolysis Targeting Chimeras (PROTAC) was first proposed by Crews et
al. in 2001, which can induce the degradation of protein of interest (POI)
through the ubiquitin degradation pathway. PROTAC consist of three parts: a
“warhead” ligand binds to POI, a ligand for E3 ligase (E3 ligase binder), and a
linker that bridges the two.
PROTAC can recruit E3 ligase and POI to form a ternary complex so that POI
can be recognized and degraded by the proteasome after ubiquitination. So far,
more than 100 target proteins including kinases, nuclear receptors and
epigenetic target-related targets have been successfully degraded. Compared
with traditional small-molecule inhibitors, it greatly expands the range of
druggable protein targets, and after degrading the target protein, all its
functions can be eliminated until the protein is resynthesized. In addition,
PROTAC acts in the way of degrading target proteins, which can minimize
potential drug resistance, and can be recycled to take effect under catalytic
dose, thus improving drug safety. It is one of the most popular modality in the
pharmaceutical field at present.
As of March 22, 2023, 25 PROTAC molecules have advanced to clinical trials,
and ARV-471 (Vepdegestrant), which has the fastest progress, has launched a
phase III clinical trial for breast cancer. PROTAC expands the range of
druggable targets and is a potential new drug development technology.

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Table 1. Clinical progress of PROTAC technology
2. Covalent PROTACs Mainly Covalently Bound To Cysteines
On Proteins

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PROTAC molecules, like traditional small-molecule inhibitors, can also be
divided into reversible non-covalent PROTAC, irreversible covalent PROTAC
and reversible covalent PROTAC. Among them, reversible non-covalent
PROTAC is the one that has been studied intensively. This is mainly because
irreversible covalent PROTAC cannot regenerate after target protein
degradation and loses the catalytic function of PAOTAC. Therefore, the
degradation effect is not as good as that of reversible non-covalent PROTAC.
Currently, all PROTACs in clinical trials are reversible and non-covalent, while
preclinical research on covalent PROTACs is more active. As the name
implies, covalent PROTAC means that the PROTAC molecule can be
covalently bind to the protein, which can produce stronger binding ability and
induce more protein degradation. The material basis of covalent binding
comes from the electrophilic warheads (such as acrylamide and
cyanoacrylamide) on PROTAC molecules and nucleophilic residues (such as
cysteine and lysine) on proteins, which undergo nucleophilic addition or
nucleophilic substitution reactions to form covalent bonds, similar to covalent
inhibitors. The covalent PROTAC in the present study mainly binds to cysteine
residues (Cys) on the protein to help form ternary complexes of
POI-PROTAC-E3 ligases.
Figure 1. The principle of covalent bond
Compared with non-covalent PROTACs, covalent PROTACs combine the dual
theoretical advantages of covalent inhibitors and PROTACs, which can not
only bind POI with high affinity, but also efficiently catalyze the degradation of
POI and can target more non-patent proteins. More and more research is
being devoted to the development of covalent PROTACs. Although covalent
binding to POIs may cause PROTACs to lose their catalytic properties, it is still

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of great benefit to the development of new PROTACs. In short, covalent
binding can degrade POIs due to lack of well-defined binding pockets or
excessive affinity for endogenous ligands, while reversible covalent PROTACs
are expected to restore the catalytic properties of PROTACs, allowing them to
remain active after one round of POI degradation induction. It can be recycled
again to improve the degradation efficiency of PROTAC molecules.
3. Development of Covalent PROTACs
The significant advantages of covalent PROTACs have attracted many
scientists to explore the possibility of covalent PROTACs. The first PROTAC
molecule reported by Professor Crews in 2001 used ovalicin to bind MetAP-2
(an E3 ligase), which was the first proof-of-concept of a covalent PROTAC.
Figure 2. A brief timeline of covalent drug discovery and PROTAC
developments
Source: References [2]

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(1). HaloTag (HT) technology. In 2015, Professor Crews et al. successfully
introduced HaloTag (HT) technology developed by Promega Company into
PROTAC and developed HaloPROTAC series compounds. HT is an
engineered bacterial dehalogenase that can be covalently bonded to
chlorinated alkanes. HaloPROTAC consists of VHL ligands conjugated to
chlorinated alkanes, which induce the degradation of HT fusion proteins. This
is also an example of a really successful covalent PROTAC.
(2). Bioorthogonal reaction. Due to the large molecular weight of PROTAC,
some researchers hope to form a complete PROTAC molecule through click
chemistry in cells through two prodrugs. The Heightman team developed
ERK-CLIPTAC 6 using ERK1/2 covalent inhibitors. Although this trial
demonstrates that intracellular click-formed PROTACs (CLIPTACs) can
overcome the problem of poor membrane permeability of PROTACs, in
practice CLIPTACs require separate clinical trials of the 2 chemical entities,
making such PROTACs less clinically viable.
(3). Targeting mutant proteins. In 2020, the KRASG12C degrader LC-2
developed by the Crews team based on the KRASG12C covalent inhibitor
MRTX849 can effectively degrade endogenous KRASG12C mutant proteins,
and the DC50 in different tumor cell lines is only 250-590 nM.
4. PROTACs Covalently Bound to POI
In fact, the first PROTAC molecule reported by Crews et al. in 2001 was a
covalent PROTAC, which used ovalicin, a covalent inhibitor of methionine
aminopeptidase-2, as the POI ligand, and the other end was a phosphopeptide
ligand targeting the F-box protein SKP2, successfully inducing degradation of
methionine aminopeptidase-2. However, most of the PROTAC molecules
reported later are non-covalent, because covalent binding may cause
PROTAC molecules to lose their catalytic properties. According to the
research of GlaxoSmithKline Tinworth et al., the irreversible covalent PROTAC
targeted by BTK (with acrylamide as the electrophilic warhead) cannot
degrade BTK protein, but non-covalent PROTAC after reducing the double
bond of acrylamide can efficiently degrade BTK protein. In addition, Dong Lu
and others from Baylor College of Medicine in the United States also came to
similar conclusions. However, based on the advantages of covalent binding
with high affinity and targeting difficult-to-drug pockets, covalent PROTACs are
still under continuous research.

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Figure 3. Structure of irreversible covalent PROTAC
Figure 4. Structure of reversible covalent PROTAC

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According to the covalent PROTAC activity data summarized by Nir London et
al. (Table 2), it can be found that the degradation efficiency of reversible
covalent PROTAC is generally stronger than that of irreversible covalent
PROTAC and non-covalent PROTAC, but in fact the greatest potential of this
type of molecule is still lies in the ability to degrade POI without the need for
effective POI ligands, and its potential to enhance degradation selectivity.
Table 2. Potency Comparison of Covalent PROTACs and Similar
Non-covalent PROTACs
5. PROTACs Covalently Bound to E3 Ligase
So far, the E3 ligases used for targeted protein degradation are still only a very
small part of the E3 ligase family, such as CRBN, MDM2, and VHL, and mainly
use non-covalent E3 ligase ligands. Compared with PROTAC molecules
covalently bound to POI, PROTAC covalently bound to E3 ligase has more
advantages in degradation efficiency. After POI is ubiquitinated and degraded
by proteasome, the covalent complex of E3 ligase-PROTAC can directly
participate in the next round of POI binding, simplifying the original process of
forming a ternary complex to forming a binary complex, accelerating the next
round of protein degradation.
Several PROTACs that covalently bind E3 ligases have been developed. The
targeted E3 ligases include RNF4, RNF114, KEAP1, DCAF16, FEM1B, etc.,
and the degradation of BRD4, ERRα, BCR-ABL, FKBP12, CDK9, ALK and
other POI has been successfully achieved at the cellular level, with the
degradation efficiency up to 94%. But in general, the efficacy of PROTAC
using non-covalent CRBN/VHL ligand is still inferior to that of Protac. Their
degradation efficiency and selectivity still need to be further optimized.

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Figure 5. PROTACs covalently bound to E3 ligase
Conclusion
As a branch of PROTAC technology, the research on covalent PROTAC is still
in its infancy. Covalent binding can improve the affinity with proteins, and is
expected to realize the targeting of difficult-to-drug proteins and expand the
range of potential targets. When combined with PROTAC technology, it is
expected to develop better protein degrader. Judging from the literature, most
of the covalent degrader reported so far are PROTACs, among which
reversible covalent PROTACs have demonstrated efficacy and safety
advantages over non-covalent PROTACs in preclinical trials, but their in vivo
activity needs to be further demonstrated.
Some key problems of covalent PROTAC need further study. The first is the
role of covalent bonds in POI degradation. Secondly, because some covalent
PROTACs are consistent in their ability to degrade wild-type and cysteine
mutant POIs, some covalent PROTACs may act through non-covalent

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mechanisms. Considering the complex mechanism of covalent PROTAC, it is
necessary to establish a more accurate and effective platform to analyze the
covalent bond formation and POI degradation kinetics.
In general, covalent PROTACs represent a class of emerging targeted protein
degradation technologies with great application potential, and it is worthy of
further extensive and in-depth research to find candidate compounds for
clinical application as soon as possible.
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References:
[1] Gabizon R, London N. The rise of covalent proteolysis targeting chimeras. Curr Opin
Chem Biol. 2021 Jun;62:24-33.
[2] Kiely-Collins H, Winter GE, Bernardes GJL. The role of reversible and irreversible
covalent chemistry in targeted protein degradation. Cell Chem Biol. 2021 Jul
15;28(7):952-968. doi: 10.1016/j.chembiol.2021.03.005. Epub 2021 Mar 30. PMID:
33789091.
[3] Lu D, Yu X, Lin H, Cheng R, Monroy EY, Qi X, Wang MC, Wang J. Applications of
covalent chemistry in targeted protein degradation. Chem Soc Rev. 2022 Nov
14;51(22):9243-9261.
[4] Grimster NP. Covalent PROTACs: the best of both worlds? RSC Med Chem. 2021 Jul
15;12(9):1452-1458.
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