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Covalent PROTACs, An Emerging Protein Degradation Technology

Release time:2023/4/10 17:26:37
Author:Huateng Pharma

In recent years, a new technology that combines covalent inhibitors and PROTACs has emerged—covalent PROTAC 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

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.


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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 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.

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Figure 2. A brief timeline of covalent drug discovery and PROTAC developments
Source: References [2]

(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

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Figure 4. Structure of reversible covalent PROTAC

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.


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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


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 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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[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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