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Abbreviation (ISO4): Prog Chem      Editor in chief: Jincai ZHAO

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Progress in Chemistry >

2025 , Vol. 37 >Issue 2: 185 - 194

DOI: https://doi.org/10.7536/PC240202

Review

Bifunctional Small Molecules for Targeted Protein Degradation

  • Zuyi Huang ,
  • Xueqiang Tan , * ,
  • Jimin Zheng , *
Expand
  • Department of Chemistry, Beijing Normal University, Beijing 100091, China
* e-mail: (Xueqiang Tan);
(Jimin Zheng)

Received date: 2024-02-05

  Revised date: 2024-08-15

  Online published: 2025-02-07

Supported by

National Natural Science Foundation of China(21773014)

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Abstract

Bifunctional small molecules are a sort of small molecules that engage multiple targets. They are subdivided into two categories: bifunctional small molecules with linkers and without linkers. Targeted protein degradation (TPD) is a currently emerging strategy hijacking cellular protein degradation systems, namely ubiquitin-proteasomal system and lysosomal system, to induce the degradation of targeted protein for drug development. Distinct from the traditional mechanism of action based on inhibition, TPD inhibits the function of targeted protein through targeted clearance, thus is advantageous in long-term inhibition and targeting undruggable proteins. With a unique mechanism of action, bifunctional small molecules are capable of binding degradation-associated protein and targeted protein simultaneously, and therefore used widely in the realm of TPD. This review summarizes the recent development of bifunctional molecules in TPD. Proteolysis targeting chimeras (PROTACs), molecular degraders of extracellular proteins through the asialoglycoprotein receptors (MoDE-As), and autophagy targeting chimeras (AUTACs) which based on bifunctional small molecules with linkers, and molecular glue degraders (MGDs) and autophagosome-tethering compounds (ATTECs) which based on bifunctional small molecules without linkers are introduced, with their clinical application highlighted. Finally, the challenges that the two categories of bifunctional small molecules respectively face in the realm of TPD as well as prospects and suggestions for their development are proposed.

Contents

1 Introduction

2 Bifunctional small molecules with linkers for TPD

2.1 PROTACs

2.2 AUTACs

2.3 MoDE-As

2.4 Challenges for bifunctional small molecules with linkers in TPD

3 Bifunctional small molecules with linkers for TPD

3.1 MGDs

3.2 ATTECs

3.3 Rational design strategy for bifunctional small molecules without linkers

4 Conclusion and outlook

Key words： small-molecule drugs; bifunctional small molecules; targeted protein degradation; cellular protein degradation system

Cite this article

Zuyi Huang , Xueqiang Tan , Jimin Zheng . Bifunctional Small Molecules for Targeted Protein Degradation[J]. Progress in Chemistry, 2025 , 37(2) : 185 -194 . DOI: 10.7536/PC240202

1 Introduction

Bifunctional molecules are a class of molecules capable of simultaneously interacting with multiple targets. Such molecules are widely present in nature, with scaffold proteins being a typical example. Scaffold proteins can bind to both enzymes and their substrates at the same time, greatly enhancing enzymatic reaction rates by inducing co-localization of the two, increasing efficiency thousands of times[1-2]. Due to their unique properties, bifunctional molecules have become an important strategy in drug design. Bispecific antibodies[3] and antibody-recruiting molecules[4] fall within the category of bifunctional molecules. Bifunctional small molecules are small compounds possessing bifunctional characteristics, which can be divided into two categories based on whether their structure contains a linker: linker-containing bifunctional small molecules and linker-free bifunctional small molecules. Linker-containing bifunctional small molecules are typically composed of two ligands connected via a covalent linker, and their structures clearly reflect their bifunctional characteristics, as shown in Figure 1a. The bifunctional characteristics of linker-free bifunctional small molecules are less obvious compared to the former, and their bifunctionality often depends on the compound-protein interface formed with one of the interacting targets, as illustrated in Figure 1b.
图1 （a）含连接子双功能小分子与（b）无连接子双功能小分子作用机制示意图

Fig. 1 Schematic diagrams of bifunctional small molecules (a) with linkers and (b) without linkers

The ubiquitin-proteasome system (UPS) and the lysosomal system are the two major protein degradation systems in eukaryotic cells. The proteasome is a giant protein complex with various proteolytic enzyme activities[5], and ubiquitin is a small molecule protein composed of 76 amino acids with a molecular weight of approximately 8.5 kDa. Proteins to be degraded can be covalently modified by ubiquitin under the catalysis of ubiquitin ligase (E3), thereby being recognized and degraded by the proteasome[6]. The lysosome is a vesicle-like organelle with a single-layer membrane structure containing more than 60 types of acidic hydrolases such as lipase, protease, and glycosidase[7]. The lysosome has three transport pathways: endocytosis, phagocytosis, and autophagy[8]. Extracellular proteins and membrane proteins are transported to the lysosome for degradation via endocytosis, while intracellular proteins are transported to the lysosome for degradation via autophagy[9].
Targeted protein degradation (TPD) is an emerging drug development strategy that achieves the targeted degradation of proteins by hijacking eukaryotic cell protein degradation pathways. TPD drugs typically act as catalysts in mediating protein degradation[10], without the need for prolonged occupation of the protein's active site, thus enabling the targeting of undruggable proteins such as transcription factors[11]. On the other hand, since the target protein needs to be resynthesized after being degraded, TPD drugs can produce a more sustained inhibitory effect compared to traditional inhibitor drugs[12]. Bifunctional small molecules, due to their ability to interact with multiple targets, can bind to the target protein while simultaneously hijacking degradation mechanism-related proteins, and have the advantages of oral bioavailability and membrane permeability, making them a major class of TPD drugs. This article categorizes the main TPD bifunctional small molecules based on whether they contain a linker structure, summarizing their development history, recent research progress, and clinical applications, and provides an outlook on the future development of these technologies. Finally, this article discusses the challenges faced by two classes of bifunctional small molecules when used for targeted protein degradation and offers corresponding prospects and suggestions for their development.

2 Bifunctional Small Molecules with Linkers for Targeted Protein Degradation

2.1 Protein Degradation Targeting Chimeras

PROTACs are bifunctional small molecules with linkers in TPD, which were first developed by the Crews and Deshaies group in 2001. They consist of an E3 ubiquitin ligase ligand, a target protein ligand, and a linker connecting the two ligands. PROTACs bind to E3 and the target protein respectively through the two ligands, forming an E3-PROTAC-target protein ternary complex, which induces the target protein to be ubiquitinated by E3, thereby degrading it via the proteasome.
The first PROTAC developed by the Crews and Deshaies groups[13] targeted methionine aminopeptidase 2 (METAP2), named PROTAC-1 (Fig. 2a). PROTAC-1 utilized ovalicin as METAP2 and phosphopeptide IκBα as the ubiquitin ligase β-TrCP ligand, successfully achieving METAP2 degradation, confirming the feasibility of using bifunctional molecules for targeted protein degradation, and laying the foundation for the development of PROTACs and other TPD bifunctional molecules. In 2002, the peptide hypoxia-inducible factor-1α (HIF-1α) was identified as a ligand for the ubiquitin ligase VHL[14]. Building on this, in 2004, VHL-PROTACs were developed using the HIF-1α and VHL binding peptide as the E3 ligand[15], as shown in Fig. 2b, revealing the feasibility of applying novel ubiquitin ligases to PROTAC development. In the same year, the cis-imidazoline analog nutlins were identified as ligands for the ubiquitin ligase MDM2[16]. Using nutlins as the E3 ligand and a polyethylene glycol-based linker connected to selective androgen receptor modulators (SARM), the Crews group[17] developed the SARM-nutlin PROTAC targeting non-steroidal androgen receptors in 2008 (Fig. 2c), verifying its ability to mediate androgen receptor-targeted degradation and confirming the feasibility of using linker-containing bifunctional small molecules for TPD. This group also improved VHL-PROTACs based on the VHL ubiquitin ligase ligand HIF-1α, developing small-molecule VHL-PROTACs with HIF-1α mimetic peptide small molecules as the E3 ligand[18]. With the discovery of more small-molecule E3 ligands, ubiquitin ligases such as cIAP[19] (Fig. 2d), CRL4CRBN[20], DCAF15[21], DCAF16[22], RNF114[23], and RNF4[24] have also found applications in PROTACs.
图2 （a）PROTAC-1，（b）VHL-PROTAC，（c）SARM-nutlin PROTAC，（d）cIAP-PROTAC的化学结构

Fig. 2 Chemical structures of (a) PROTAC-1, (b) VHL-PROTAC, (c) SARM-nutlin PROTAC, (d) cIAP-PROTAC, ligands for E3s are marked in red

The action of PROTACs in targeted protein degradation can be used to eliminate cancer-related proteins, thus making cancer treatment the main direction of PROTACs research. Currently, various orally active PROTACs for cancer treatment have entered clinical trials. ARV-471, introduced by Arvinas, is the first PROTACs drug to enter clinical trials and can treat patients with locally advanced/metastatic breast cancer by targeting the degradation of estrogen receptors[25], and it has now progressed to phase III clinical trials. Other PROTACs drugs that have entered clinical trials include KT-333, developed by Kymera, which targets the transcription regulator STAT3 for treating T-cell malignancies and solid tumors[26], and KT-253, which targets MDM2 for treating r/r advanced myeloid malignancies and solid tumors[27]; CFT8634, developed by C4 Therapeutics, which targets BRD9 for treating synovial sarcoma, and CFT8919, which targets the L858R mutation in EGFR for treating non-small cell lung cancer.
In addition, PROTACs can also be applied to the treatment of neurodegenerative diseases, immune diseases, cardiovascular and cerebrovascular diseases, etc.[28]. The abnormal phosphorylation of microtubule-associated protein tau is an important inducement of tauopathies such as Alzheimer's disease. The PROTAC molecule based on VHL ubiquitin ligase, C004019, has shown the ability to target and clear tau protein in both in vitro and in vivo experiments, and is expected to be used for the treatment of tauopathies[29]; PROTAC(H-PGDS)-1 can target hematopoietic prostaglandin D synthase and can be used for the treatment of allergic diseases and Duchenne muscular dystrophy[30]; HMGCR-PROTAC can target HMGCR, the rate-limiting enzyme of the mevalonate pathway, limiting cholesterol synthesis, thereby treating atherosclerotic dyslipidemia[31]. The emergence of PROTACs provides a new option of targeted protein degradation for the treatment of these diseases.

2.2 Autophagy-Targeting Chimeras

AUTACs are linker-containing bifunctional small molecules composed of an autophagy-targeting ligand and a target protein ligand connected via a linker, achieving targeted degradation of the target protein by hijacking lysosome selective autophagy. Selective autophagy can be divided into ubiquitin-dependent and non-ubiquitin-dependent selective autophagy based on whether the specific recognition of cargo is achieved through ubiquitination labeling[32]. As shown in Figure 3, during the selective autophagy process, the cargo can be recognized by the selective autophagy receptor p62 and form liquid-like biomolecular condensates with p62 that induce phagophore formation[33]. The phagophore can recruit microtubule-associated protein light chain 3 (LC3), and the p62 bound to the cargo interacts with LC3 localized on the phagophore[34], allowing the biomolecular condensates formed by p62 and the cargo to be selectively isolated by the autophagosome extended from the phagophore. Finally, the autophagosome fuses with the lysosome, degrading the cargo within the lysosome. In the above process, p62 and LC3 play important roles in the selection of autophagic cargo; therefore, AUTACs can induce lysosomal degradation of target proteins by hijacking such proteins.
图3 自噬机理示意图

Fig. 3 Schematic diagram of autophagy

In 2019, the Arimoto group[35] developed AUTACs using para-fluorobenzylguanine (FBnG) as an autophagy-targeting ligand based on the process of pathogen autophagy. The group's earlier work discovered that during pathogen autophagy, endogenous 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) can react with cysteine residues on bacterial surface proteins through S-guanylation[36]. Experiments on Group A Streptococcus (GAS) found that this modification promotes K63 polyubiquitination on the bacterial surface, indicating that S-guanylation may play a role in recruiting selective autophagy tags during selective autophagy. In summary, 8-nitro-cGMP is an endogenous autophagy inducer. Building on this, the Arimoto group connected FBnG to the target protein ligand via a polyethylene glycol linker, developing FBnG-AUTACs. After binding to the target protein, the FBnG moiety can mimic the function of endogenous protein S-guanylation modification, inducing lysosomal degradation of the target protein. The group designed AUTAC1-3 molecules targeting intracellular proteins MetAP2, FKBP12, and BRD4, verifying the targeted protein degradation capability of AUTACs and confirming the feasibility of inducing lysosomal targeting degradation of target proteins using bifunctional small molecules containing linkers.
Moreover, the Arimoto group[37] further investigated the structure-activity relationship of the cysteine substructure and degradation tag of FBnG-AUTACs, as shown in Figure 4. The results indicated that the FBnG degradation tag did not require optimization. On the other hand, optimizing the cysteine substructure to a pyrazole structure significantly enhanced the activity of FBnG-AUTACs, enabling them to exhibit the highest targeted degradation activity at concentrations less than 1 μmol·L-1. Therefore, the group defined FBnG-AUTACs with the pyrazole substructure as second-generation FBnG-AUTACs.
图4 第一代FBnG-AUTACs与第二代FBnG-AUTACs的化学结构

Fig. 4 Chemical structures of first generation FBnG-AUTAC and second generation FBnG-AUTAC

In 2019, Li et al[38] discovered linkerless bifunctional small molecules that interact with LC3 and mutant Huntington protein (mHTT) through high-throughput screening methods for the development of Huntington's disease (HD) therapies (see 3.2). Building on this, in 2021, Pei et al[39] used these small molecules as LC3 ligands, connected them to BRD4 ligand JQ1 via a polyethylene glycol-based linker, designed linker-containing bifunctional small molecules LC3-AUTACs, and experimentally verified the targeted degradation effect of LC3-AUTACs. In 2023, Dong et al connected the LC3 ligand ispinesib with nicotinamide phosphoribosyltransferase (NAMPT) inhibitor via a polyethylene glycol linker, designed LC3-AUTACs targeting NAMPT, and conducted a structure-activity relationship study on the length of the polyethylene glycol linker[40-41].
In 2022, Kwon et al[42] developed p62-AUTACs, which achieve lysosomal targeting degradation of target proteins by hijacking the selective autophagy receptor p62. In their early work, Kwon et al found that proteins with specific N-terminal residues can bind to the zinc finger domain of p62 and switch p62 to an activated conformation. This transformation can accelerate p62 oligomerization through disulfide bonds and promote the transfer of p62 and its cargo to autophagosomes via the interaction between p62 and LC3[43]. Therefore, Kwon et al hypothesized that bifunctional molecules capable of activating p62 while binding to target proteins could induce lysosomal degradation of the target proteins. Based on the presumed binding pocket of the zinc finger domain in the full-length 3D structural model of p62, Kwon et al conducted a virtual screening of a database containing 540,000 compounds and identified two small molecules with potential binding affinity for the p62 zinc finger domain. They experimentally demonstrated the oligomerization property of p62 after activation. On this basis, through 3D modeling and structure-activity relationship studies, Kwon et al further optimized and obtained p62 zinc finger domain ligands such as YT-8-8, YOK-1304, YOK-2204, and YTK-105 (Figure 5). By linking the aforementioned ligands to the target protein ligands using polyethylene glycol linkers, they obtained the bifunctional small molecules with linkers, p62-AUTACs. Kwon et al designed p62-AUTACs targeting ERβ, AR, and MetAP2, respectively, and validated their targeted degradation capabilities.
图5 p62锌指结构域配体化学结构

Fig. 5 Chemical structure of p62 ZZ domain ligand

In neurodegenerative diseases such as Alzheimer's disease, misfolded tau proteins and marker proteins like HTT tend to form degradation-resistant protein aggregates, leading to their accumulation within cells. Targeting the highly aggregative P301L tau mutant (tauP301L) and mutant HTT (mHTT), Kwon et al. utilized 4-phenylbutyric acid (PBA), which can selectively recognize hydrophobic domains in misfolded proteins, and Anle138b, which can selectively identify protein aggregates associated with neurodegenerative diseases, as target protein ligands connected to p62 ligands, achieving the targeted clearance of tauP301L and mHTT by p62-AUTACs. Therefore, p62-AUTACs have significant potential for application in clearing pathological proteins related to neurodegenerative diseases. Kwon et al.[44] further applied p62-AUTACs to the study of therapies for Parkinson's disease (PD). The p62-AUTACs based on Anle138b successfully achieved the targeted degradation of pathologic aggregates of α-synuclein, a key pathological marker of PD, and verified this effect in PD mice.

2.3 ASGPR-Based Small Molecule Extracellular Protein Degraders

MoDE-As were developed by the Spiegel group and consist of a tri-N-acetylgalactosamine (tri-GalNAc) linked to a target protein ligand through a linker, forming a bifunctional small molecule[45]. MoDE-As achieve targeted protein degradation by hijacking receptor-mediated endocytosis[46]. As shown in Figure 6, specific receptors on the cell surface bind with cargo bearing corresponding ligands to form complexes that internalize into transport vesicles. These vesicles then fuse with endosomes, forming large vesicles with acidic contents[47]. In the acidic environment, the cargo separates from the receptor, with the cargo being transported to lysosomes for degradation while the receptor is recycled back to the cell surface. Lysosome-targeting receptor proteins (LTRs) are a class of receptors involved in protein endocytosis, capable of recognizing specific glycan modifications on glycoproteins, thereby directing glycoproteins to lysosomes for degradation[48]. ASGPR is a lysosome-targeting receptor protein specifically expressed in the liver, which can recognize glycoproteins labeled with galactose or N-acetylgalactosamine (GalNAc). MoDE-As use tri-GalNAc as an asialoglycoprotein receptor (ASGPR) ligand to induce the targeted degradation of the desired protein via endocytosis to lysosomes[49]. As shown in Figure 6, the Spiegel group designed D-MoDE-A targeting α-DNP antibodies and M-MoDE-A targeting macrophage migration inhibitory factor (MIF) (Figure 7), verifying the occurrence of endocytosis and the targeted degradation of the target protein in vitro. They also confirmed the ability of D-MoDE-A and M-MoDE-A to degrade corresponding target proteins in mice within a mouse model.
图6 受体介导的内吞作用机理示意图

Fig. 6 Schematic diagram of receptor mediated endocytosis

图7 MoDE-As的化学结构。tri-GalNAc和目的蛋白配体分别用红色和蓝色标记

Fig. 7 Chemical structures of MoDE-As. Tri-GalNAc and ligands for target proteins are respectively marked in red and blue

2.4 Challenges Faced by Bifunctional Small Molecules with Linkers in TPD

Bifunctional small molecules with linkers, due to their relatively complex ligand-linker-ligand structure, have larger molecular weights, which are not conducive to their oral bioactivity and pharmacokinetic properties such as absorption, distribution, metabolism, and excretion[50]. Therefore, it is necessary to develop and summarize rational optimization principles and experiences for bifunctional small molecules with linkers in order to improve their drug-like properties[51-52]. In addition, the ternary system formed by the bifunctional small molecules with linkers, degradation mechanism-related proteins, and target proteins may exhibit a hook effect at higher concentrations of bifunctional molecules, potentially leading to reduced activity[53]. Developing design strategies to mitigate or overcome the hook effect can facilitate the development of TPD drugs based on bifunctional small molecules with linkers[54].

3 Linkerless Bifunctional Small Molecules for Targeted Protein Degradation

3.1 Molecular Glue Degraders

MGDs are a class of linkerless bifunctional small molecules that can simultaneously interact with ubiquitin ligase E3 and target proteins, promoting the ubiquitination of target proteins and thereby inducing their degradation through the proteasome. Immunomodulatory drugs (IMiDs) are the earliest discovered and main class of MGDs. In 2010, in studies on the teratogenicity of the IMiD thalidomide, Hiroshi Handa's group found that thalidomide could bind to CRBN, which acts as a substrate adapter in the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex[55-56]. In 2014, Fischer et al.[57] crystallized DDB1-CRBN complexes with thalidomide and its derivatives lenalidomide and pomalidomide, and analyzed the crystal structures, confirming the ability of CRL4CRBN to bind IMiDs. The anti-proliferative and immunomodulatory effects of IMiDs are considered to be related to IMiD-mediated ubiquitination and degradation of the transcription factors IKZF1 and IKZF3 by CRL4CRBN[58].
In the field of cancer treatment, MGDs drugs have great application potential[59]. The immunomodulatory drugs thalidomide, lenalidomide, and pomalidomide have been approved by the US Food and Drug Administration (FDA) for clinical treatment of multiple myeloma (MM) targeting IKZF1/3 degradation[60]. On the other hand, CRBN modulator drugs (CELMoDs) based on CRBN ubiquitin ligase developed from IMiDs are an important class of MGDs that have entered clinical trials in recent years[61], as shown in Figure 8. CC-92480 is the first CELMoDs to enter clinical research. In preclinical studies of MM treatment targeting IKZF1/3, CC-92480 exhibited activity more than 1000 times higher than pomalidomide, a type of IMiDs drug, in lenalidomide-resistant cells[62], and demonstrated good tumor inhibition effects in lenalidomide-resistant mouse xenograft models[63]. Currently, CC-92480 has entered phase III clinical trials for the treatment of relapsed or refractory MM[64]. Another CELMoDs targeting IKZF1/3, CFT-7455, due to its cellular level activity far exceeding traditional IMiDs and other CELMoDs in preclinical studies, is called the new generation of CELMoDs. Currently, CFT-7455 is in phase I/II clinical trials[65]. Meanwhile, CELMoDs targeting other types of protein targets are continuously being developed, such as CC-90009, which has entered phase II clinical trials for the treatment of relapsed or refractory acute myeloid leukemia targeting GSPT1[66], and recently developed sickle cell anemia treatments dWIZ-1 and dWIZ-2 targeting WIZ[67], broadening the application scope of MGDs drugs.
图8 CELMoDs的化学结构

Fig. 8 Chemical structures of CELMoDs

3.2 Autophagosome-Coupled Compounds

HD is a currently incurable neurodegenerative disease caused by the accumulation and aggregation of mHTT in cells[68]. Li et al.[38] hypothesized that compounds selectively mediating the interaction between LC3 and mHTT might induce mHTT targeted degradation. Through high-throughput screening methods, as shown in Figure 9, they discovered candidate compounds 10O5, 8F20, AN1, and AN2 from 3,375 compounds and verified their ability to degrade mHTT. These compounds can interact with both LC3 and mHTT simultaneously and do not possess linker structures, belonging to the category of linkerless bifunctional small molecules. Li et al.[69] defined the above compounds as autophagosome-tethering compounds (ATTECs), which are linkerless bifunctional small molecules that promote the lysosomal degradation of pathogenic proteins by inducing their co-localization with LC3. Li et al. speculated that the ability of ATTECs to bind mHTT is related to its abnormally extended polyglutamine tract (polyQ). Therefore, they tested the targeted degradation ability of ATTECs on ataxin-3, which also contains polyQ, and exogenously expressed green fluorescent protein with different lengths of polyQ. The results showed that ATTECs can recognize extended polyQ, indicating their potential application value in treating polyQ-related diseases such as Huntington's disease and spinocerebellar ataxia.
图9 ATTECs的化学结构

Fig. 9 Chemical structures of ATTECs

3.3 Rational Design Strategy for Linkerless Bifunctional Small Molecules

Due to the lack of clear structural characteristics of linker-containing bifunctional small molecule ligand-linker-ligand, the rational design of linker-free bifunctional small molecules faces challenges. Currently, the development of novel TPD without linkers mainly adopts a strategy of high-throughput screening followed by mechanism verification, which limits the efficiency of their discovery. To address this issue, the Nomura group[70] proposed a strategy to convert protein-targeting ligands into covalent MGDs via a covalent handle. As shown in Figure 10a, the group attached different chemical handles to the exit vector portion of the CDK4/6 inhibitor Ribociclib. Testing the ability of the resulting Ribociclib analogs to reduce CDK4 activity revealed that the analog EST1027, which is linked to para-trifluoromethyl cinnamamide, significantly reduced CDK4 levels, while the analog EST1036 with a similar structure, para-trifluoromethyl propionamide, did not exhibit targeted degradation activity. Therefore, it was concluded that the activity of EST1027 is related to the 1,4-addition of the cinnamamide moiety to cysteine sulfate anions. The group further studied the structure-activity relationship of the handle linked to EST1027 and optimized to obtain the minimal covalent chemical handle based on fumaric acid, validating ubiquitin ligase RNF126 as the initial target protein for this handle. Transplanting the minimal covalent chemical handle onto the exit vector portions of targeting ligands for different proteins such as BRD4, AR, and BTK (Figure 10b) successfully achieved targeted degradation of the corresponding proteins. Additionally, the group successfully applied this strategy to the ubiquitin ligase DCAF16, demonstrating the transferability of this strategy[71].
图10 基于共价手柄策略的MGDs理性设计：（a）最小共价手柄的鉴定过程；（b）基于最小共价手柄理性设计的分子胶

Fig. 10 Covalent handle-based MGDs rational design: (a) Identification of minimal covalent handle. (b) MGDs rationally designed based on the minimal covalent handle

4 Conclusion and Prospect

In recent years, bifunctional molecules have developed rapidly, especially TPD bifunctional small molecules. Common small molecule drugs exert their inhibitory function by occupying the active site of the target protein, while TPD bifunctional small molecules achieve targeted degradation of the target protein by hijacking the ubiquitin-proteasome system and the lysosome system, thus achieving a longer-lasting inhibitory effect and having a broader target spectrum. On the one hand, TPD bifunctional small molecules can be used for cancer treatment to target and eliminate cancer-related proteins such as BRD4 and CDK6; on the other hand, they can be used for the treatment of neurodegenerative diseases marked by abnormal protein accumulation, such as Alzheimer's disease, by clearing abnormal protein monomers or their oligomers and aggregates, which is of great value in clinical applications.
In future development, there are still some issues with TPD bifunctional small molecules that need to be improved and resolved: (1) Although the strategy of using bifunctional small molecules to target and degrade proteins can extend the inhibition time and achieve better therapeutic effects, it may also cause greater toxicity when off-target effects occur. Therefore, by utilizing tissue-specific expression of degradation mechanism-related proteins such as ubiquitin ligase E3, selective autophagy receptors, lysosome-targeting receptors, or preparing bifunctional small molecules as prodrugs, developing TPD bifunctional small molecules with tissue specificity or controllable mechanisms is of great significance for their clinical application as drugs. (2) The structural characteristics of TPD bifunctional small molecules containing linkers make them have a large molecular weight, which is an unfavorable factor for drug-likeness. Research on rational design strategies for linkers will help reduce their molecular weight by optimizing linker length. In terms of selecting ligands for target proteins, under the premise of comprehensively considering the impact on degradation activity, ligands with moderate binding activity but smaller molecular weight can be chosen. (3) The development of TPD bifunctional small molecules without linkers mainly adopts the strategy of high-throughput screening followed by mechanism verification, lacking in rational design principles, which limits their development and application as drugs. Currently, although a rational design strategy for MGDs based on covalent chemical handles has emerged, the potency and selectivity of MGDs designed through this strategy still need to be improved, and because one end of their ligand is covalent, there are problems of varying degrees of cytotoxicity. Combining artificial neural networks and machine learning methods to analyze protein structures, and on this basis, conducting virtual screening of small molecule libraries or designing TPD bifunctional small molecules without linkers may improve their development efficiency.

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