Targeted protein degradation (TPD) technology has been developed rapidly in recent years, especially proteolysis targeting chimeras (PROTACs). To date, a large number of degraders have been discovered against more than 70 disease targets. TPD technology greatly expands the selection of drug targets and provides a powerful solution to solve difficult problems that traditional small molecule inhibitors cannot solve. This article reviews the structure and biological activity of small molecule degraders and the mechanism of action of emerging TPD technology. At the same time, the current challenges in the field of TPD are also described.

Background

The ubiquitin-proteasome system (UPS) is the major pathway for the degradation of most cellular proteins. Ubiquitin is a highly conserved peptide consisting of 76 amino acid residues. The main mechanism of action is as follows: First, in the presence of ATP, the carboxyl terminal of ubiquitin combines with the sulfhydryl group on E1 activating enzyme to form a thioester. Then, the E1 activating enzyme interacts with the E2 coupling enzyme and transfers ubiquitin to E2 through a transthioesterification reaction. Finally, E3 ligases catalyze the transfer of ubiquitin to substrates through the formation of isopeptide bonds. This substrate can undergo polyubiquitination through multiple E1-E2-E3 stages. Polyubiquitinated substrates are recognized by the 26S proteasome and cleaved into polypeptides, which are hydrolyzed to amino acids by other intracellular proteases. Humans have 2 species of E1, about 40 species of E2, and more than 600 species of E3.

In recent years, great progress has been made in UPS-induced protein degradation, especially the proteolysis-targeting chimera (PROTAC) technology. A PROTAC is a bifunctional molecule consisting of two “warheads” connected by a linker, one warhead binds to a protein of interest (POI) and the other binds to an E3 ligase. After the formation of the ternary complex (POI:PROTAC:E3), POI is polyubiquitinated and degraded by the proteasome. PROTACs can be recycled and continue to catalyze another degradation process. The formation of ternary complexes is the key step to play the role of PROTAC.

Research Progress of PROTAC Ligands

E3 ligand: A PROTAC molecule consists of POI ligand, E3 ligand and linker. E3 ligand is one of the important determinants of PROTAC potency and target selectivity. Cereblon protein (CRBN), VHL, MDM2, and IAP ligands are the most popular E3 ligands in PROTAC development.

• Cereblon protein (CRBN) ligands

Thalidomide and its analogues, also known as immunomodulatory drugs (IMiDs), are now used to treat blood cancers: including initially diagnosed multiple myeloma (thalidomide), refractory multiple myeloma (from lenalidomide and pomalidomide) and 5q deletion-associated myelodysplastic syndrome. Thalidomide targets the E3 ligase, cereblon (CRBN). CRBN is a component of the E3 ligase complex CUL4-RBX1-DDB1-CRBN (CRL4CRBN) that functions as a substrate acceptor.

• VHL ligands

VonHippel-Lindau (VHL) is the substrate-acceptor component of the E3 ligase complex CUL2-RBX1-ElonginB-VHL-VHL (CRL2VHL) and has been widely used as an E3 ligand in PROTAC research.

• MDM2 ligands

MDM2 is an E3 ligase that accelerates the degradation of the tumor suppressor p53. MDM2 is an important target for tumor therapy. To disrupt the MDM2-p53 protein-protein interaction (PPI) and upregulate p53 expression, Nutlin-3 was identified as an MDM2 ligand with inhibitory activity against MDM2 (IC50 = 90 nM).

• IAPs ligands

IAPs are closely related to chemotherapy resistance, disease progression, and poor prognosis, and their members include XIAP, cIAP1, cIAP2, LIVIN/ML-IAP, and ILP, and can also serve as E3 ligases. IAP ligand-based PROTACs, known as specific and non-genetic IAP-dependent protein erasers (SNIPERs), are effective degraders targeting ER, BRD4, and BCR-ABL.

Research progress of PROTACs targeting kinases

• PROTACs targeting BCR-ABL

BCR-ABL fusion protein is involved in the occurrence and development of chronic myeloid leukemia. Combining BCR-ABL inhibitor imatinib, bosatinib or dasatinib with CRBN ligand pomalidomide or VHL ligand can synthesize a series of PROTACs. PROTAC34 can effectively inhibit the proliferation of K562 cell line with IC50 of 4.4 nM. Furthermore, by changing BCR-ABL or E3 ligands (VHL or CRBN ligands), the selectivity of PROTACs can be significantly altered. Subsequently, PROTAC35 discovered using the allosteric inhibitor GNF-5 against BCR-ABL1 has submicromolar degradative activity on BCR-ABL1 (DC50 = 340 nM).

Recruitment of RNF114 E3 ligase by PROTAC44 synthesized using nimbolide as E3 ligand. This PROTAC has higher selectivity between BCR-ABL and c-ABL. This suggests that the ligase recruited to E3 is one of the key determinants of PROTAC selectivity.

• PROTACs targeting Bruton’s tyrosine kinase (BTK)

BTK is encoded by the BTK gene, which is expressed in the hematopoietic system except for T lymphocytes. BTK is associated with the proliferation, differentiation and survival of B cells, making it an important target for the treatment of non-Hodgkin’s lymphoma (NHL).

Ibrutinib-based PROTAC46 potently induces BTK degradation in HBL1 cells (DC50 = 6.3 nM) and other NHL cell lines. This PROTAC can also significantly inhibit the proliferation of HBL1 cell line with a half-inhibitory concentration value of 1.5 nM. Importantly, the resistant BTKC481SHBL1 cell line also responded significantly to 46.

PROTAC47 synthesized using BTK inhibitor CGI1746 as BTK ligand had strong degradative activity on BTK in Ramos cells and BTKC481S in TMD8 cells. In addition, this PROTAC is also effective against IKZF1 and IKZF3 and accelerates their degradation.

• PROTACs targeting cyclin-dependent kinases (CDKs)

CDKs are a family of serine/threonine protein kinases that regulate the cell cycle (CDK1, CDK2, CDK4, CDK6) and gene transcription (CDK7-13). CDK has 20 members, many of which have been shown to be closely related to the occurrence and development of tumors, making them important targets for cancer therapy.

Palbociclib is a dual inhibitor of CDK4 and CDK6 with comparable inhibitory activity between these two targets. palbociclib-based PROTACs are selective CDK6 degraders.

PROTACs 73 and 74 were synthesized on the basis of pan-CDK inhibitors AT-7519 and FN-1501. 73 selectively induced the degradation of CDK2 at a concentration of 1000 nM and showed cellular activity against PC‐3 cell line (IC50= 840 nM), similar to AT‐7519. In contrast, 74 was able to induce the degradation of both CDK2 (DC50 = 62 nM) and CDK9 (DC50 = 33 nM) in the PC-3 cell line. The IC50 value for 74 pairs of PC-3 cell line is 120 nM.

• PROTACs targeting EGFR

PROTACs based on the first epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs): EGFR mutation and overexpression are closely related to the occurrence and development of non-small cell lung cancer (NSCLC), which makes EGFR a NSCLC important target for treatment.

Using the first-generation EGFR-TKI gefitinib as the EGFR ligand, the VHL-recruited PROTAC 77 had good degrading activity on EGFRdel19 in HCC827 cells (DC50 = 11.7 nM). PROTAC 78 synthesized on the basis of lapatinib can induce the degradation of EGFRWT (DC50 = 39.2 nM) in OVCAR8 cells, but has less activity on EGFRexon20ins (DC50 = 736.2 nM) in Hela cells.

PROTACs based on second- or third-generation EGFR-TKIs: Afatinib is a second-generation EGFR-TKI. PROTAC82 synthesized based on afatinib has moderate degradation activity on EGFRL858R/T790M in H1975 cell line (DC50 = 215.8 nM).

PROTAC85 using the second-generation EGFR-TKI Canertinib as the EGFR ligand has strong degradation activity on both EGFRdel19 (DC50=30-100nM) and EGFRL858R/T790M (DC50

Using the third-generation EGFR-TKI as the warhead, a series of PROTACs were synthesized by changing the linker and E3 ligand. In H1975 cells, the most potent PROTAC83 has a DC50 value of 5.9 nM against EGFRL858R/T790M. Osimertinib-derived PROTAC84, at a concentration of 10000nM, can promote the degradation of EGFRdel19 in PC9 cells.

• PROTACs targeting ALK

ALK is a receptor tyrosine kinase. Aberrant activation of ALK has been implicated in a variety of tumors, including ALCL, NSCLC, diffuse large B-cell lymphoma, breast cancer, colorectal cancer, inflammatory myofibroblastic tumor, esophageal squamous cell carcinoma, and renal cell carcinoma. Furthermore, point mutations in ALK are associated with neuroblastoma. Therefore, AKL is one of the most important targets for the treatment of cancer.

The ALK inhibitor ceritinib-derived PROTAC91 effectively reduced the levels of NPM-ALK (DC50 = 3 nM) in SU-DHL-1 cells and EML4-ALK (DC50 = 34 nM) in H2228 cells.

Brigatinib is a second-generation ALK inhibitor. PROTAC95, as a potent ALK degrader, effectively kills ALK in SR cells (DC50 = 7.0 nM), and significantly inhibits SR (50% inhibitory concentration = 1.7 nM) and H2228 (50th inhibitory concentration = 46 nM) cell lines proliferation.

• PROTACs targeting B-RAF

B-RAF belongs to the RAF family and is an important node in the RAS-RAF-MEK-ERK signaling pathway. Mutation and overexpression of B-RAF are thought to promote tumorigenesis and progression. The B-RAF inhibitor BI882370-derived PRTOAC99 was able to significantly reduce the level of B-RAFV600E (DC50 = 15 nM) in A375 cells while retaining wild-type B-RAF (B-RAFWT).

Research progress of PROTACs targeting nuclear receptors (NR)

NRs is a family of ligand-activated transcription factors, including about 48 members, responsible for the regulation of gene transcription. NRs are closely related to many physiological and pathological processes, such as metastasis, reproduction, aging and cancer. Androgen receptor (AR) and estrogen receptor (ER) have attracted much attention in the treatment of prostate cancer and breast cancer.

• PROTACs targeting ER

The ERα-targeting PROTAC124, consisting of estradiol as an ERα ligand and an IκBα phosphopeptide (DRHDSGLDSM), recognizes and binds to the SCFβ-TRCP E3 ligase. 124 promotes the ubiquitination of ERα and effectively induces the degradation of ERα at a concentration of 10,000 nM.

• PROTACs targeting AR

AR has a great effect on the occurrence and development of prostate cancer, making it an important target for prostate cancer treatment.

• PROTACs targeting the estrogen-related receptor (ERRα)

ERRα, as a member of NR, is a regulator of gene expression related to metabolism and energy homeostasis.

PROTACs targeting other proteins

• PROTACs targeting the BET family

BET proteins serve as epigenetic “readers” that can specifically interact with acetylated lysine residues of histones and non-histones to regulate chromatin dynamics and gene transcription. BET proteins (including BRD2, BRD3, BRD4 and BRDT) contain two bromodomains (BD) and one extraterrestrial domain (ET).

Utilized OTX015 as a BRD4-targeting PROTAC 152. 152 exhibited significant degradation activity against BRD4 (DC50 JQ1-based PROTAC 153 can completely degrade BRD4 in MV4;11 cells by hijacking CRBN and proteasome.

• PROTACs targeting the B-cell lymphoma 2 (BCL-2) family

The CL-2 gene family encodes more than 20 proteins that regulate apoptosis. Among these members, antiapoptotic proteins including BCL-2, BCL-XL, and MCL-1 have been shown to be key targets for cancer therapy.

ABT263 is a dual BCL-2/BCL-XL inhibitor. A potent BCL-XL degrader PROTAC171 was found in MOLT-4 cells using ABT263, with a DC50 value of 63nM and a Dmax value of 90.8%. PROTAC 176 based on the MCL-1 inhibitor a-1210477 was also found to be a potent MCL-1 degrader.

• PROTACs targeting histone deacetylases (HDACs)

HDACs are responsible for regulating histone and non-histone acetylation levels and serve as important antitumor targets.

HDAC6 inhibitor nexturastat A-based PROTAC 184 can accelerate the rapid degradation of HDAC6 in MM1S cells with a DC50 value of 3.8 nM. Subsequently, the structure of 184 was further optimized to obtain PROTAC 185, which showed DC50=3.2nM in MM1S cells (similar to 184) and high selectivity for HDAC6. Linking the HDAC inhibitor SAHA to statin to generate PROTAC 186 significantly reduces the levels of HDAC1, HDAC6 and HDAC8 at a concentration of 4 nM.

• PROTACs targeting SHP2

SHP2, a phosphatase encoded by the PTPN1 gene, acts as a regulator of T cell activation and many signaling pathways, including RAS-ERK, PI3K-AKT, and JAK-STAT.

CRBN-recruiting PROTAC 191 based on the SHP2 allosteric inhibitor TNO155. 191 was shown to promote CRBN-dependent degradation of SHP2 (DC50 = 6.02 nM in MV4;11 cells). CRBN-recruited PROTAC 192 from the SHP2 allosteric inhibitor RMC-4550. 192 is potent at concentrations as low as 10 nM with high specificity. The SHP2 inhibitor SHP099-derived PROTAC 193 is an active SHP2 degrader with nanomolar degradation activity.

• PROTACs targeting KRAS

KRAS, a GTPase encoded by the kirsten rat sarcoma virus oncogene homolog (KRAS) gene, is frequently mutated in cancer. Some KRAS mutations lead to its persistent activation, leading to tumorigenesis and progression.

Pomalidomide was combined with quinazoline-containing compounds to synthesize PROTAC 194. 194 was shown to effectively induce GFP-KRASG12C and CRBN dimerization and promote the depletion of GFP-KRASG12C fusion protein without affecting endogenous KRASG12C.

Research progress of HaloPROTACs

HaloTag (HT) is a modified haloalkane dehalogenase that removes halides from aliphatic hydrocarbons. During catalysis, a covalent ester bond is formed between the aspartic acid in the enzyme and the hydrocarbon substrate. In general, HaloPROTAC is a bifunctional molecule consisting of a chloroalkane chain group and an E3 ligand group capable of covalently binding to HT. After binding to HalT and E3, HaloPROTAC will accelerate the ubiquitination of HT fusion to the target protein and subsequent proteolytic degradation.

Research progress of dTAG system

Generally, POI is fused to F36V-mutated fk506-binding protein-12 (FKBP12F36V), and the resulting fusion protein is degraded by FKBP12F36V-targeted PROTACs to achieve POI degradation.

Other techniques for targeting proteolysis

• Lysosome-targeted degradation chimera (LYTAC) and antibody-based PROTAC (AbTAC)

PROTAC must pass through the cell membrane and enter the cell in order to achieve the purpose of protein degradation. LYTAC and AbTAC are bifunctional molecules with extracellular functional effects, which can be used to deal with extracellular POIs or some POIs that are difficult to permeate the membrane.

• Autophagy targeting chimera (AUTAC) and autophagy-tethering complex (ATTEC)

The autophagy-lysosome system is another important pathway for the degradation of damaged or wasted cellular components and plays a key role in cellular homeostasis.

AUTAC consists of POI ligand, cyclic guanosine (cGMP), or its analog moiety and linker. After binding to AUTAC, POI will be labeled by the autophagy tag cGMP or its analogs and degraded by the autophagy-lysosome system. ATTEC is able to induce POI degradation through autophagy.

Research progress of molecular glue

Molecular Glue is a protein-protein interaction (PPI) stabilizer that binds to proteins, enabling them to interact with novel substrates.

Cyclosporin A (CsA) is an immunosuppressant that acts as a molecular glue. Rapamycin, another molecular glue, is used to prevent organ rejection in patients. Rapamycin can bind to FKBP12 to form the FKBP12-rapamycin complex, which interacts with the mammalian target of rapamycin (mTOR) to exert its immunosuppressive function. The plant hormone auxin is a molecular glue that can induce SCFTIR1 E3 ligase to interact with auxin/indole-3-acetic acid (Aux/IAA), degrade Aux/IAA, and regulate plant growth.

PROTACs-induced degradation of light-regulated proteins

There are two main types of PROTACs. One type is light-responsive uncaged PROTACs with photocleavable protecting groups, which employ a photosensitive group to first occlude the active site of the molecule. When the molecule is transported to a specific site and tissue, light is used to cut off the photosensitive group to restore the activity of PROTACs and start the degradation of the target protein. The other is photoswitchable PROTACs with photosensitive groups, whose conformation can be changed by photostimulation, allowing PRTOACs to switch between active and inactive conformations.

Research progress of hydrophobic labeling technology

Hydrophobic tags can mimic partially misfolded proteins, which can be recognized by chaperones and undergo proteasomal degradation. Therefore, labeling POIs with high-fat-containing tags would be an effective approach to treating TPD.

Hydrophobic labeling technology expands the application range of TPD. Targets that can be degraded by PROTACs can be addressed by hydrophobically labeled small molecules. However, the poor solubility and permeability resulting from strong hydrophobicity often limit the biological activity of these degraders.