Peptide and Small Molecule Based PROTACs Technology

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The degradation or intervention of pathogenic proteins has become one of the most effective therapeutic strategies. Targeted protein degradation (TPD) utilizes the naturally occurring protein degradation systems within cells to achieve specific and efficient degradation of disease-associated proteins, thereby achieving therapeutic effects. Proteins within cells are constantly synthesized and degraded, with the ubiquitin-proteasome system (UPS) and lysosomal degradation pathways being the two main ways eukaryotic cells degrade proteins.

In the degradation method based on UPS, Proteolysis Targeting Chimeras (PROTACs) are the most mature system studied. PROTACs are dual-functional molecules consisting of ligands for binding target proteins (POI ligand) and ligands capable of recruiting E3 complexes (E3 ligand), as well as a linker connecting the two. PROTACs facilitate ubiquitination modification of the POI by connecting the ligands at both ends via the linker, thereby bringing the POI and E3 closer together. When PROTACs enter the cytoplasm, their dual-functional groups form a ternary complex with POI and E3, recruited by the E3 ligand, leading to ubiquitination of the target protein by E3, while PROTACs dissociate. Eventually, the polyubiquitinated POI is recognized and degraded by the proteasome, while the detached PROTAC enters the next degradation cycle.

Peptide Based PROTACs

In 2001, the Crews team and the Deshaies team first reported the PROTAC technology. Their designed PROTAC-1, utilizing the IκBα phosphorylated peptide to recruit Skp1-Cullin-F boxβ-TRCP (SCFβ-TRCP) E3 ubiquitin ligase, induced the degradation of Methionine aminopeptidase-2 (METAP-2). In this study, the vascular endothelial growth inhibitor targeting METAP-2, Ovalicin, covalently bound with the IκBα phosphorylated peptide to form PROTAC-1, which could rapidly degrade METAP-2 extracellularly. However, PROTAC-1 is far from being a drug; it can only be regarded as a scientific attempt.

• Based on Cell-penetrating Peptides (CPPs)

The cell membrane has selectively permeable characteristics, and the peptide form of the E3 ligand leads to PROTACs with a large molecular weight, which cannot freely pass through the cell membrane, greatly reducing the application potential of PROTACs drugs. The introduction of Cell-penetrating peptides (CPPs) to mediate the entry of large biomolecular drugs into cells is one of the hotspots in the field of biomedicine research. Schneekloth et al. conferred cell permeability to PROTACs by fusing polylysine cell-penetrating peptides at the linker of PROTACs and demonstrated at the cellular level that the molecule could spontaneously enter cells and degrade target proteins. It is worth noting that in this study, the E3 ligand selected was the shortest peptide segment in hypoxia-inducible factor 1α (HIF-1α) that could be recognized by E3, which could recruit von Hippel-Lindau (VHL) protein in the E3 ligase complex VBC-Cul2, becoming one of the most commonly used E3 complexes in subsequent PROTAC applications. Meanwhile, the target protein ligand part was composed of the small molecule AP21998, targeting the F36V mutant (FKBP12^F36V) of FK506 binding protein 12 kDa (FKBP12) associated with various cardiovascular diseases. Although CPPs can promote the entry of peptide PROTACs into cells to a certain extent, the CPPs system itself also has the problem of poor delivery efficiency. Therefore, establishing an efficient CPP system will become one of the urgent tasks for the development of peptide PROTACs.

Small Molecule Based PROTACs

Although peptide PROTACs have high biocompatibility and low in vivo toxicity, which gives them a huge advantage in safety, they face issues such as poor cell permeability, weak stability, and large molecular weight. In contrast, small molecule PROTACs are more easily absorbed by the body, making them promising candidates for drug development. In addition, the protein ligand part of small molecules can be replaced by clinically approved small molecule drugs, which significantly reduces the development cycle and cost.

Small Molecule PROTACs Recruited by MDM2

The Mouse double minute (MDM) E3 ligase family plays an important role in tumor occurrence and development, especially in regulating the function of p53. The imidazoline derivative Nutlins, with its high affinity for MDM2 and the ability to disrupt the binding between MDM2 and p53, is commonly used in clinical cancer therapy. In 2008, the first fully small molecule form of PROTAC, synthesized by linking Nutlins with the small molecule Selective androgen receptor modular (SARM) targeting AR, was proven to effectively degrade AR protein in intracellular experiments. However, the effective dose of SARM-Nutlins was very high, only effective in the millimolar range for degrading target proteins. Therefore, the screening of small molecule ligands capable of efficiently recruiting other E3 complexes became an important breakthrough in the subsequent development of PROTAC technology.

Small Molecule PROTACs Recruited by VHL

Research on VHL E3-based PROTACs primarily focuses on finding suitable small molecule substitutes for the HIF-1α peptide fragment. In 2012, the Crews team and the Ciulli team identified the first small molecule ligand capable of binding to VHL E3 using a bioinformatics approach, and the affinity between the two was significantly higher than that of HIF-1α. Based on this small molecule ligand, the first small molecule PROTACs recruited by VHL E3 were designed and synthesized in 2015. Experimental results indicate that they can degrade almost all target proteins (>90%) within the nanomolar range, and results from mouse models also show that PROTAC-ERRα can lead to the degradation of approximately 40% of Estrogen-related Receptor-α (ERRα). To date, VHL E3-based PROTACs have been used for the targeted degradation of various disease-related proteins, such as BCR-ABL, Anaplastic Lymphoma Kinase (ALK), and Focal Adhesion Kinase (FAK), among others.

Small Molecule PROTACs Recruited by CREN

Research on Cereblon (CRBN) E3 complexes made a significant breakthrough in 2010. Researchers studying the mechanism of immunomodulatory drugs (IMiDs) such as thalidomide discovered that their primary target was the CRBN component of the Culin-RING ubiquitin ligase (CRL) complex. Subsequent studies revealed that besides thalidomide, other phthalimide drugs such as pomalidomide and lenalidomide also induced the degradation of different target proteins like IKAROS family zinc finger 1 and 3 (IKZF1 and 3) by binding to CRBN.

Crews et al. connected the small molecule inhibitor of BRD4, OTX015, with pomalidomide to synthesize ARV-825. Experimental results showed that treatment with nanomolar concentrations of ARV-825 for around 2 hours led to the degradation of over 50% of BRD4 in Burkitt lymphoma cells, eventually achieving complete degradation. Moreover, compared to OTX015 alone, ARV-825 could more effectively inhibit tumor cell proliferation. Another pan-BET inhibitor, JQ1, was also utilized in PROTAC research. Researchers connected JQ1 with phthalimides to synthesize dBET molecules. Through large-scale proteomic methods, dBET1 was confirmed to efficiently and specifically induce the degradation of all BRD family proteins, including BRD4, in mice, exhibiting significant tumor suppression effects and delaying the progression of leukemia. Subsequently, PROTACs synthesized using CRBN E3 ligands and BET small molecule inhibitors, such as BETd-246, QCA570, and BETd-260, have also been employed in cancer therapy.

Small Molecule PROTACs Recruited by IAPs

Methylbestatin (MeBS) can bind to the cellular inhibitor of apoptosis protein 1 (cIAP1) within the inhibitor of apoptosis proteins (IAPs) E3 family. The first PROTAC recruiting cIAP-1 E3 ligase was named SNIPER (Specific and non-genetic inhibitor-of-apoptosis proteins-dependent protein eraser). SNIPER targets cellular retinoic acid binding proteins-Ⅰ/Ⅱ (CRABP-Ⅰ/Ⅱ) through all-trans retinoic acid (ARTA), which plays a crucial role in acute promyelocytic leukemia and neuroblastoma. In cellular experiments, SNIPER could induce rapid degradation of CRABP-Ⅰ/Ⅱ in neuroblastoma cells IMR-32, and SNIPER-treated IMR-32 showed significantly slowed migration. Subsequently, MeBS derivatives MV1 and LCL161 were used as ligands for IAP E3, showing different substrate specificities. So far, the SNIPER technology has been successfully used for the degradation of various disease-associated proteins, such as ER, AR, Bromodomain-containing protein (BRD), and TACC3 (Transforming acidic coiled-coil-3). However, due to the fact that IAP-based PROTACs mostly work in the micromolar range and may cause degradation of IAP itself while inducing degradation of target proteins, there may be significant risks in their use as therapeutic drugs.

PROTACs Based on Covalent Binding to E3

The discovery of small molecules capable of covalently binding to E3 ubiquitin ligase complexes introduces more types of E3 complexes, such as DCAF16, Ring finger protein 114 (RNF114), and RNF4, into the application of PROTACs. For example, the small molecule nimbolide can form covalent bonds with the cysteine residue at the N-terminus of the RNF114 E3 ligase complex, disrupting RNF114's recognition and degradation of substrates such as tumor suppressors p21 and p57, thereby achieving therapeutic effects. Combining nimbolide with JQ1 indeed effectively binds to RNF114 and leads to the degradation of the BET family. Compared to the traditional non-covalent binding between E3 and its ligands, the formation of irreversible binding between E3 and its ligands seems to be more advantageous in terms of kinetics, as they only require a single reaction between the substrate protein and the ligand to initiate the degradation process of the target protein.