Proteolysis targeting Chimera (PROTAC) has been emerged by hijacking the endogenous ubiquitin-proteasome system (UPS) to specifically degrade protein of interests (POI) in 2001. PROTAC are drug-like molecules that induce the degradation of proteins, and they work by tightly binding E3 ligases and target proteins (POI), inducing ubiquitination of the POI and its subsequent degradation via the proteasome. Based on their mechanism of action, PROTAC can potentially target any type of protein including previously “undruggable” class of protein therapeutic targets. As a next blockbuster therapeutic modality, there can be new challenges, for example the ability to identify and investigate clinical safety risks using suitable preclinical models.

1. Overview of the involved technology

PROTACs exploit the natural ubiquitin proteasome system for chemically induced protein degradation. The ubiquitin-proteasome system (UPS) is the primary intracellular mechanism for destruction of damaged proteins or those no longer required. The 76-residue protein ubiquitin is attached to proteins via a lysine isopeptide bond as a post-translational modification (PTM) via a cascade of three enzymes: an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase. Free ubiquitin is activated by an E1 in an ATP-dependent process during which it is converted to a C-terminal thioester. Trans-thioesterification passes the ubiquitin from the E1 unto an E2. Finally, an E3 complex facilitates transfer of ubiquitin, either directly or indirectly, to a substrate protein lysine. Enzymes involved in protein ubiquitination are present in the human genome in increasing numbers as they progress from E1, of which there are only 2, to E3, with more than 600 postulated E3 family members. E3 ligases serve to recruit substrates and facilitate transfer of ubiquitin from an E2 conjugating enzyme to the target protein. When a protein is tagged with polyubiquitination and recognized by the 26S proteasome, the ubiquitin chains are removed by proteasome associated deubiquitinating enzymes (DUBs) and recycled, whereas the protein substrate is unfolded and degraded.

The PROTAC technology allows the UPS system to be chemically co-opted and aimed to degrade a specific target protein. This approach employs E3 ligase ligands, fused via a flexible chemical linker to a targeting element for the protein of interest, to elicit ectopic ubiquitination, resulting in protein degradation. The technology is routinely used in cultured cells and in vivo and has even entered clinical trials, and function against a wide range of protein classes and in different subcellular locations, including the cytosol, the nucleus, and the plasma membrane.

2. The drug development of PROTACs

2.1. The first generation peptide-based PROTACs

The use of PROTACs began in 2001, when Sakamoto et al. [1] published a chemical and peptide-based degrader for the methionine aminopeptidase-2, which paved the way for a new strategy in drug design. The first PROTAC, named PROTAC-1, recruits the SCFβ-TrCP E3 ligase to dictate the ubiquitination and degradation of MetAP-2 in Xenopus egg extracts. Structurally, PROTAC-1 consisted of two moieties and a linker region, amongst which the IκBα phosphopeptide was responsible for recruiting SCFβ-TrCP E3 ligase, while ovalicin bond with the POI, MetAP-2. This study initiates a new era for conditional inactivation of specific proteins with PROTACs, especially for those so-called non-druggable targets. An estradiol-based PROTAC was synthesized by using a similar strategy that recruits SCFβ-TrCP to promote the destruction of estrogen receptor alpha (ERα), and a dihydroxytestosterone (DHT)-based PROTAC to degrade androgen receptor (AR), which might be helpful for the breast and prostate cancer therapy, respectively. However, the high molecules weight of PROTACs led to low cell permeability efficacy, which can only be microinjected into cell, and the phosphopeptide is also susceptible to intracellular phosphatases, all of which limit their practical usage in clinic.

To overcome the cell permeability and stability shortages derived from phosphopeptide-based PROTACs, the first cell permeable peptide-based, VHL-based PROTAC was added 8 tandem arginine at the end of the E3 ligase ligand. By adopting a hydroxyl-proline peptide derived from the degron in HIF1 (ALAP-OHYIPA) as the ligand to recruit the VHL E3 ligase, those VHL-based PROTACs with either a FKBP12 ligand or DHT as warheads, promote the degradation of FKBP12 and AR protein in cells, respectively. Structurally, ALAPYIP is responsible for recruiting the VHL E3 ligase, while the tandem arginine sequence mimics HIV TA T motif to increase cell permeability. Similar hydroxyl-proline peptide (MLAP-OHYIPM) has hijacked the VHL E3 ligase, and this VHL-based PROTAC against ERα can enter into cells and inhibit cell proliferation via degrading ERα. Then, the linker was optimized location onto the C7α position of estradiol that generated a PROTAC with the highest affinity to ER and the most efficiency in degradation rate for ERα. Using this VHL-based PROTACs concept, the PROTAC-A (with DHT as AR ligand) and PROTAC-B (with estradiol as ER ligand) were synthesized by modifying the hydroxyl-proline peptide into a pentapeptide degron (ALAP-OHY) to inhibit the proliferation of hormone-dependent prostate and breast cancer cells in vitro through specifically destructing AR and ERα, respectively.

Although with the shortage of high molecular weight, peptide-based PROTACs still have several advantages compared to small molecular drug, such as larger contact interface with POI and more choices of modifications on the drug, thus it is still of option to further explore peptide-based PROTACs with other optimizing ways, for example, to adopt a synthesized peptide with better cell-permeability and chemical stability.

To be continued in Part II…

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