1.
Proteolysis-Targeting Chimeras as Tools in Drug Development
(I)
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. Overviewof 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 (as shown in Figure 1).

2.
Figure 1. Schematic Representation of the Ubiquitin Cycle
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-OH
YIPA) 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 sequencemimics HIV TAT motif to increasecell permeability.
Similar hydroxyl-proline peptide (MLAP-OH
YIPM) 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, thePROTAC-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-OH
Y) to inhibit the proliferation of hormone-dependent prostate and breast cancer
cells in vitro through specifically destructingAR 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…