Leading Edge

Primer

Proteolysis-Targeting Chimeras as Therapeutics

and Tools for Biological Discovery
George M. Burslem1 and Craig M. Crews1,2,*
1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA
2Departments of Chemistry and Pharmacology, Yale University, New Haven, CT, USA
*Correspondence: craig.crews@yale.edu
https://doi.org/10.1016/j.cell.2019.11.031

New biological tools provide new techniques to probe fundamental biological processes. Here we
describe the burgeoning field of proteolysis-targeting chimeras (PROTACs), which are capable of
modulating protein concentrations at a post-translational level by co-opting the ubiquitin-protea-
some system. We describe the PROTAC technology and its application to drug discovery and pro-
vide examples where PROTACs have enabled novel biological insights. Furthermore, we provide a
workflow for PROTAC development and use and discuss the benefits and issues associated with
PROTACs. Finally, we compare PROTAC-mediated protein-level modulation with other technolo-
gies, such as RNAi and genome editing.

Introduction tude of biological functions that are still being investigated

Technological advances often lead to major biological discov-
eries (Botstein, 2010; Editorial, 2000; Fields, 2001; van Steensel,
2015), which, in turn, drive new technology development.
The search for new technologies to answer biological ques-
tions has given rise to the field of chemical biology (Altmann
et al., 2009), and perhaps one of the most exciting technologies
to arise is targeted protein degradation by proteolysis-targeting
chimeras (PROTACs). In this Primer, we will review this
technology, its applications to drug discovery, and its use in
the exploration of fundamental biology. Additionally, we will
outline the workflow involved in successfully employing
PROTACs for experimentation and compare PROTACs with
other techniques.
Overview of the Technology
The Ubiquitin-Proteasome System
The ubiquitin-proteasome system (UPS) is the primary intracel-
lular mechanism for destruction of damaged proteins or those
no longer required (Amm et al., 2014). This has been extensively
reviewed elsewhere (Kleiger and Mayor, 2014); however, a
brief explanation of the UPS is provided here as it pertains to
PROTACs. 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 (Figure 1). Free ubiquitin is activated by an E1 in an
ATP-dependent process during which it is converted to a C-ter-
minal thioester (Figure 1). Trans-thioesterification passes the
ubiquitin from the E1 unto an E2. Finally, an E3 complex facili-
tates transfer of ubiquitin, either directly or indirectly, to a sub-
strate protein lysine.
Ubiquitin itself can, in turn, be ubiquitinated on one or more
of its seven surface lysine residues. These PTMs have a multi-
(Komander and Rape, 2012; Yau and Rape, 2016), but the
canonical role of K48 polyubiquitin is to signal a protein for
destruction via the proteasome (Grice and Nathan, 2016).
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 postu-
lated E3 family members (Ardley and Robinson, 2005). 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 poly-K48 ubiquitination and recognized
by the proteasome, the ubiquitin chains are removed by protea-
some-associated deubiquitinating enzymes (DUBs) and re-
cycled, whereas the protein substrate is unfolded and degraded
(Figure 1).
Hijacking the UPS System
The PROTAC technology allows the UPS system to be chemi-
cally 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
(Figure 1). Beginning with proof-of-concept experiments in cell
lysates with peptidic ligands (Sakamoto et al., 2001), the technol-
ogy has matured and is routinely used in cultured cells and
in vivo and has even entered clinical trials. Importantly, the tech-
nology is now comprised of all-synthetic modular compounds
(Bondeson et al., 2015; Winter et al., 2015) that function against
a wide range of protein classes and in different subcellular
locations, including the cytosol, the nucleus, and the plasma
membrane (Burslem et al., 2018b). As we come to better under-
stand this new technology, it is clear that protein-protein con-
tacts between the neo-substrate and E3 complex are a crucial
determinant of PROTAC success, as shown in Figure 2 (Bonde-
son et al., 2018; Gadd et al., 2017; Nowak et al., 2018; Smith

102 Cell 181, April 2, 2020 ª 2019 Elsevier Inc.


et al., 2019; Zorba et al., 2018). Farnaby et al. (2019) and Roy
et al. (2019) have been instrumental in the structural and bio-
physical characterization of co-operativity in this area.
Only a handful of E3 ligases have been employed in the
PROTAC technology (Table 1), with most of the reported com-
pounds using either cereblon (CRBN) or Von Hippel-Lindau
(VHL) because of the availability of drug-like small molecules
that recruit them (Buckley et al., 2012; Winter et al., 2015).
cellular inhibitor of apoptosis (cIAP) ligands have also been
used but often lead to auto-ubiquitination and degradation of
the E3 ligase itself, making them less attractive (Sekine et al.,
2008). The use of nutlin compounds to recruit MDM2 played a
key role in the development of the first all-small-molecule PRO-
TAC (Schneekloth et al., 2008) and has recently re-emerged as a
potent method for targeted protein degradation (Hines et al.,
2019). With more than 600 postulated members of the E3
superfamily, it is exciting to watch ligands emerge for new E3
ligases and their application to targeted protein degradation
(Ottis et al., 2017; Spradlin et al., 2019). Indeed, some of these
new ligands allow degradation only in specific cellular compart-
ments because of their restricted localization (Zhang et al.,
2019b). Given the importance of E3/target protein-protein
interactions in PROTAC-mediated degradation, more E3s
Figure 1. Schematic Representation of the
Ubiquitin Cycle and How It Can Be Co-opted
by PROTACs
Shown are ubiquitin-E1 thioester (UBA1 Ub com-
plex, PDB: 3CMM), ubiquitin-E2 thioester
(UBE2D3-UbDha complex, PDB: 5IFR), the ubiq-
uitin-E2-E3-substrate complex (model composed
of PDB: 1LQB, 5N4W, and 5IFR), the ubiquitin-E2-
E3-PROTAC-neosubstrate complex (model
composed of PDB: 5T35, 5N4W, and 5IFR), and
the proteasome (PDB: 5I4G).
available for recruitment provide a
greater chance of successful PROTAC
development.
Applications
PROTAC technology has applications in
both biological discovery and drug discov-
ery. In many ways, PROTACs represent
the chemical equivalent of small interfering
RNA (siRNA), albeit allowing removal of a
protein at a post-translational level rather
than silencing at a post-transcriptional
level. Therefore, they are a useful tool for
studying the roles of a protein in biological
systems in the laboratory. Additionally, the
small-molecule nature of PROTACs cir-
cumvents issues associated with delivery
and biodistribution that hinder clinical
applications of siRNA, eliciting great inter-
est from the pharmaceutical industry.
PROTACs in Drug Discovery
Unsurprisingly, given its small-molecule
nature, PROTAC technology is progress-
ing into the clinic for numerous indications. Initial studies have
focused on degradation of hormone receptors (Flanagan and
Neklesa, 2019), specifically androgen receptors (Neklesa et al.,
2019) and estrogen receptors (Flanagan et al., 2019). Notably,
these trials are for orally bioavailable PROTACs, highlighting
the benefits of PROTACs over therapeutic RNAi (Setten et al.,
2019) or traditional selective estrogen downregulators (SERDs)
(Patel and Bihani, 2018).
Androgen Receptor
Androgen receptor (AR) antagonists, such as enzalutamide
(Rathkopf and Scher, 2013), have been used therapeutically
with great benefits for prostate cancer patients; however, resis-
tance often arises. AR PROTACs have been shown repeatedly
to outperform enzalutamide, particularly in resistance contexts
of increased androgen levels or mutations in AR that convert
antagonists into agonists (Han et al., 2019; Salami et al., 2018).
The AR PROTAC ARV-110 is currently in clinical trials for meta-
static castration-resistant prostate cancer.
Estrogen Receptor
The SERDs validated induced estrogen receptor (ER) degrada-
tion as a therapeutic strategy, with fulvestrant being Food
and Drug Administration (FDA) approved to treat breast cancer
(Howell et al., 2004). However, ER PROTACs induce more
Cell 181, April 2, 2020 103
Figure 2. Composition of a PROTAC for BRD-4
Left: structure of VHL ligand bound to VHL (PBD: 6FMI). Center: PROTAC (MZ-1)-induced ternary complex between VHL and BRD-4 (PDB: 5T35). Right: structure
of JQ-1 bound to BRD-4 (PDB: 3MXF).
efficient degradation than fulvestrant and possess improved
pharmacological properties (Flanagan et al., 2019; Hu et al.,
2019). The ER PROTAC ARV-471 is currently in clinical trials
for advanced or metastatic ER+/Her2 breast cancer.
BRD4
The bromodomain and extraterminal (BET) domain epigenetic
reader protein BRD4 is perhaps the most commonly PROTAC-
targeted protein (reviewed in Yang et al., 2019) and is often
used as a test substrate for technological advances in protein
degradation (Martin et al., 2019; Spradlin et al., 2019; Ward
et al., 2019; Zhang et al., 2019b). Targeting BRD4 using PRO-
TACs is particularly attractive because its degradation results
in more robust loss of c-Myc relative to simple BRD4 inhibition,
resulting in enhanced apoptosis of various cancer cells both
in vitro and in vivo (Lu et al., 2015; Piya et al., 2019; Winter
et al., 2015). Indeed, BRD4-targeting PROTACs appear to be po-
tential therapeutics for secondary acute myeloid leukemia (AML)
(Saenz et al., 2017, 2019), multiple myeloma (Zhang et al., 2018),
mantle cell lymphoma (Sun et al., 2018), diffuse large B cell lym-
phoma (Jain et al., 2019), and castration-resistant prostate can-
cer (Raina et al., 2016) in preclinical models. Interestingly, BRD4
PROTACs have also been employed as a test bed for PROTAC
resistance mechanisms, revealing that resistance to VHL-re-
cruiting PROTACs can arise from loss of Cul2 and that CRBN-re-
cruiting PROTAC-treated cells can lose CRBN or its cognate E2,
UBE2G1 (Ottis et al., 2019; Zhang et al., 2019a). These mecha-
nisms of resistance should be considered when developing
PROTACs into therapeutics.
Targeting Aggregated Proteins
A challenging but potentially very exciting class of targets for
protein degradation are aggregation-prone proteins such as
alpha-synuclein, transthyretin, and tau (Iadanza et al., 2018).
Degradation of these proteins, either in their monomeric or
104 Cell 181, April 2, 2020
aggregated state, via direct recruitment to E3 ligases by
PROTACs is an enticing approach to treating protein aggrega-
tion diseases. Several PROTAC studies have begun to address
this challenge by inducing tau degradation (Chu et al., 2016; Silva
et al., 2019).
Biological Discoveries Enabled by PROTACs
Beyond serving as potential drugs, PROTACs are unique
tools for the study of protein function, combining the advantages
of small molecules with those of other gene silencing and/or ed-
iting techniques. Perhaps most importantly, they enable the
study of acute protein loss rather than the gradual selection of
cells that survive without a given protein, avoiding issues arising
from compensatory pathways or reprogramming. PROTACs can
also be employed in contexts, such as patient samples, where
other techniques may not be amenable. Below are examples
where PROTACs enabled discovery or confirmation of biological
insights.
BCR-ABL-Independent CML Stem Cells
The development of imatinib, followed by other BCR-ABL
kinase inhibitors, has transformed the lives of chronic myeloid
leukemia (CML) patients. However, kinase inhibition is not cura-
tive in most patients (Druker, 2009). A population of CML stem
cells survives despite inhibition of oncogenic kinase activity
(Corbin et al., 2011), and it has been postulated that they depend
on kinase-independent roles of BCR-ABL for survival. Unfortu-
nately, this has proven to be challenging to study, especially in
patient samples, because of the inherent selection requirements
following genetic knockdown. Our laboratory and others have
developed PROTACs capable of inducing degradation of BCR-
ABL (Lai et al., 2016; Shibata et al., 2017, 2019). Recently, a
collaborative study with Brian Druker (Burslem et al., 2019), em-
ploying allosteric BCR-ABL PROTACs, revealed that CML
Table 1. E3 Ligases Currently Used in PROTACs
E3 Adaptor Native Substrate Ligand Key References
VHL hydroxylated HIF-1a VHL peptidomimetics Bondeson et al., 2015; Buckley et al., 2012;
Schneekloth et al., 2004
CRBN glutamine synthetase MEIS2 IMiDs Lu et al., 2015; Winter et al., 2015
MDM2 p53 idasanutlin Hines et al., 2019; Schneekloth et al., 2008
b-TRCP b-catenin phosphorylated peptide Sakamoto et al., 2001
cIAP second mitochondrion-derived activator bestatin esters Itoh et al., 2010
of caspases (SMAC) MV-1
LCL161 (SMAC mimetics)
RNF4 poly-sumoylated proteins CCW-16 (covalent fragment) Ward et al., 2019
RNF14 p21 nimbolide (natural product) Spradlin et al., 2019
DCAF16 unknown KB02 (covalent fragment) Zhang et al., 2019b

patient stem cells are not dependent on the presence of
BCR-ABL protein, suggesting that another mechanism drives
their proliferation and that alternative treatments should be
explored.
FLT-3 ITD Plays Non-kinase Roles in AML
Internal tandem duplication (ITD) of key FLT-3 subdomains are
validated driver mutations in acute myeloid leukemia, but clinical
trials have demonstrated that constant and complete kinase
inhibition is required for therapeutic benefits (Grunwald and
Levis, 2013; Pratz et al., 2009; Smith et al., 2012). We postulated
that degradation of mutant FLT-3 ITD could provide a potential
therapeutic approach because degradation results in complete
and sustained lack of signaling. Conversion of the phase 3
clinical candidate quizartinib (AC220) (Zarrinkar et al., 2009)
into a PROTAC resulted in a compound with enhanced antipro-
liferative effects relative to quizartinib despite being a less
potent inhibitor in vitro and in cellulo, revealing non-kinase roles
of FLT-3 ITD in AML (Burslem et al., 2018c).
TRIM24 as a Key Dependency in AML
Similar to BRD4 PROTAC development, PROTACs have also
been used to study the bromodomain-containing transcriptional
coactivating protein TRIM24. Conversion of the TRIM24 ligand
IACS9571 (Palmer et al., 2016) into a PROTAC (Gechijian et al.,
2018) generated a potent TRIM24 degrader. Interestingly,
although IACS9571 inhibits TRIM24 binding to hyperacetylated
chromatin, it fails to induce a strong transcriptional response.
However, TRIM24 degradation via PROTAC-mediated VHL
recruitment significantly upregulates tumor suppressor genes
in AML cells, demonstrating the TRIM24 dependency of acute
leukemias.
BRD9 Is Critical for Synovial Sarcoma
Having developed exquisitely selective BRD9 PROTACs
(Remillard et al., 2017) to study this target’s role in the BRG1/
BRM-associated factor (BAF) complex, the degrader was em-
ployed to confirm the dependence of synovial sarcoma on
the non-canonical BAF complex associated with expression
of the SS18-SSX oncogenic fusion protein and/or loss of
SNF5 (Brien et al., 2018). Interestingly, malignant rhabdoid
tumors also lack SNF-5 and rely on BRD9-containing non-ca-
nonical BAF complexes, as evidenced by PROTAC-mediated
BRD9 depletion (Michel et al., 2018). This demonstrated
that, upon loss of SNF-5, cancer cells remodel the BAF com-
plex. PROTACs for other components of the BAF complex
have also been reported recently (Farnaby et al., 2019).
FAK Scaffolding Roles Are Crucial for Cell Migration but
Not Proliferation
Given that patient studies with focal adhesion kinase (FAK) inhib-
itors have failed to live up to their preclinical promise, Cromm
et al. (2018) incorporated the FAK inhibitor defactinib into a
PROTAC to assess the effect of FAK degradation versus inhibi-
tion (Cromm et al., 2018). Cell culture studies revealed that,
although FAK kinase inhibition is unable to prevent cell migration
in wound healing assays and invasion in trans-well assays,
FAK degradation was efficacious, underscoring the kinase-inde-
pendent functions of FAK. Interestingly, both this work and a
concurrent study from Boehringer Ingelheim (Popow et al.,
2019) failed to phenocopy the antiproliferative effect of short
hairpin RNA (shRNA)-mediated FAK depletion (McDonald
et al., 2017).
Tag-Based Systems
Development of a bespoke PROTAC for a potential target may
be beyond the means of many academic laboratories; therefore,
tag-based methods have been developed to combine genetic
modification with the power of PROTAC technology. The two
most commonly used PROTAC systems are discussed below.
HaloPROTACs
The HaloPROTAC system utilizes the HaloTag protein, an
engineered bacterial dehalogenase that allows orthogonal,
covalent conjugation of a chloroalkane-labeled molecule to a
fusion protein of interest (Los et al., 2008). Both VHL-recruiting
(Buckley et al., 2015; Tovell et al., 2019) and cIAP-recruiting
HaloPROTACs (Tomoshige et al., 2015, 2016) have been re-
ported to induce degradation of various HaloTag fusion sub-
strates, including cytosolic (ERK, MEK, and GFP), endosomal
(VPS34 and SGK3) and nuclear-localized (CREB1) proteins.
The HaloPROTAC system has also afforded biological insights
into multiple systems, as detailed below.
The Role of PNPLA3 in Fatty Liver Disease
BasuRay et al. (2019) used HaloPROTAC3 to degrade HaloTag
fused with patatin-like phospholipase domain-containing
protein 3 (PNPLA3) in vivo. An I148M mutation in PNPLA3 is
associated with non-alcoholic fatty liver disease, which leads
to steatosis via accumulation of triglycerides into lipid droplets

Cell 181, April 2, 2020 105

(Smagris et al., 2015). The I148M mutation results in an approx-
imately 80% reduction in triglyceride hydrolase activity, but,
surprisingly, the presence of the reduced or non-catalytic
PNPLA3 protein is required for development of hepatic
steatosis. PROTAC-mediated in vivo degradation of I148M
PNPLA3 returned liver triglycerides in mice to normal, providing
additional evidence that mutant PNPLA3 accumulation is
responsible for hepatic steatosis.
Kinetics of WASH Complex Formation
The HaloPROTAC system has also been used to confirm that
heat shock factor binding protein 1 (HSBP1) is an assembly
factor for the Wiskott–Aldrich syndrome protein and SCAR ho-
molog (WASH) complex, which plays a key role in endosomal
sorting. Degradation of a HaloTag:WASH fusion protein and sub-
sequent siRNA knockdown of HSBP1 during PROTAC washout
demonstrated that HSBP1 is the assembly factor required for re-
modeling of CCDC53 homotrimers into the WASH complex (Vis-
weshwaran et al., 2018). This approach highlights the use of
PROTACs to enable temporal control of protein levels, enabling
pulse-chase experimentation that may otherwise be challenging.
dTAG
The dTAG system works in a similar way as HaloPROTACs, but
instead of tagging protein with the HaloTag protein, the F36V
FKBP mutant protein (Clackson et al., 1998) is fused to the pro-
tein of interest. The dTAG system uses an F36V-selective
‘‘bump’’ ligand, tethered to a immunomodulatory imide drug
(IMiD) derivative to recruit CRBN and subsequently degrade
the FKBP fusion protein (Nabet et al., 2018). This system has
been shown to be amenable to in vivo experimentation via intra-
peritoneal (i.p.) injection at 25 mg/kg. Proof-of-principle studies
have shown dTAG to be amenable to a wide variety of proteins,
including HDAC1, MYC, EZH2, PLK1, and KRASG12V, and the
system has been employed to address biological questions, as
exemplified below.
Basal-like Breast Cancer Cells Are Not MELK Dependent
Previous studies using shRNA silencing suggested that basal-
like breast cancer (BBC) cells depend on maternal embryonic
leucine zipper kinase (MELK) expression for survival (Hebbard
et al., 2010; Touré et al., 2016). However, a thorough study em-
ploying selective MELK inhibitors, CRISPR, and PROTAC-medi-
ated degradation discovered that BBC cells are agnostic to
MELK levels (Huang et al., 2017). Huang et al. (2017) employed
CRISPR gene editing to knock out MELK and observed no effect
on proliferation. Similarly, MELK inhibitors exhibited no antipro-
liferative activity. Concerned that compensatory signaling arose
during the selection and/or cloning procedures to explain their
observations, they also introduced a dTAG version of MELK prior
to knockout of endogenous MELK so that MELK was constantly
expressed, circumventing any impetus for compensation to
arise. Employment of a dTAG PROTAC to rapidly and selectively
induce dTAG-MELK degradation showed no effect on prolifera-
tion despite acute MELK loss, confirming that BBC cells are not
MELK dependent.
Cytosolic Mutant Nucleophosmin Is Crucial for
Leukemogenesis
Brunetti et al. (2018) used CRISPR/Cas9 gene editing to demon-
strate that mutant nucleophosmin (NPM1) relocalization, via nu-
clear export sequence disruption, could suppress cell prolifera-
106 Cell 181, April 2, 2020
tion and induce differentiation of hematopoietic stem cells. This
occurs via disruption of the HOX/MEIS1 transcriptional program,
consistent with the hypothesis that HOX/MEIS1 genes are
master regulators of the hematopoietic lineage (Argiropoulos
et al., 2007). To further confirm that lack of cytosolic NPM1 in-
duces this phenotype, Brunetti et. al. (2018) employed the
dTAG system to rapidly deplete NPM1 (>85% loss in 4 h). This
resulted in the same phenotype, confirming direct correlation
between HOX/MEIS1 expression downregulation and lack of
cytosolic NPM1.
The dTAG system has also been used to confirm the addiction
of acute myeloid leukemia cells to ENL(YEATS) domain-contain-
ing protein 1 following its identification in a genome wide
CRISPR screen (Erb et al., 2017) and to demonstrate that degra-
dation of SNF5 allows re-association of cMyc with chromatin
(Weissmiller et al., 2019). It has also been used to demonstrate
that YY1 has no direct role in the modulation of replication
timing (Sima et al., 2019) and to demonstrate that OCT4 is crucial
for localization of Mediator condensates at super-enhancers in
embryonic stem cells (Boija et al., 2018).
HaloPROTAC versus dTAG
Both HaloPROTAC and dTAG are powerful approaches and
could conceivably be used simultaneously because of their
orthogonality. However, there are subtle differences between
the two systems that may determine which system is most
suitable for each application. For example, dTAG requires a
smaller tag (FKBP12F36V, 12 kDa) to be incorporated into the
target protein than HaloTag (33 kDa), which may favor the use
of dTAG in congested systems where tags may perturb pro-
tein-protein interactions. However, the commercially availability
and multitude of other uses for HaloTag fusions (England et al.,
2015) makes it the perfect choice when, for example, one
wishes to look at subcellular localization of a protein as well
as induce its degradation. Furthermore, the HaloPROTAC sys-
tem does not exhibit the ‘‘hook effect’’ observed with dTAG.
Additionally, the use of VHL ligands in the HaloPROTAC
approach enables the use of diastereomer controls and avoids
issues associated with the use of IMiD-based PROTACs, dis-
cussed below.
Workflow
To use the PROTAC system, it is necessary to either develop
de novo a PROTAC capable of targeting the protein of interest
or to modify the latter with a tag (described above) to enable
degradation via HaloPROTAC or dTAG degrader molecule.
Indeed, it may be advantageous to perform preliminary studies
with the tagged target protein to establish proof of concept prior
to developing a PROTAC to target the endogenous protein.
Given the recent advances in genome editing, it is relatively
easy to tag a protein of interest at an endogenous locus to
enable its ligand-induced degradation without the necessities
of target overexpression or developing novel ligands (Brand
and Winter, 2019). Displayed in Figure 3 is a typical workflow
to develop a PROTAC. The workflow begins, obviously, with se-
lecting a target protein to degrade.
Target Selection
Some ideal PROTAC targets are (1) proteins without enzymatic
function that cannot be modulated with traditional small

Figure 3. PROTAC Development Workflow
molecules, (2) proteins that have additional roles beyond enzy-
matic activity (e.g., scaffolding roles of FAK or BCR-ABL),
or (3) proteins that, upon inhibition, undergo compensatory
upregulation, making it difficult to achieve maximal loss of
protein function. It may be useful to look at previous knockdown
experiments (e.g., phenotypic genome-wide CRISPR screens
or RNAi) or for cell type-specific dependencies during target
selection to gather information about the target protein (Tsher-
niak et al., 2017).
There are some proteins for which inhibition is sufficient to
abrogate all function and degradation adds little in terms of
biology, but these proteins are likely few and far between. In
these cases, PROTACs can nevertheless be beneficial in
terms of reduced dosage and duration of effect. More excitingly,
PROTACs can extend the druggable proteome to any protein
that is ligandable. Of the approximately 17,470 observed
proteins (Omenn et al., 2018), only 10%–15% are considered
druggable (Hopkins and Groom, 2002), and many more have
been shown to be ligandable (Backus et al., 2016; Parker et al.,
2017), but many of these small-molecule binding events enact
no function. The ability to impart activity to otherwise biologically
inert ligands via PROTAC conversion greatly expands the
druggable proteome.
PROTAC Development
Subsequently, a thorough literature investigation for known
target ligands should be performed. If a ligand is available,
then inspection of either a co-crystal structure or known
structure-activity relationships may reveal a suitable location
for linker attachment. At this point, PROTAC conversion can
begin, employing synthetic and medicinal chemistry to append
a linker and E3 recruiting element to the targeting ligand. If no
ligand has been reported previously, or if the reported com-
pounds have questionable structures, then a campaign to iden-
tify a ligand for the target protein may be initiated; alternatively,
it may be simpler to initially use the HaloPROTAC or dTAG
approach. We recommend use of multiple linker lengths and
compositions when developing PROTACs, especially when
the synthetic approach allows modularity. Linker length
and composition can have profound effects; for example,
HaloPROTACs with an ethylene glycol unit removed have no
ability to induce degradation (Buckley et al., 2015), BCR-ABL
PROTACs with an ether replacing an amide demonstrate
more cell permeability (Burslem et al., 2019), and lapatinib-
based PROTACs can be either dual EGFR/Her2 degraders or se-
lective EGFR degraders, depending on linker length (Burslem
et al., 2018b).
When the PROTAC candidate molecules or cell line ex-
pressing tagged proteins are available, it is crucial to confirm
first that they do indeed induce target degradation. Most
commonly this is achieved by immunoblotting, although mass
spectrometry or flow cytometry can be used, depending on
the target protein properties (Buckley et al., 2015). Iterative
rounds of screening may be necessary to synthesize a suffi-
ciently potent PROTAC, or it may be necessary to explore
several tagging approaches and/or sites of tag incorporation
into the target for maximal degradation. We generally recom-
mend an initial PROTAC treatment time of 24 h, but kinetic char-
acterization often reveals that degradation occurs much more
rapidly. Recently, bespoke technologies for the study of PRO-
TACs in cells have been developed that may prove to be instru-
mental in guiding their development (Riching et al., 2018). The
synthesis and assay of candidates is normally the rate-limiting
step for use of a PROTAC and depends on the quality of the
chemical matter available for the target. Starting with a well-
characterized small molecule provides a distinct advantage,
and as more become available via the Target 2035 initiative
(Carter et al., 2019), the rate and ease of PROTAC development
will no doubt increase.
PROTAC Validation
When a PROTAC is identified, it is crucial to carefully charac-
terize the degradation event via control experiments—e.g., via
qPCR confirmation that protein loss occurs at a post-transla-
tional level and not by decreases in mRNA (Bondeson et al.,
2015)—prior to its use as a tool. Some small molecules induce
apparent degradation of their target protein but actually act at
the transcriptional level (Field et al., 2017). It is also crucial to
confirm that degradation is occurring via the UPS. To do this,
cells can be co-treated with pharmacological modulators of
the UPS. We specifically recommend co-treatment with a
proteasome inhibitor (epoxomicin [Kim and Crews, 2013] or bor-
tezomib [Paramore and Frantz, 2003]), which should preserve
Cell 181, April 2, 2020 107
protein levels at untreated levels. Additionally, co-treatment with
MLN-4924 (pevonedistat), a NEDD8-activating enzyme inhibitor,
will confirm that an active E3 ligase is involved in degradation
(Cullin ligases require neddylation for activity) (Brownell et al.,
2010). Because of the toxicity of these control compounds, it is
often necessary to use the shortest incubation time required
for significant degradation (e.g., 5 h versus 24 h).
Importantly, degradation by application of a small molecule
enables use of an inactive analog as a control compound.
This is crucial for discovery of novel biology when employing a
PROTAC derived from an inhibitor. Typically, an inactive
PROTAC can be created by discrete modification of the E3 ligase
ligand structure to compromise its activity; the resulting
molecule is incapable of inducing degradation but equally
potent as the PROTAC at inhibiting the target. We favor use of
the VHL-recruiting ligand for careful interrogation of inhibition
versus degradation because the simple inversion of stereocen-
ters on the VHL ligand yields a compound with equal inhibitory
activity and pharmacological properties, such as cell perme-
ability, enabling it to function as a true control (Burslem et al.,
2018b). Inversion of stereocenters can also be employed to
generate inactive compounds as controls for MDM2-recruiting
PROTACs (Hines et al., 2019). It is possible to compromise the
CRBN-recruiting IMiD ligands via omission of a carbonyl or by
methylation of the imide functionality; however, this does subtly
modulate its physicochemical properties (Burslem et al., 2018a;
Huang et al., 2018). Care must be taken when using IMiD
analogs because they may still induce degradation of zinc-finger
neo-substrates associated with IMiD pathophysiology (Ishoey
et al., 2018).
Inactive PROTACs are important experimental controls that
can confirm or disprove the PROTAC mechanism of degrada-
tion, given that ligand binding alone could destabilize the target
without necessarily recruiting an E3 ligase (Huang et al., 2017),
such as is the case with hydrophobic tagging or SERDs (Gustaf-
son et al., 2015; Neklesa et al., 2011; Wardell et al., 2011).
An additional, optional experiment to conduct at this point is
a proteomic characterization of the effect induced by PROTAC
treatment. Mass spectrometry or reverse-phase protein arrays,
for example, can enable the user to acquire selectivity data
for the PROTAC. Quantitative proteomics is a powerful tool
to identify other proteins, beyond that targeted, that are downre-
gulated by PROTAC treatment. This may, of course, be a biolog-
ical result of loss of the target protein, such as with c-Myc loss
when BRD4 is degraded (Lu et al., 2015; Winter et al., 2015), or
a pharmacological result, such as the unexpected degradation
of p38a by foretinib-based PROTACs (Bondeson et al., 2018;
Smith et al., 2019).
Advantages and Disadvantages of PROTACs
Advantages
Catalytic Mechanism of Action. Because PROTACs operate via
an event-driven rather than an occupancy-driven mechanism,
they act as catalysts for selective protein degradation in that
one molecule of PROTAC induces the ubiquitination of multiple
molecules of target protein (Bondeson et al., 2015). Indeed,
recent studies demonstrate that as little as 10% E3 ligase
occupancy is able to efficiently induce target degradation
108 Cell 181, April 2, 2020
(Zhang et al., 2019b), albeit with covalent modification of the
E3, creating a permanently reprogrammed E3 ligase. Limited
occupancy on the targeting ligand side has also been demon-
strated to be sufficient via use of low-affinity ligands (Crew
et al., 2018; Smith et al., 2019) or co-treatment with a compet-
itive agonist (Salami et al., 2018). This catalytic nature of
PROTACs mimics RNAi.
Small-Molecule Nature. Their small-molecule nature makes
PROTACs as simple to use in experiments as inhibitors. No
specialized transfection reagents, culture conditions, or viruses
are required for PROTAC application. Indeed, with the exception
of tag-based systems, use of PROTACs requires no genetic
modification of the model system, allowing interrogation of
completely endogenous systems. This allows direct comparison
of degradation versus inhibition with the same methodology,
resulting in an enhanced biological understanding of protein
function and abrogating the risks of perturbing complex biolog-
ical systems with, for example, transfection reagents that may
either induce effects of their own or mask subtle effects of
RNAi (Jacobsen et al., 2009).
Another advantage arising from the small-molecule nature of
PROTACs is control over the concentration of compound,
enabling quantitative control of protein levels. Rather than
employing a ‘‘digital’’ on/off switch, such as observed with
gene editing, a PROTAC can be used in an ‘‘analog’’ fashion to
modulate protein levels between 0%–100%. This may be partic-
ularly powerful to address proteins that are overexpressed in a
disease state but nevertheless essential in a normal or healthy
state. Following a dose-response experiment, it is possible to
treat at a dose that induces sub-maximal degradation, poten-
tially restoring overexpressed protein levels down to normal.
Temporal Control. PROTACs allow exquisite temporal control
of protein levels (degradation can be observed in as little as 1 h),
allowing study of acute protein loss in a way not possible with
many other techniques. This prevents biological compensation
from arising upon deletion of a key protein during selection, as
happens with various RNAi techniques as well as CRISPR/
Cas9. A common CRISPR technique is to employ homology-
directed repair (HDR) to insert GFP or resistance genes at the
perturbed loci (Leonetti et al., 2016), allowing selection of cells
that were successfully edited. Although undoubtedly powerful,
this multi-day selection could conceivably select a cellular
population with an alternative pathway driving proliferation or
circumnavigation of issues arising from loss of a particular
protein. PROTACs are able to rapidly deplete a protein in an
entire cellular population, providing a more accurate method to
interrogate functions of a protein in a native context.
Additionally, PROTAC withdrawal allows rapid restoration of
target protein levels as fast as protein re-synthesis permits.
This provides a reversibility enabling elegant experiments,
such as those described above for the WASH complex. Although
some PROTACs may survive in cells after washout of compound
and continue to enact degradation, concurrent addition of
excess E3 ligase ligand can competitively prevent any additional
degradation (Burslem et al., 2018b).
Portability. A further benefit of the PROTAC approach lies in its
portability. Generally, if a PROTAC induces ubiquitination and
degradation of a protein within one system, then it can be applied
more broadly, provided the new system has the required
machinery (E3 ligase, etc.). This enables rapid screening of pro-
tein roles in different cell types without requiring their genetic
modification. Furthermore, it enables study of proteins in con-
texts not amenable to other techniques, such as unculturable
patient cells; e.g., CML stem cells, discussed above (Burslem
et al., 2019).
Another advantage of portability lies in the transition to in vivo
experiments and, potentially, clinical translation. Application of
PROTACs in vivo requires no genetic manipulation of the ani-
mals, enabling more rapid progression of experimentation and
the ability to probe unperturbed systems. Although PROTACs
may need optimization of their physico-chemical properties for
in vivo work, there are many examples of PROTACs functioning
in vivo, including in mammals (Bondeson et al., 2015; Burslem
et al., 2018c; Jain et al., 2019; Nabet et al., 2018; Winter et al.,
2015) and non-human primates (Sun et al., 2019). Despite
any potential pharmacokinetic challenges with PROTACs,
their small-molecule nature and portability make them readily
and directly translational in a way other techniques are not. It
is important to note that PROTACs often exhibit better pharma-
cokinetic properties than would be anticipated from their molec-
ular weight. In fact, is has been possible to develop PROTACs
with oral bioavailability in humans, as evidenced by recent re-
ports of phase 1 clinical trials of ARV-110 and ARV-471, which
achieved exposures in the efficacious range observed in preclin-
ical studies when administered orally once per day.
Disadvantages
Discovery Phase. Despite all of the advantages outlined above
for PROTACs, they are not without associated challenges. One
major disadvantage is the lead time for PROTAC development,
which can be relatively lengthy compared with designing
and ordering an siRNA or guide RNA. As outlined in Figure 3,
PROTACs require not only a ligand for the protein of interest
but also its conversion into a PROTAC. This can be a time-
consuming process involving substantial synthetic and medici-
nal chemistry. The tag-based systems (HaloPROTACs and
dTAG) bypass this discovery phase but also abrogate some of
the advantages of the PROTAC system concerning portability
and lack of genetic manipulation.
Off-Target Effects. As with other techniques, off-target effects
with PROTACs are possible. Following PROTAC treatment, the
use of proteomics to quantify proteins, including those ex-
pressed at low levels, is a powerful tool to assess the off-target
effects of PROTACs (Savitski et al., 2018). Even when the recruit-
ing element selectivity is known, proteomics can reveal surpris-
ing new PROTAC substrates, likely resulting from the additive
effects of protein-protein interactions between the protein in
question and the E3 ligase (Bondeson et al., 2018). In theory,
ligand binding to an E3 ligase could perturb binding of endoge-
nous substrates. Fortuitously, much higher ligand concentra-
tions than typically employed with VHL-recruiting PROTACs
(Frost et al., 2016) are required to stabilize its endogenous target,
HIF-1a (Burslem et al., 2017); however, off-target effects could
be more problematic with other E3 ligases for which the native
substrates currently are poorly characterized or unknown. The
off-target effects of CRBN-recruiting PROTACs must be very
carefully assessed because the IMiD components, alone or
incorporated into PROTACs, can induce degradation of zinc-
finger CRBN neo-substrates (Fischer et al., 2014; Ishoey et al.,
2018; Krönke et al., 2014; Matyskiela et al., 2016).
Hook Effect. PROTACs and other bifunctional molecules
exhibit an initially curious but entirely rational phenomenon
whereby higher concentrations do not always result in more ef-
fect (Douglass et al., 2013). This so-called hook effect results
from formation of unproductive dimers at high PROTAC concen-
tration rather than the productive trimeric complex required for
degradation. This leads to concerns surrounding pharmacoki-
netic/pharmacodynamic (PK/PD) relationships and dosing regi-
mens. However, it should be noted that favorable protein-protein
interactions between the E3 and target protein appear to in-
crease the maximum concentration that can be used before
the hook effect is observed (Buckley et al., 2015; Tovell
et al., 2019).
Not All Proteins or Subcellular Locations Are Amenable Yet.
Finally, because PROTACs are still an emerging technology,
they have not yet been demonstrated to function against every
protein class or in every subcellular compartment (Figure 4).
Cytosolic and nuclear proteins can routinely be degraded (Bon-
deson et al., 2015); indeed, some E3 ligases allow selective nu-
clear degradation (Zhang et al., 2019b). Several examples of re-
ceptor tyrosine kinase degradation have been reported (Burslem
et al., 2018b, 2018c), but no examples of more than single-pass
transmembrane proteins have been reported. HaloPROTACs
have been employed to degrade proteins localized to the
membranes of endosomes (Tovell et al., 2019). Despite hydro-
phobic tagging experiments having validated unfolded protein
responses in both the Golgi and the endoplasmic reticulum
(ER) (Hellerschmied et al., 2019; Raina et al., 2014; Serebrenik
et al., 2018), degradation of proteins via PROTAC has yet to be
reported. Bespoke ligands for ligases localized to these organ-
elles will likely be required to enact PROTAC-mediated degrada-
tion in these subcellular locations (Smith et al., 2011).
Comparison with Alternatives
In this section we will briefly compare PROTACs with other cur-
rent technologies that enable the study of protein function and
discuss applications where one technique may be preferable.
RNAi
RNAi has become ubiquitous in modern biological research
and is an excellent tool to study protein function (Agrawal
et al., 2003; Setten et al., 2019). PROTACs in many ways func-
tion as chemical siRNA equivalents, although they do differ
slightly. PROTACs have the advantage of being experimentally
less complex than siRNA because no transfection reagents
are required. However, the ease of PROTAC use is juxtaposed
with their rigor of development. Thus, for large target libraries,
siRNA may be advantageous, whereas, for study of one
particular protein, PROTACs have the advantage. Both tech-
niques are ‘‘knockdown’’ approaches and suffer from potential
off-target effects, but given the common use of proteomics to
characterize PROTACs, the off-targets may be better known.
Indeed, PROTACs have been used to invalidate hits from
siRNA experiments (Huang et al., 2017; Nunes et al., 2019; Po-
pow et al., 2019). It may also be possible to achieve greater
overall reduction in protein levels, particularly for long-lived
Cell 181, April 2, 2020 109

proteins, with PROTACs compared with RNAi because PRO-
TAC activity targets the protein rather than preventing addi-
tional expression.
Gene Editing
CRISPR/Cas9 and related gene editing techniques are certainly
efficient in experiments where long-term protein depletion
is required. However, the requisite selection is both time
consuming and allows biological compensation of the protein
loss. PROTACs provide an advantage in terms of kinetics, allow-
ing study of acute protein loss to expedite workflow and poten-
tially provide more relevant details about the endogenous
system. Gene editing provides protein knockout as long as
each copy of the gene is edited. This is challenging in polyploid
cells (e.g., HeLa cells), but because PROTACs act post-tran-
scriptionally, this is not a concern with their use.
Post-Translational Protein Degradation
There are many other methods for small-molecule-induced
post-translational protein degradation (for example, auxin-
induced degradation and the Shield-1 system), which we
have reviewed in detail elsewhere (Burslem and Crews,
2017). However, these rely on the expression of tagged
proteins and/or expression of other endogenous proteins;
PROTACs do not. Recently, the Trim-Away system was re-
ported (Clift et al., 2017), employing antibodies to target
the E3 ligase TRIM21 to proteins, leading to their degradation.
However, this requires the expression of TRIM21 protein and
for the antibody to be microinjected or electroporated into
the cell, thus lacking the small-molecule advantages of
PROTACs.
Conclusions
In this Primer, we demonstrate, with examples and an expla-
nation of the technology, the power of PROTACs as tools to
probe biological systems as well as to be potential therapeu-
110 Cell 181, April 2, 2020
Figure 4. Diagram of a Typical Mammalian
Cell, Denoting Locations of Proteins that
Are Susceptible to PROTAC-Mediated
Degradation
Solid lines represent locations that have been
targeted with PROTACs. Dashed lines represent
locations that have not yet been targeted with
PROTACs.
tics. This targeted protein degradation
approach adds a new tool to the exper-
imental biology toolbox, combining the
benefits of RNAi and small-molecule in-
hibitors and providing complementary
orthogonality to the pre-existing tools.
We hope that PROTACs will become a
mainstay in protein function investiga-
tion, and we believe that they enable
us, as a community, to address biolog-
ical questions that are currently intrac-
table. For example, the reversibility
and kinetic advantages of PROTACs
provide tools to temporally control
protein levels with much higher resolution that other ap-
proaches. This provides the opportunity to compare acute
versus chronic loss of a protein and to study the effect of
reintroducing that protein. The ability to deplete a protein
for a defined time and then restore it to endogenous levels
as fast as transcription allows may prove to be useful in
the study of protein complex assembly, particularly with
respect to order of association. As the PROTAC technology
continues to mature, the disadvantages described above
will become less significant. For example, currently the
largest challenge in the use of PROTACs is the length of the
discovery phase. As we continue to develop enhanced knowl-
edge of the UPS, of the requirements for potent PROTACs,
and of ligands for protein targets, the discovery phase will
be significantly shorter. Similarly, as ligands for additional E3
ligases become available, the likelihood of successful
PROTAC development increases, and more cellular locations
become available.
ACKNOWLEDGMENTS
G.M.B. and C.M.C. thank John Hines, PhD, for editing. G.M.B. is a Fellow of
The Leukemia and Lymphoma Society. C.M.C. gratefully acknowledges the
American Cancer Society and the NIH for support (R35CA197589).
DECLARATION OF INTERESTS
C.M.C. is a founder, consultant, and shareholder in Arvinas, which supports
research in his lab. C.M.C. is an inventor of the following patents: 9500653
(Small-Molecule Hydrophobic Tagging of Fusion Proteins and Induced Degra-
dation of Same), 9632089 (Small-Molecule Hydrophobic Tagging of Fusion
Proteins and Induced Degradation of Same), 10145848 (Small-Molecule Hy-
drophobic Tagging of Fusion Proteins and Induced Degradation of Same),
9938264 (Proteolysis-Targeting Chimera Compounds and Methods of Prepar-
ing and Using Same), 7041298 (Proteolysis-Targeting Chimeric Pharmaceu-
tical), 7208157 (Proteolysis-Targeting Chimeric Pharmaceutical), and
10071164 (Estrogen-Related Receptor Alpha-Based PROTAC Compounds
and Associated Methods of Use).
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