REVIEW
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Cell Research (2016) 26:484-498.
www.nature.com/cr
Drugging the undruggables: exploring the ubiquitin
system for drug development
Xiaodong Huang
1
, Vishva M Dixit
2
1
Department of Discovery Oncology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA;
2
Department of Physio-
logical Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA
Dynamic modulation of protein levels is tightly controlled in response to physiological cues. In mammalian cells,
much of the protein degradation is carried out by the ubiquitin-proteasome system (UPS). Similar to kinases, com-
ponents of the ubiquitin system are often dysregulated, leading to a variety of diseases, including cancer and neuro-
degeneration, making them attractive drug targets. However, so far there are only a handful of drugs targeting the
ubiquitin system that have been approved by the FDA. Here, we review possible therapeutic intervention nodes in
the ubiquitin system, analyze the challenges, and highlight the most promising strategies to target the UPS.
Keywords: ubiquitin-proteasome system; drug target; degrader; PROTAC; hydrophobic tag; SERD
Cell Research (2016) 26:484-498. doi:10.1038/cr.2016.31; published online 22 March 2016
Correspondence: Xiaodong Huang
a
, Vishva Dixit
b
a
E-mail: huang.xiaodong@gene.com
b
E-mail: dixit@gene.com
Introduction
Ubiquitination is a post-translational modification,
where a small protein, ubiquitin, is covalently attached
to lysine residues on a substrate protein [1]. This mod-
ication is carried out sequentially by a cascade of en-
zymatic reactions involving an intimate collaboration
between E1 activating, E2 conjugating and E3 ligating
enzymes. Ubiquitin is first activated by E1 and enters
into a thioester linkage with the catalytic cysteine; it is
then transferred through a trans-esterification reaction
to an E2 conjugating enzyme. Subsequently, E3 ligases
either behave as bona fide enzymes (HECT E3s), or a
“matchmaker” (RING E3s), to transfer ubiquitin from a
charged E2 to substrates, facilitating the formation of an
isopeptide bond between the C terminal glycine of ubiq-
uitin and substrate lysine residue [1] (Figure 1).
Since ubiquitin itself has seven lysine residues, this
modication can be dispersed and propagated, by trans-
ferring additional ubiquitin molecules to one of the seven
lysine residues or the N-terminal amino group, to form
eight homogeneous or multiple mixed or branched chain
types [1]. Depending on the chain topology, ubiquitina-
tion can lead to dierent biological outcomes. For exam-
ple, K48 and K11 chains are related to degradation by
the proteasome [2-4], whereas K63 and linear ubiquitin
chains have a scaolding role for signaling assemblies
and play a prominent role in many biological processes,
including inammation [3, 5].
Like other post-translational modications, ubiquitina-
tion is reversible and countered by ~100 deubiquitinases
(DUBs) encoded in the human genome [6, 7]. DUBs
are proteases composed of five sub-families, including
ubiquitin carboxyl-terminal hydrolases (UCH), ubiquitin
specific proteases (USP), ovarian tumor like proteases
(OTU), JAMM/MPN metalloproteases and Machado-Ja-
cob-disease proteases (MJD). All DUBs are cysteine pro-
teases other than the JAMM/MPN metalloproteases [6].
Since ubiquitination regulates a variety of complex
cellular processes ranging from protein degradation to
modulating protein-protein interactions, from endocyto-
sis to cell cycle progression, from activating to inactivat-
ing substrates, it is not surprising that one or more com-
ponents in the system could go awry, leading to a variety
of diseases, including cancer and neurodegeneration [8].
For example, mutations in PARKIN, an E3 ligase, are
known to cause a familial form of Parkinson’s disease [9];
and chromosomal translocation of USP6 gene is linked
to aneurysmal bone cyst, a local aggressive osseous le-
sion [10].
The success of the kinase inhibitors in the last two
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decades has prompted the pharmaceutical industry to at-
tempt the same strategy in targeting the ubiquitin system
[11, 12]. However, progress has been slow. So far, only
a handful of small molecules have been successfully de-
veloped. This is largely because most components of the
ubiquitin system do not carry out a readily identiable
enzymatic function with a well-dened catalytic pocket,
making them dicult small molecule targets; secondly,
ubiquitination depends on the dynamic rearrangement
of multiple protein-protein interactions that traditionally
have been challenging to disrupt with small molecules.
In spite of this complexity, with advances in technol-
ogy and better understanding of ubiquitination biology,
industry remains committed to drug development in this
area. Below we will review the involvement of ubiquiti-
nation system in human diseases and the progress that
has been made to target the ubiquitin system. In addition
to inhibitors, we also discuss advances in activating ubiq-
uitination to degrade the most dicult targets.
Targeting E1 activating enzymes
Ubiquitin activating enzymes (UBEs or E1 enzymes)
are at the apex of the ubiquitination cascade. As an ATP-
dependent step, E1 enzymes catalyze the formation of a
thioester bond between the C-terminal carboxyl group
of ubiquitin and the cysteine residue of E1 itself [13]. To
date, there are two ubiquitin E1 enzymes identified in
humans, UBA1 and UBA6, which control ubiquitination
of all downstream targets [14].
Figure 1 Summary of the ubiquitin system and possible intervention nodes. Ubiquitination is an ATP-dependent process
carried out by three classes of enzymes. E1 activating enzymes form a thioester bond with ubiquitin, followed by subsequent
binding of ubiquitin to E2 conjugating enzymes, and ultimately the formation of an isopeptide bond between the carboxyl-ter-
minal glycine of ubiquitin and a lysine residue on the substrate protein, which requires E3 ubiquitin ligases. Multiple interven-
tion nodes in the reaction cascade have been proposed to either block or enhance ubiquitination.
PYR-41 was the rst identied cell permeable inhib-
itor for UBA1 [15]. The structure of PYR-41 suggests it
is an irreversible inhibitor since it is subject to nucleop-
hilic attack and potentially could covalently modify the
active cysteine (Cys
632
) of UBA1 [15]. Similar to PYR-
41, PYZD-4409 is another UBE1 inhibitor based on a
pyrazolidine pharmacophore [16]. Although both PYR-
41 and PYZD-4409 preferentially induce cell death in
malignant cell lines and primary patient samples, the
precise mechanism of action of these compounds and
o-target activities are currently incompletely character-
ized.
In addition to ubiquitin, there are more than a dozen
ubiquitin-like molecules (Ubls) in mammals that are all
activated by an equivalent enzymatic cascade for con-
jugation to their cognate substrates [17]. One of these
Ubl-conjugation pathways involves NEDD8, an Ubl
molecule that shares ~60% sequence similarity with
ubiquitin. Like ubiquitination, neddylated substrates, in
particular cullins – the regulatory scaold of multi-sub-
unit E3-ligases – play a critical role in cell proliferation.
Therefore, a NEDD8 activating enzyme (NAE) inhibitor
was expected to possess anti-cancer therapeutic poten-
tial. The most promising NAE inhibitor, MLN4924, is
currently being evaluated in several phase II studies with
promising preliminary results [18]. MLN4924 induces
cell death due to uncontrolled DNA synthesis during
S-phase of the cell cycle, leading to DNA damage and
induction of apoptosis, suggesting that proliferating tu-
mor cells are more susceptible to NAE inhibition [18].
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MLN4924 interacts with the nucleotide-binding site
within NAE and forms a covalent adduct that mimics
NEDD8-AMP, but cannot participate in subsequent reac-
tions, resulting in the blockage of NAE function [19].
Among all neddylated proteins, cullin family mem-
bers, the core scaffolds of SCF (SKP, Cullin, F-box)
E3 ligases, are best characterized. Neddylation of cul-
lin changes the conformation of the cullin C-terminal
domain and enables ubiquitin transfer [20]. Indeed,
MLN4924 treatment disrupted CRL (Cullin RING ligase)
-mediated protein turnover, resulting in the accumulation
of both oncoproteins as well as tumor suppressors such
as NRF2, p27, and IκB [21-23]. Therefore, the mecha-
nisms of action of MLN4924 is intimately linked to the
attenuation of a multitude of cullin RING E3 ligases.
Although many E1 inhibitors have been developed,
except for MLN4924, none has entered clinical trials,
most likely due to issues of specificity or poor drug-
like properties. Importantly, E1 inhibitors should not be
considered equivalent to proteasome inhibitors, which
induce accumulation of ubiquitinated substrates.
Targeting E2 conjugating enzymes
The E2 ubiquitin conjugating enzymes interact with
numerous downstream E3 ligases to transfer charged
ubiquitin molecules that are in labile thioester linkage
onto substrate proteins [24]. Traditionally, E2 enzymes
were treated as “ubiquitin carriers”, but recent work sug-
gests that these enzymes not only dictate ubiquitin chain
linkage and length of chain, but in many cases also de-
termine substrate specicity [17]. Furthermore, there are
~38 E2 enzymes in mammals, making it a class of targets
with potentially more specicity than E1 enzymes [24].
Given this, targeting E2 enzymes should provide more
selectivity than E1 enzymes.
A compound CC0651, identied to potently inhibit the
ubiquitination of p27
KIP1
by the E3 ligase SCF
SKP2
was
instead discovered to be an allosteric inhibitor of an E2
enzyme, CDC34 [25]. Mechanistically, CC0651 inserts
into a cryptic binding pocket in CDC34 distant from the
catalytic site, causing conformational rearrangement that
interferes with the discharge of charged ubiquitin to ac-
ceptor lysine residues. Despite promising data in vitro,
however, further development of CC0651 has largely
failed due to difficulties in optimization (http://www.
nature.com/scibx/journal/v4/n28/full/scibx.2011.784.ht-
ml#B1).
The UBE2N-UBE2V1 heterodimer is an E2 enzyme
that catalyzes the synthesis of K63-specific poly-ubiq-
uitin chains. UBE2N is the active subunit, whereas
UBE2V1 is an E2 variant that lacks the active site cys-
teine residue [26]. NSC697923 is a small molecule that
inhibits the formation of UBE2N~Ub thioester conju-
gates, thereby blocking transfer of ubiquitin to substrates
[27]. Another UBE2N inhibitor BAY 11-7082 was rst
thought to inhibit IKK since it blocked IκB-α phosphor-
ylation in cells [28], but a recent study suggests that
BAY 11-7082 actually exerts these eects by covalently
modifying the reactive cysteine residues of UBE2N and
possibly several other E2 enzymes [29].
Targeting E3 ligases
The ubiquitin E3 ligase family is the largest family
in ubiquitin signaling with ~700 members identied or
predicted to possess ligase activities [30]. There are three
subfamilies of E3 ubiquitin ligases: RING E3s, which
act as scaolding molecules to bring ubiquitin-charged
E2 enzymes in close contact with their substrates; HECT
E3s, which catalyze the transfer of ubiquitin to their own
cysteine residues and subsequently to substrates, and
a third subfamily, RING-Between-RING (RBR) E3s,
which include PARKIN and ARIH1, and mechanistically
behave as hybrids between RING and HECT [31-33]. As
E3s are a large family of enzymes that use distinct cat-
alytic mechanisms, targeting E3s is anticipated to yield
better specificity, less toxicity and be a more superior
option. It is impossible to cover all eorts to target E3
ligases; instead we will focus on several most promising
examples listed below.
SCF
SKP2
The F-box protein SKP2 forms a complex with CUL1,
SKP1, and a RING nger protein RBX1, together termed
SCF
SKP2
[34]. SKP2 was rst identied as a critical cell
cycle regulator because it ubiquitinates several important
cell cycle regulators, including p27
KIP1
and p21
CIP1
, both
are critical CDK inhibitors [35-37]. SKP2 also plays a
critical role in EGFR-mediated AKT ubiquitination and
membrane recruitment [38]. The oncogenic potential of
SKP2 was suggested by its overexpression in a variety of
human cancers [39, 40]. Importantly, this overexpression
of SKP2 showed an inverse relationship with p27
KIP1
[41,
42]. Furthermore, the protein levels of SKP2 could serve
as a prognostic biomarker, with higher levels predicting
poor patient survival [38, 41, 43].
Given the importance of SKP2 in regulating degrada-
tion of tumor suppressors and its clear oncogenic poten-
tial, inhibiting SKP2 may represent a unique opportunity
for the treatment of dierent types of tumors. Unfortu-
nately, unlike kinases, SCF
SKP2
is a large multi-subunit
complex, and does not possess any obvious cavity for
targeting by small molecules. However, the success of
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developing GDC-199 (venetoclax), an inhibitor for a
protein that does not have enzymatic activity (BCL2) has
convinced many that the time for disrupting protein-pro-
tein interactions might nally be here [44]. Indeed SKP2
does have several potential protein interaction interfaces
that could be explored by small molecules to disrupt in-
teraction with either p27 or SKP1 (Figure 2A).
Pocket 3, for example, is formed jointly by SKP2 and
CKS1, which is essential for p27 binding and ubiquitina-
tion by SKP2 (Figure 2A). The pocket was interrogated
in a virtual screen and 96 hits were confirmed in bio-
chemical and biophysical studies [45]. These compounds
selectively inhibit SKP2-p27
KIP1
interaction, and there-
fore block the degradation of p27
KIP1
.
In another in silico screening eort, compound 25 was
identied to selectively suppress SCF
SKP2
, but not other
SCF E3 ligase activities [46]. Mechanistically, com-
pound 25 disrupts the interaction between SKP1-SKP2
and thus abrogates SCF
SKP2
ligase activity. Although no
crystal structure is available, compound 25 presumably
occupies pocket 1, but not pocket 2 of SKP2, both of
which are critical for SKP1-SKP2 interaction (Figure
2A). However, careful analysis of pocket 1 and com-
pound 25 suggests that the ligand might not fully occupy
the pocket. Interestingly, the available structure of SKP2
lacks the N-terminal 96 amino acids [47]. A potential
explanation is that the missing N-terminal segment could
fold back to buttress compound 25 to ensure a snug t
in the pocket. Despite this caveat, compound 25 exhibits
potent antitumor activities in multiple animal models and
synergistically inhibits tumor survival with chemothera-
peutic agents (Figure 2B) [46], conrming that inhibiting
SCF
SKP2
is potentially benecial for cancer patients.
MDM2
As the guardian of the genome, p53 is arguably one of
the most important tumor suppressors that controls the
regulation and expression of many genes that mediate
cell cycle arrest, DNA repair and apoptosis [48]. Under
physiological conditions, newly synthesized p53 quickly
undergoes ubiquitination and degradation, resulting in
a “futile cycle” and a very low “steady-state” level of
protein. This is largely controlled by a RING nger E3
ligase, MDM2 (murine double minute 2, HDM2 in hu-
man) [49]. In addition to being a transcriptional inhibitor
of p53, MDM2 also tightly interacts with p53 protein
itself by recognizing the N-terminal transactivation do-
main (TAD), allowing p53 to undergo ubiquitination
and subsequent proteasomal degradation [50, 51]. As a
negative regulator of p53, MDM2 is overexpressed in
many cancers by either gene amplication or transcrip-
tional up-regulation [52]. Furthermore, overexpression
Figure 2 SCF
SKP2
as a possible anti-cancer target. (A) The crystal structure of SCF
SKP2
highlights potential interfaces (pockets
1-3) that small molecule inhibitors can bind to and block its E3 ligase function. (B) Compound 25 has been identied to be a
selective SCF
SKP2
inhibitor. It blocks ubiquitination and degradation of p27, as well as ubiquitination and activation of AKT. To-
gether, this compound exhibits potent antitumor activities in multiple animal models.
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of MDM2 has been linked to worse prognosis in dier-
ent types of tumors, correlating with altered p53 protein
levels, although it has not been conrmed whether these
tumors have wild-type or mutant p53 [53, 54].
Its oncogenic potential as well as being a negative
regulator of p53 warrants consideration of MDM2 as an
attractive drug target. Among all the small molecules that
inhibit MDM2, Nutlins, a family of cis-imidazoline ana-
logues identied by high-throughput screening, holds the
greatest potential and is currently being tested in clinical
trials (Figure 3). Importantly, Nutlin treatment showed a
dose-dependent anti-proliferative and cytotoxic activity
that diered between cell lines depending on their p53
status [55]. Furthermore, as anticipated, Nutlin treatment
induced accumulation of wild-type, but not mutant p53
protein.
In addition to Nutlins, a couple of other small mole-
cules have also been identied to disrupt MDM2-p53 in-
teraction. For example, similar to Nutlins, MI-219 binds
MDM2 and blocks its interaction with p53, leading to in-
duction of cell cycle arrest and selective apoptosis in tu-
mor cells [56]. Another promising molecule, RITA (reac-
tivation of p53 and induction of tumor cell apoptosis) has
been shown to prevent the interaction of p53 and MDM2
and to induce p53 accumulation in tumor cells [57]. In
contrast to Nutlins, RITA binds p53 but not MDM2;
therefore it might inhibit many other interactions of p53
that have little to do with ubiquitination of p53.
Although the small molecules disrupting MDM2-p53
interaction hold great potential in restoring p53 function,
one caveat is that they are only efficacious in tumors
harboring wild-type p53, as most p53 mutants are no
longer subject to ubiquitination by MDM2 and become
stabilized [58]. Instead, molecules aiming to restore the
native conformation of p53 mutants and reactivate their
tumor-suppressor function may be of more benet to a
broader spectrum of cancers. For instance: PRIMA-1
and its analog APR-246 covalently modify p53 mutants
through the alkylation of thiol groups, restoring wild-
type conformation and function to mutant p53 [59].
Inhibitor of apoptosis proteins
Inhibitors of apoptosis proteins (IAPs) are a family
of anti-apoptosis proteins that function in part by inhib-
iting caspases. In humans, there are at least eight IAP
family members [60]. All IAP proteins have one to three
baculoviral IAP repeat (BIR) domains that participate in
binding caspases [60]. Most IAPs have a RING domain
at their C-terminus that is required for ubiquitination of
their substrates as well as auto-ubiquitination of some
members including c-IAP1, c-IAP2 and X-linked inhibi-
tor of apoptosis (XIAP) [61, 62]. IAP proteins are impli-
cated in various cancers and attempts are being made to
target them using small molecule inhibitors or antisense
oligonucleotides [63].
SMAC/DIABLO is a mitochondrial protein that is re-
leased to bind and inhibit IAPs during apoptosis, thereby
freeing caspases to activate apoptosis [64, 65]. Initial ef-
fort to generate IAP antagonists was aimed at mimicking
the four amino-terminal residues of mature active SMAC
Figure 3 MDM2 as a potential anti-cancer target. p53 is rapidly ubiquitinated by MDM2 and degraded via the proteasome.
Nutlins (and other MDM2/p53 interaction inhibitors) disrupt the interaction between MDM2 and p53, resulting in accumulation
of p53 and its anti-tumor eect.