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Current Pharmaceutical Design, 2013, 19, 000-000
1
Emerging Role of the Ubiquitin-proteasome System as Drug Targets
Gozde Kar1, Ozlem Keskin1, Franca Fraternali2 and Attila Gursoy1,*
1
Center for Computational Biology and Bioinformatics and College of Engineering, Koc University, Rumelifeneri Yolu, 34450 Sariyer Istanbul, Turkey; 2Randall Division of Cell and Molecular Biophysics, New Hunt's House, King's College London, Guy's Campus,
SE1 1UL London UK
Abstract: The ubiquitin-proteosome system (UPS) regulates a wide range of cellular processes including protein degradation, DNA repair, transcription regulation, and cell signaling. Alterations and mutations in UPS components give rise to various human diseases, most
prominently cancer and neurodegenerative disorders. Here, we review recent advances in UPS-based drug discovery highlighting the
emerging relationships between the UPS and disease and discuss potential future therapeutic interventions. In particular, we focus on recent structural approaches in UPS and explain how the knowledge of protein structural details can guide the design of specifically targeted inhibitors.
Keywords: Ubiquitin-proteosome system, disease, drug design.
1. INTRODUCTION
The ubiquitin-proteosome system (UPS) is essential for the
protein turnover and has a wide variety of functions ranging from
cell-cycle control and transcription to development [1]. Ubiquitin is
a small 76-aminoacid protein that can reversibly bind to and label
other proteins in a highly specific manner. Ubiquitin attachment
takes place via a sequential enzymatic route involving ubiquitin
activation (by E1 enzymes), ubiquitin conjugation (by E2 enzymes), and ubiquitin substrate tagging (by E3 enzymes). In the
first step, ubiquitin is activated by an ATP-dependent E1 and E1
delivers the activated ubiquitin to E2 through a transthiolation reaction. E3s recognize specific target substrates and catalyze the ubiquitin transfer from the E2 to the substrate. Ubiquitin can be conjugated to the substrate as a monomer on one (monoubiquitination) or
more substrate lysines (multiubiquitination) or as a polymer
(polyubiquitination) by forming ubiquitin chains [2]. Seven lysine
residues of ubiquitin can participate in ubiquitin conjugation, leading to ubiquitin chains of different lengths, topologies and functions. Substrates that are tagged by ubiquitin chains conjugated
through the lysine 48 of ubiquitin are targeted by proteosome and
undergo degradation process, whereas chains linked at lysine 63 are
associated with signaling and trafficking events [3]. Importantly,
ubiquitination is a reversible process; the deubiquitinating enzymes
(DUBs) can in fact remove ubiquitins from modified substrates,
thus rescuing the substrate from degradation.
Considering the crucial multiple biological roles of UPS enzymes, it is expected that malfunctioning of any component participating in the regulation of these enzymes would result in disease.
There is in fact accumulating evidence of altered functions of UPS
enzymes in numerous cancer types [4], cardiovascular and viral
diseases [5, 6] and neurodegenerative disorders [7]. In treating such
diseases, developing efficient small-molecule inhibitors targeting
the UPS pathway rather than the single molecules is of crucial importance and has elicited significant interest in recent drug design
studies. In this review, we first summarize role of UPS in human
diseases along with the current approaches in the development of
small-molecule inhibitors targeting UPS components. We then
discuss the potential future therapeutic interventions, focusing
*Address correspondence to this author at the Center for Computational
Biology and Bioinformatics and College of Engineering, Koc University,
Rumelifeneri Yolu, 34450 Sariyer Istanbul, Turkey;
Tel:/Fax: ???????????????????????; E-mail: [email protected]
1381-6128/13 $58.00+.00
particularly on targeting protein-protein interactions and interfaces
of UPS components in disease with the ultimate purpose of designing more efficient and selective inhibitors.
2. TARGETING UPS IN DISEASE
Ubiquitin attachment to proteins (ubiquitination) is a major
post-translational modification that can have profound effects on
protein stability, localization or interaction pattern [8]. Several proteins regulated by ubiquitination control cell signaling events, and
hence are crucial for cell-cycle progression, proliferation and apoptosis [8]. Such signaling processes are frequently altered in cancer
with several tumor suppressors and oncogenes representing the
enzymes of the ubiquitin conjugation and deconjugation pathways.
The first inhibitor targeting the proteosomal degradation of proteins
in cancer is bortezomib, which has illustrated a clinical success and
stimulated further research in the design of specific UPS-based
inhibitors. Subsequently, other UPS enzymes such as E1, E2 and E3
have started to be exploited as targets in disease. Below, we start by
describing the role of each enzyme classes (E1, E2, E3, DUBs and
proteasome) in the UPS pathway and in disease and by listing all
the so far available inhibitors and therapeutic approaches. The UPS
pathway and the various possibilities for therapeutic intervention
are displayed in (Fig. 1).
Targeting E1 Activating Enzymes
The first step of the UPS pathway is ubiquitin activation, performed by E1 enzymes. Ubiquitin (Ub) or ubiquitin-like proteins
(Ubl) are adenylated at their C-terminal glycine residue and react
with the E1 active site thiol to become charged as a thiol ester.
Then, Ub or Ubl are transferred to E2 conjugating enzymes also
through a thiol ester bond. There are eight structurally and functionally similar E1s that can activate Ub and Ubls. Although Ubactivating E1s can charge a variety of E2s, Ubl-activating E1s appear to charge only a single E2 [1].
For inhibiting ubiquitin activation, there are a couple of points
to target (Fig. 1). One of them is to prevent ATP from binding to E1
and hence to inhibit Ub-adenylate formation using small-molecule
inhibitors. Another possibility is to target the E1 active site thiol for
blocking the formation of E1-Ub complex. One example is PYR41
which is a pyrazone derivative designed for inhibiting HDM2dependent p53 ubiquitination. The nitrogen dioxide group of
PYR41 covalently modifies the active site cysteine of E1 while,
notably, it does not inhibit other thiol-dependent enzymes including
many E2s [9]. The downstream effects of PYR41 treatment are NF© 2013 Bentham Science Publishers
2 Current Pharmaceutical Design, 2013, Vol. 19, No. 00
Kar et al.
Fig. (1). UPS mechanism and several potential UPS-based drug targets. Ubiquitin attachment takes place via a sequential enzymatic route. In the first step,
ubiquitin is activated by an ATP-dependent E1 and E1 delivers the activated ubiquitin to E2 through a transthiolation reaction. E3s recognize specific target
substrates and catalyze the ubiquitin transfer from the E2 to the substrate. Substrates that are tagged by ubiquitin chains conjugated through the lysine 48 of
ubiquitin are targeted by proteosome and undergo degradation process, whereas monoubiquitination is associated with signaling and localization events [3].
The deubiquitinating enzymes (DUBs) can remove ubiquitins from modified substrates. There are several potential drug targets in UPS in disease: the activity
of E1, E2, E3 enzymes, DUB and proteasome can be inhibited. Additionally, inhibition by blocking the interface regions of E1-E2, E2-E3 and E3-susbtrate
interactions would provide an efficient means in treating UPS-associated diseases.
B activation, stabilization of p53 and induction of p53-dependent
transcription [9]. Besides PYR41, the nitric oxide (NO)-producing
drug JS-K also inhibits E1-ubiquitin thioester formation through an
interaction between the E1 active-site thiol and NO [10]. Similarly,
this compound results in an increased p53 expression and decreased
levels of total ubiquitinated proteins. E1s can also be inhibited
through adduct formation. One example of such compounds is
Nedd8-activating enzyme (NAE-E1) inhibitor; MLN4924, which is
an adenosine sulphamate analogue [11, 12] that binds to Nedd8
thioster form of NAE-E1 and then forms a covalent adduct with
Nedd8. Blocking NAE by MLN4924 results in the inhibition of
CRL (Cullin-Ring Finger Ligase) activity and thus stabilization of
CRL substrates (such as CDT1), leading to deregulation of DNA
synthesis during the S phase of the cell division cycle and apoptosis
in proliferating cells [11]. MLN4924 has entered Phase II clinical
trials for treating multiple myeloma and non-Hodgkin’s lymphoma.
(~500 or more have been proposed to exist in human) [18], which
suggests that one E2 can recognize several different E3s [19]. The
principles determining the E2-E3 selectivity are unclear and in
many cases it is not known which E3s interact with which E2s [20].
Since the knowledge about the functions of E2s is also limited, it is
difficult to design selective E2 inhibitors. Despite these challenges,
there are emerging approaches for inhibiting E2s (Fig. 1). One example for blocking E1-E2 interaction is a synthetic peptide called
UBC12N26, which was shown to compete for UBC12-NAE binding, hence blocking transthiolation of Nedd8 to UBC12 and preventing the transfer of Nedd8 to the substrate targets [21]. For some
E2s such as UBE2G2, E3 binding can allosterically activate ubiquitination activity, then another potential mode of inhibition may be
the blocking E2-dependent ubiquitin transfer using allosteric effectors. An additional approach is to inhibit catalytic activity of E2s by
targeting the conserved asparagine residue of E2s.
Targeting E2 Conjugating Enzymes
E2s are involved in ubiquitin conjugation and ubiquitin transfer
to the substrates. E2s mediate ubiquitin chain assembly and ubiquitin linkage topology, therefore together with E3s, leading to determination of the fate of the substrates. E2s have been implicated
in many diseases including cancer. For example, UBCH10 was
observed in chromosomal instability [13], UBE2T in lung cancer
[14], UBE2Q2 in head and neck squamous cell carcinoma [15] and
the SUMO E2 UBC9 in ovarian carcinoma and breast cancer [16,
17].
Currently, there is a limited number of known E2s (~40 in human); however the number of known E3s is increasing rapidly
Targeting E3 Ubiquitin Ligases
E3s recognize specific target substrates and catalyze the ubiquitin transfer from the E2 to the substrate. E3s are represented by
three main classes: Homologs of E6AP Carboxy Terminus (HECT),
Really Interesting New Gene (RING), and the UFD2 homology (Ubox) family proteins. HECT is a domain of ~350 amino acids; its
N-terminal lobe contains the E2-binding site, and the C-terminal
lobe confers catalytic activity. The conserved Cys residue in the Cterminal lobe forms thioester complexes with ubiquitin [22]. The
RING finger domain contains a short motif rich in histidine and
cysteine residues that coordinate zinc atoms in a cross-brace structure, characterized by a central -helix and variable-length loops
separated by small beta strands [23]. The U-box domain is similar
Emerging Role of the Ubiquitin-proteasome System
to the structure of the RING domain with the exception that it lacks
the conserved histidine and cysteine residues [24]. The ubiquitination mechanisms for these three E3 classes differ. For HECT domain E3s, the ubiquitin is first transferred from E2 to the active site
residue of HECT E3, with subsequent transfer from E3 to the substrate protein. For RING and U-box type E3s, ubiquitin is directly
transferred from E2 to the substrate without an E3 intermediate
linkage.
Although the human genome encodes numerous E3 ligases [25]
and there are several efforts in identifying functions of E3s and
their association to human disease, progresses in this field are still
in its infancy. In the UPS, E3 ligases are probably the most attractive drug targets since the substrate selectivity relies mainly on the
specificity of E3s. Potential points to target are: E2-E3 binding
interface, E3-substrate recognition and the activity of the E3 itself
(Fig. 1). Below, we present E3s, their alterations in diseases, the
available inhibitors targeting these E3s; a summary is provided in
Table 1.
RING-finger Domain Type E3s
Mdm2 is a RING-finger E3 ligase that mediates ubiquitination
of tumor suppressor protein p53. Mdm2 is an oncogenic protein
whose gene amplification and overexpression are prominently present in human cancers including breast cancer, lung cancers and
many other tumor types [26, 27]. There have been so far a numerous approaches that have proposed compounds disrupting the interaction between E3 and its substrates. One of such inhibitors is Nutlin /R7112, which has entered clinical trials. Nutlin /R7112 targets
the interface of E3 ligase MDM2 (HDM2) and its substrate p53 to
prevent p53 degradation and thus to elevate p53 expression [28].
Similarly, the compound RITA is used to promote p53 stabilization
[29]. Known disease associations include various cancers; breast
and lung, blood cancers and multiple myeloma [30].
Another ligase class that can be targeted in multiple cancers and
disorders linked to the NF-B pathway is cullin-RING E3 ligases
(CRLs). SCFSkp2 is a CRL that ubiquitinates p27 targeting it for
proteosomal degradation [31]. Elevated levels of SCFSkp2 and decreased levels of p27 are observed in some human cancers. Chen et
al. [32] have identified a compound that is active against leukemia
cells and prevent the formation of the functional SCF complex,
which stabilizes p27, induce G1/S cell cycle arrest, and thereby
triggers cell death [32]. SCFTRCP is a CRL that mediates the degradation of IB; the inhibitory component of the proinflammatory
transcription factor NF-B [33]. An inhibitor of SCFTRCP was
found as well, which prevents the polyubiquitination and subsequent degradation of IB [33]. Another example is the inhibitor of
Cdc4, the yeast ortholog of the mammalian Cullin RING E3 ligase
Fbw7, that was identified in a biochemical screen and disrupts binding of Cdc4 to its substrate SIC1 [34]. Inhibition is achieved
through an allosteric mechanism: the inhibitor binds to Cdc4 at a
site distant from SIC1 binding, which induces a large conformational change resulting in a distortion of the substrate binding
pocket and impediment of the substrate recognition [34]. Although
these inhibitors of CRL have not entered into clinical trials yet, they
represent a strong new avenue for treating several human diseases.
The Inhibitors of Apoptosis Proteins (IAPs) are also actively
targeted E3 ligases in disease. They mediate degradation of several
substrates involved in apoptosis and signaling and are linked to
several cancer types such as liver and lung, ovarian carcinoma,
solid and lymphoid tumors [30]. There are a few antagonists of
IAPS such as GDC-0152, LCL161, YM155 and TL32711 that have
entered clinical trials. These compounds are mimetics of SMAC
proteins that antagonize IAPs and promote IAP auto-ubiquitination
and degradation, leading to the death of cancerous cells through
TNF pathway [35].
BRCA1 (Breast cancer type 1 susceptibility protein) is an E3
ligase with tumor suppressor activity that is involved in DNA dam-
Current Pharmaceutical Design, 2013, Vol. 19, No. 00
3
age repair and cell cycle check point control processes. It is mutated
in more than 50% of inherited breast cancers [36] and its mutation
leads to early-onset breast and ovarian cancers and to the development of a malignant phenotype [37]. BRCA1 heterodimerizes with
another RING finger protein, BRCA1 Associated Ring Domain 1
(BARD1) increasing its ubiquitin ligase activity [38].
The Parkin gene encodes a RING finger E3 that can ubiquitinate itself and the -synuclein interacting protein, Synphilin 1. Familial associated mutations in Parkin leads to a deficiency in E3
activity and thus to Parkinson’s disease [39]. Histopathological
hallmark of Parkinson’s disease are the so-called Lewy bodies
which are abnormal protein aggregates developing inside nerve
cells. Lew bodies contain ubiquitin and filamentous aggregates of
denatured proteins including -synuclein [40].
Another E3 ubiquitin ligase is VHL (von Hippel-Lindau) that is
mutated in a familial cancer susceptibility syndrome, von HippelLindau syndrome. This disease is characterized by the development
of various highly angiogenic tumors such as renal cell carcinoma,
pancreatic cysts and tumors [41]. VHL ligase functions as a substrate recognition subunit in the VCB-Cullin 2-VHL complex and
mediates ubiquitination of HIF-1 (Hypoxia-inducible factor-1)
protein [42]. HIF-1 functions in oxygen homeostasis and is a regulator of energy metabolism and angiogenesis processes [42]. Under
normal oxygen levels, HIF-1 is hydroxylated, recognized by VHL
and ubiquitinated by VCB-Cullin 2-VHL complex leading to its
proteasomal degradation. However, hypoxia decreases the capacity
of prolyl hydroxylases to hydroxylate HIF-1, and in turn HIF-1
escapes from VHL-induced degradation, which promotes expression of hypoxia inducible genes such as Glucose Transporter 1 and
Vascular Endothelial Growth Factor (VEGF) [4]. Mutations in
VHL are observed to prevent degradation of HIF-1 which leads to
upregulation of HIF-1-induced genes and to high levels of tumor
vascularization [43]. Consequently, reactivating the VHL ligase
would be a possible strategy to treat VHL-associated tumors. In one
such effort, a bioengineered VHL complex is exploited to increase
HIF-1 degradation [44].
HECT Domain Type E3s
E6-Associated Protein (E6-AP) is a member of HECT E3 ubiquitin ligase family that catalyzes ubiquitination and subsequent
degradation of p53 upon infection by Highrisk human papillomaviruses (HPV) [45]. HPV 16 and 18 are associated prominently with
cervical cancer together with genital and head and neck cancers
[46]. Additionally, mutations in E6-AP are associated with Angelman’s syndrome [47].
ARF-BP1 (HUWE1) is another HECT-type E3 ligase that can
induce p53 ubiquitination and degradation [48]. ARF-BP1 is highly
expressed in breast cancer cell lines and therefore appears to have a
potential role in breast cancer tumorigenesis [49].
Another HECT type E3 is Nedd4-1, belonging to Nedd4-like
subgroup, negatively regulates tumor suppressor Phosphatase and
Tensin Homolog protein (PTEN) by ubiquitinating it for degradation [50]. Overexpression of Nedd4-1 was detected in multiple
human cancer samples and the depletion of Nedd4-1 inhibited
xenograft tumor growth in a PTEN-dependent manner [50]. On the
contrary, one study reported no dysregulation of PTEN protein
level upon Nedd4-1 knockout [51]. PTEN is frequently mutated in
human tumors [52]. A more exact explanation of the role of Nedd41 in regulating PTEN is therefore necessary to elucidate mechanisms of tumor formation and progression.
Smurf2 is also a HECT E3 and has a key function in mediating
TGF signaling pathway [53, 54]. It mediates ubiquitination of
Smad1, Smad2 and TGF receptor 1. High-level expression of
Smurf2 has been observed to correlate with tumor development,
lymph node metastasis and a poor survival rate in esophageal
squamous cell cancer [55].
4 Current Pharmaceutical Design, 2013, Vol. 19, No. 00
Table 1.
Kar et al.
E3 ligases and Disease
E3
Substrate
Associated disease
Alterations in disease
Inhibitor/Treatment
Brca1
NPM1 [170],
RBBP8 [171],
RNA polymerase
[172],
ESR1 [173]
Breast and ovarian cancer [37]
Promoter hypermethylation [174],
genomic deletions and duplications [175]
-
Cervical, genital, head and neck
carcinomas [45, 177], Angelman’s
syndrome [47]
Mutations associated with Angelman’s syndrome [47], infection by
high-risk HPV in cancer [45, 177]
mRNA decay factor TTP promotes
rapid decay of E6-AP and stabilizes p53 in cervical cancer [178]
(RING-finger)
E6-AP
p53 [45],
(HECT)
p27 [176]
IAP
Several substrates
Various cancers including liver,
lung, ovarian carcinoma, lymphoma
Overexpression of IAPs in various
cancers [179]
Many inhibitors targeting IAPs
such as GDC-0152, AEG35156,
YM155, LCL161, AT-406
p53 and p27 [180]
Various cancers including breast and
lung
Overexpression and amplification
[27], point mutations and nucleotide insertions in human tumors
[181]
Nutlins, RITA, Parthenolide
PTEN [50]
Multiple cancers, including bladder,
prostate, colorectal and gastric cancer [50, 182]
Overexpression and mutations in
cancer [50, 182]
-
PARKIN, Synphilin
1 [39]
Parkinson’s disease [39]
Familial associated mutations [39]
(RING-finger)
Nitric oxide to inhibit Parkin’s
ligase activity [183]
SCFTRCP
IB [184]
Multiple cancers and NF-B related
disorders
Overexpression
Inhibitor to prevent SCFTRCP activity [33]
(RING-finger)
Notch [185], c-Myc
[186], c-Jun [187],
cyclin E [188] and
mTOR [189]
Colon, stomach cancer, T-cell acute
lymphocytic leukemia [190]
Mutations in substrate recognition
domain [190]
Allosteric inhibitor of its yeast
ortholog, Cdc4 impedes recognition of the substrate [34]
SCFSkp2
p27 [191]
Multiple cancers including breast
and colon [192], gastric, lung, prostate, ovarian cancer [191]
Gene amplification and overexpression in cancer [193]
Inhibitor to prevent SCFSkp2 activity and stabilize p27 [32]
Esophageal squamous cell cancer
[55]
Overexpression [55]
-
(HECT)
Smad1 [53], Smad2
[54], TGF receptor
1 [194]
VHL
HIF-1 [42]
Renal cell carcinoma, pancreatic
cancer, VHL syndrome [195]
Germline deletion, mutations [43]
Bioengineered VHL complex to
increase HIF-1 degradation [44]
p53 [57], p63 [196],
KLF5 [197],
ERBB4 [198]
Breast and prostate cancer [56]
Overexpression [56]
-
MEKK2, AIF,
TGF-activated
kinase [199, 200]
Breast cancer and acute myeloid
leukaemia [199, 201]
Overexpression [202]
Inhibitor compound AEG35156 to
inhibit XIAP [203]
(RING-finger)
Mdm2
(RING-finger)
Nedd4-1
(HECT)
Parkin
(RING-finger)
SCFFbw7
(RING-finger)
Smurf2
(RING-finger)
Wwp1
(HECT)
Xiap
(RING-finger)
E3 ligases together with their substrates, associated disease, alterations and available inhibitors/treatment are listed.
Wwp1 is a HECT E3 that is aberrantly regulated in cancer.
Overexpression of Wwp1 is observed in breast and prostate cancer
samples [56]. Wwp1 has been shown to regulate p53 ubiquitination
tanslocating p53 to the cytoplasm and diminishing its transcriptional activity [57]. Inactivation of Wwp1, therefore, could activate
p53, which may provide a means for treating cancer.
The HECT E3 ligases may be more amenable to smallmolecule intervention than other classes of E3s as they form cova-
lent thioester intermediates with ubiquitin before transferring ubiquitin to the substrate. Since this step requires a large conformational
change in the HECT domain, it may be possible to block this transition by small-molecule inhibitors [1].
Targeting Deubiquitinating Enzymes
Deubiquitinating enzymes regulate various ubiquitin-mediated
biological processes such as proteosome-dependent degradation,
Emerging Role of the Ubiquitin-proteasome System
Current Pharmaceutical Design, 2013, Vol. 19, No. 00
protein localization and endocytosis by reversing the action of the
ubiquitin conjugation mechanism [58]. Deubiquitinases belong to
five different gene families; four are cysteine peptidases (the ubiquitin-specific proteases (USPs), the ubiquitin C-terminal hydrolases
(UCHs), ovarian tumor domain proteins (OTU), and MachadoJoseph disease protein (MJD), and the fifth family consists of metalloproteinases from the JAMM/MPN domain family [58].
The dysregulation of DUBs is highly observed in human diseases, especially cancer. In recent years, there is a growing interest
in developing inhibitors against DUBs although there has been a
limited success in turning these into drugs [59]. The DUBs have
been extensively reviewed in [59]. Here, we classify the DUBs
according to their domain type, overview advances in targeting
these DUBs and provide a summary in Table 2.
USP Family of Deubiquitinating Enzymes
USP7 (also known as Herpes virus associated USP (HAUSP))
belongs to USP family of DUBs that has a critical role in cell proliferation and differentiation through regulating the activity of
phosphatase and tensin homolog and localization of FOXO [60].
USP7 participates in p53 regulation and catalyzes deubiquitination
of both p53 and Mdm2 [61, 62]. Therefore, USP7 can act as a tumor suppressor or an oncogene depending on whether it predominantly deubiquitinates p53 or Mdm2, respectively [63]. USP7 also
deubiquitinates a tumor suppressor PTEN leading to cytoplasmic
accumulation of this protein [60]. USP7 has been observed to be
overexpressed in prostate cancer and associated with tumor aggressiveness [60], which makes this DUB is an important target for
pharmaceutical intervention [59]. A high-throughput screen identified a small molecule USP7 inhibitor; HBX 41108 that can activate
p53 resulting in p53-dependent apoptosis [64]. A fluorescencebased multiple assay has recently characterized another inhibitor
(P022077) for USP7 [65].
Table 2.
5
Another member of USP family of DUBs is USP8 (UBPY),
which functions in receptor endocytosis and trafficking processes
and interact with several proteins including the epidermal growth
factor receptor (EGFR) [66]. The depletion of USP8 leads to accumulation of ubiquitinated EGFR in endosomes [67]. Although an
inhibitor of USP8; HBX 90397 is identified, the resulting effects
are questionable since knockout of USP8 was observed to cause a
liver failure in adult mice [67, 68].
USP2a deubiquitinates and stabilizes FAS, a metabolic oncogene overexpressed in many cancers [69]. The depletion of USP2a
was observed to destabilize FAS and leads to apoptosis [69]. USP2a
deubiquitinates Mdm2 as well, leading to stabilization of Mdm2
levels and induction of p53 degradation [70]. USP2a deubiquitinates also Mdmx, a Mdm2-like p53 repressor [71]. Interestingly,
USP2a has been observed to have dual functions in disease. While
overexpression of USP protects prostate cancer cells from apoptosis
by reducing p53 stability, on the contrary, another study has illustrated that it is downregulated in breast carcinomas suggesting that
USP2a may have an antitumor property [72].
USP28 is another cancer-associated DUB; it is overexpressed in
breast and colon carcinomas, and is essential regulating the stability
of c-Myc, a central modulator of cell growth, proliferation and
apoptosis [73]. Since c-Myc is dysregulated in many human cancers
and cancer cells require c-Myc activity for growth [74], inhibiting
USP28 may be useful in targeting such malignancies by accelerating degradation of c-Myc [59].
USP14 is a DUB that blocks the degradation of ubiquitinated
substrates. Loss of USP14 is related to neurodegenerative diseases
resulting in an ataxic neurological phenotype in mice [75]. A smallmolecule inhibitor of USP14, called IU1, has been identified [76].
IU1 did not inhibit eight other tested deubiquitinating enzymes
making it a relatively specific inhibitor of USP14 [76]. Treating the
DUBs and Disease
DUB
Substrate
Associated disease
Alterations in disease
Inhibitor
A20
RIP, RIP2, TRAF6, NEMO and
MALT1 [93-98]
Several lymphomas including Hodgkin’s
lymphoma and B cell lymphoma [204]
Promoter methylation, mutations
[204]
-
CYLD
Bcl-3, TRAF2, TRAF6,
TRAF7, RIP1, TAK1 [86-89]
Kidney, colon, B-cell malignancies, multiple myeloma, uterine cervix carcinoma and
melanoma [84]
Mutations, decreased expression
in cancer [84]
-
USP2a
MDM2, FAS and MDMX [6971]
Prostate cancer [205]
Overexpression [205]
-
USP28
c-Myc [73], 53BP1 [206]
Breast and colon cancers [73]
Overexpression, somatic mutations in cancer [73]
-
USP7
p53, MDM2, PTEN, FOXO4
[60-62, 207, 208]
Prostate, colon, non-small cell lung cancer
[60, 82]
Overexpression [60],
USP7 inhibitor,
HBX41108 activates
p53 [64] and
P022077 [65]
USP8
Several receptor tyrosine
kinases including MET, EGFR
[66]
Multiple abnormalities in RTK-dependent
pathways [209]
The depletion of USP8 leads to
accumulation of ubiquitinated
EGFR in endosomes [67]
USP8 inhibitor, HBX
90397[67]
USP9x
Several proteins including catenin, Smad4 and MCL1 [7779]
Colon, breast, small cell lung carcinoma,
lymphoma [79, 210]
Overexpression [79, 210]
-
USP14
Tau, ataxin-3 [76]
Neurodegenerative diseases, ataxia [75]
Loss of USP14 [75]
USP14 inhibitor, IU1
[76]
DUBs together with their substrates, associated disease, alterations and available inhibitors are listed.
Downregulation [82]
6 Current Pharmaceutical Design, 2013, Vol. 19, No. 00
cells with IU1 increases the degradation of several substrates including Tau and ataxin-3, both of which have been implicated in
neurodegenerative diseases [76]. Such inhibitors targeting USP14
could, therefore, potentially be used to reduce or eliminate misfolded and aggregated proteins that accumulate in neurodegenerative diseases and improve the prognosis in such disorders [25].
USP9x is another DUB from USP family that deubiquitinates
several proteins related to cancer disease such as -catenin, Smad4
and MCL1 [77-79]. -catenin is known to promote colon cancer
cell growth and it is stabilized by USP9x [77]. By an siRNA-based
screen, USP9x has been found to be important for TGF signaling
[78]. An essential component of TGF signaling pathway, Smad4,
becomes inactive by undergoing monoubiquitination. USP9x was
observed to deubiquitinate Smad4, hence, it positively regulates
TGF signaling pathway, which is related to tumor initiation and
progression [78]. USP9x also deubiquitinates MCL1, that is an antiapoptotic BCL-2 homolog leading to blocking of MCL1 degradation and stabilizing MCL1 levels, thus leading to cell survival [79].
However, having a dual function, it can also promote apoptosis by
stabilizing apoptosis signal-regulating kinase 1 [80].
Other DUBs belonging to USP family that are linked to disease
are: USP1 related to Fanconi anaemia DNA repair pathway [81];
USP6 to aneurismal bone cysts [58]; and USP7 to non-small cell
lung adenocarcinoma [82].
UCH Family of Deubiquitinating Enzymes
CYLD is a DUB belonging to UCH family and is mutated in
Familial Cylindromatosis disease, in which numerous skin tumors
develop [83]. Mutation or decreased expression levels of CYLD
have been observed in kidney cancer, colon cancer, B-cell malignancies, multiple myeloma, uterine cervix carcinoma and melanoma [84]. CYLD deubiquitinates the Bcl-3 protein, which is a
transcriptional co-activator of NF-B, blocks its nuclear translocation and hence inhibits NF-B activation [85]. CYLD also deubiquitinates TRAF2, TRAF6, TRAF7 (TNF Receptor Associated Factors), RIP1, TAK1 leading to inactivation of NF-B signaling [8689]. CYLD is also associated with Wnt signaling pathway that is
essential for embryonic development and is frequently activated in
cancer [90]. It negatively regulates Wnt signaling through deubiquitinating Dishevelled protein and activating -catenin [91]. Having
a tumor suppressor role, increasing the activity of CYLD that is lost
during tumor progression may be an efficient treatment in cancer.
OTU Family of Deubiquitinating Enzymes
A20 contains an OTU domain at the amino-terminus that possesses DUB activity. Additionally, it also functions as an E3 ligase
through its carboxyl-terminal consisting of seven zinc fingers. A20
can act as tumor suppressor by downregulating NF-B signalling
[92]. It removes K63-linked ubiquitin chains from RIP, RIP2,
TRAF6, NEMO and MALT1 [93-98]. With its E3 ligase activity, it
conjugates K48-linked polyubiquitin chains to RIP1 and TRAF2
targeting them for degradation [99]. Inactivation of A20 protein is
associated with several chronic inflammatory and autoimmune
diseases.
JAMM Family of Deubiquitinating Enzymes
The JAMM domain proteins are zinc metalloisopeptidases [58].
Members of this class include AMSH, BRCC36, CSN5 and POH1.
The activity of AMSH regulates trafficking of G protein-coupled
receptors and receptor tyrosine kinases [100]. BRCC36 has a role in
the DNA damage response. CSN5 mediates NEDD8 attachment to
cullins, regulating the cullin-RING ligase activity and stability .
Finally, POH1 functions as a part of 19S cap of the 26S proteasome
to trim polyubiquitin chains from the substrates. Though no drugs
are yet available, these DUBs are potential drug targets.
Kar et al.
Targeting Proteasome
The proteasome is a large, 26S, multicatalytic protease in which
proteolysis of short-lived or regulatory proteins from the cytoplasm,
the nucleus or the endoplasmic reticulum is carried out predominantly [101]. The 26S proteasome (proteasome holoenzyme) consists of two major subcomplexes: the 20S core particle (CP) that
contains the protease subunits and the 19S regulatory particle (RP)
that regulates the function of 20S CP [101]. The 19S regulatory
particle contains two mutisubunit structures, a lid and a base. It has
multiple roles in regulating proteasomal activity: selecting substrates, preparing them for degradation and translocating them into
the CP. The proteolytic activity of the proteasome occurs in the 20S
CP that contains two inner -rings that have the proteolytic active
sites [101]. Active sites of 20S proteasome are the caspase-like
(1), trypsin-like (2) and chymotrypsin-like (5) that use an amino
terminal threonine as the catalytic aminoacid residue. There are one
or two regulatory particles attached to the surface of the outer rings
of the 20S CP to form 26S proteaome. After degradation of the
substrate, short peptides derived from the substrate and reusable
ubiquitin are released.
Alterations in the proteasome are associated with many human
diseases including neurodegenerative disorders, cardiac dysfunction, cataract formation, cachexia and rheumatoid diseases [102].
Several antiapoptotic and proliferative signaling pathways such as
pro-oncogenic NF- pathway require proteasomal activity [4]. NF pathway is activated in many tumor types [103]. The inhibitors
of NF-s (IBs) keep NF- factors in an inactive state. Upon
phoshorylation and polyubiquitination of IBs and subsequent degradation by the proteaosome, NF- factors become activated
[103]. The first clinically validated drug to target UPS is bortezomib, which reversibly inhibits the active sites in the 20S proteasome. Bortezomib-mediated proteasome inhibition leads to downregulation of NF- signaling through blocking IB degradation
[104] and to endoplasmic reticulum stress, which in turn promotes
cell death [105]. The effects of NF- inhibition are the upregulation of the cyclin-dependent kinase inhibitors p21 and p27 and
downregulation of pro-inflammatory response genes, consequently
leading to increased apoptosis in tumor cells [106]. Bortezomib is
currently used as an anticancer drug for the treatment of multiple
myeloma, relapsed mantle cell lymphoma. It is also in many other
clinical trials including Phase III clinical trials for follicular nonHodgkin’s lymphoma and Phase II trials for diffuse large B cell
lymphoma [25].
Owing to success of Bortezomib, several new generation of
proteasome inhibitors such as Carfilzomib (PR-171), MLN9708,
CEP18770, NPI-0052, ONX0912, PR957 and IPSI-001 are currently in development [107-114]. These proteasome inhibitors target different active sites of the 20S proteasome and differ in binding
kinetics. For example, while Bortezomib inhibits the caspase-like
(1) and chymotrypsin-like (5) activities of the proteasome in a
reversible manner, NPI-0052 irreversibly blocks the trypsin-like
(2) and chymotrypsin-like (5) sites. NPI-0052 is in Phase I stage
and when combined with Bortezomib in low doses has been observed to show synergistic effects on cancer cells in vitro [115].
Another proteasome inhibitor, Carfilzomib, which has entered
Phase III trials targets mainly the 5 site of the proteasome in an
irreversible manner and has been developed for the treatment of
relapsed multiple myeloma [107]. Other proteasome inhibitors are
detailed in Table 3.
Despite the success of Bortezomib and efforts for developing
other inhibitors, targeting proteasome is somewhat problematic
since this final common step of the degradative pathways are used
by hundreds of different processes in the cell. Therefore, inhibition
of the proteasome is likely to interrupt several diverse pathways.
Emerging Role of the Ubiquitin-proteasome System
Table 3.
Current Pharmaceutical Design, 2013, Vol. 19, No. 00
7
Proteasome Inhibitors, Development Stage, Binding Kinetics and Targeted Disease
Inhibitor
Development Stage
Disease
Bortezomib
Approved
Multiple myeloma and mantle cell lymphoma
Carfilzomib
Phase III
Multiple myeloma and other cancers
Irreversible
CEP18770
Phase I
Multiple myeloma and other cancers
Slowly reversible
IPSI-001
Preclinical
MLN9708
Phase I
Multiple myeloma and other cancers
Reversible
NPI-0052
Phase I
Multiple myeloma and leukemia
Irreversible
ONX0912
Phase I
Multiple myeloma and other cancers
Irreversible
PR957
Preclinical
Autoimmune disorders
Irreversible
Multiple myeloma and other hematologic malignancies
Developing proteasome inhibitors with distinct substrate selectivity
and lower toxicity would reduce such inconveniences in treating
associated diseases.
3. IMPORTANCE OF STRUCTURES IN TARGETING UPS
INTERACTIONS
Protein-protein interactions have a central role in regulating
many biological processes, cellular and signaling pathways. Alterations in protein-protein interactions may lead to several diseases
such as cancer and neurological disorders. Therefore, proteinprotein interactions are recurrently considered as drug targets in
disease states [116-122]. Drugs targeting protein-protein interactions, ultimately head protein interfaces where two proteins come
into contact. Understanding the details and principles of protein
interfaces is, therefore, immensely essential to develop efficient
strategies in drug design. At protein interfaces there are some critical residues that account for the majority of the binding energy
called hot spots [123]. A hot spot is defined as a residue that, when
mutated to alanine, leads to a dramatic decrease in binding free
energy (Gbinding > 2 kcal/mol) [123, 124]. Since these residues
are more critical than others to the stability of the complex, targeting the hot spots may lead to improved inhibition of protein-protein
Binding Kinetics
Slowly reversible
Not reported
interactions and to designation of novel compounds. A potent inhibitor mimicking such key residue interactions can achieve favorable interaction energy; thus, can compete with the original partner
for binding [121]. One such competitive inhibitor in treating immune disorders is SP-4206 that binds to hot spot residues of cytokine IL-2 with high affinity and block the formation of the natural
complex of IL-2 and its receptor IL-2R [125]. The most widely
known example of competitive inhibitors of the UPS is nutlins that
prevent the Mdm2-p53 binding, thus enhance the tumor suppressor
activity of p53. Alanine-scanning of the Mdm2-p53 binding region
illustrated that there are three hot spot residues, Phe19, Trp23 and
Leu26, on p53 critical for binding to Mdm2 [126]. Nutlin family of
compounds such as Nutlin-2 and Nutlin-3 mimics p53 and binds to
Mdm2 even with a greater affinity than p53 [127]. Mdm2-p53 and
Mdm2-Nutlin-2 binding are visualized in (Fig. 2).
In the UPS, there are five major steps: activation, conjugation,
ubiquitin-conjugate recognition, ubiquitin removal, and substrate
degradation by the proteasome [1]. Since the greatest amount of
specificity is the conjugation step that is mainly mediated by E3s,
the interactions of E3s represent important drug targets. The E3 and
E2 proteins function in a concerted manner; however, the principles
determining E2-E3 selectivity are still not entirely understood. In
Fig. (2). Nutlin-2 targeting Mdm2-p53 interaction interface. (A) Mdm2 is an E3 ligase mediating ubiquitination and subsequent degradation of p53.
Alanine-scanning of the Mdm2-p53 binding region illustrated that there are three hot spot residues, Phe19, Trp23 and Leu26, on p53 critical for binding to
Mdm2 [126]. (B) Nutlin-2 prevents the interaction between Mdm2 and p53 by mimicking the conformation of Phe19, Trp23 and Leu26 of p53 to block Mdm2
p53-binding region [127].
8 Current Pharmaceutical Design, 2013, Vol. 19, No. 00
addition, in many cases it is not known which E3s interact with
which E2s and how they interact with each other. Identifying all E2
partners of a specific E3 is essential for inferring the principles
guiding E2 selection by an E3. In one experimental approach, all E2
partners of Brca1 ubiquitin ligase were identified by two-hybrid
experiments [128, 129]. Brca1 was found to interact with multiple
different E2s and to possess E2-specific ubiquitin-transfer properties. Gardner et al. identified the interactions of HRD1 (HMG-CoA
reductase degradation) ubiquitin ligase by in vivo cross-linking
methods [130]. Ballar et al. illustrated how HRD1 can further act as
an E3 for another E3, gp78 involved in ER-associated degradation
[131]. Two large-scale studies have addressed this E2-E3 identification problem. In a global yeast-two hybrid screen, over 300 high
quality interactions are uncovered [132]. They generated mutants of
UBE2N to mimic the interaction pattern of another E2, UBE2D2,
which resulted in new E3 interactions and hence illustrated how the
E3 interactions of the E2 specific for K63 linkages UBE2N can be
altered towards the K48-specific UBE2D2 [132]. Markson et al.
combined yeast two-hybrid screens with homology modeling methods yielding a map of human E2-E3 RING interactions [133]. Their
data includes 535 experimentally defined novel E2-RING E3 interactions and more than 1300 E2-RING E3 pairs with favorable predicted free energy values. They extended this network by including
known human E2-RING E3 pairs, hence, assembled a network of
2644 proteins and 5087 interactions [133].
Including structural details of the interactions can assist in understanding principles beyond E3 selectivity. The first structural
clues of E2-E3 interaction specificity were discovered from the
crystal structure of the E3 ligase c-Cbl RING and the E2, UBE2L3
[134] (Fig. 3A). The -helix and zinc-chelating loops of the RING
domain are in contact with loop L1 and L2 of UBE2L3. In particular, F63 residue in loop L1, P97 and A98 in loop L2 of UBE2L3
mediate its binding to c-Cbl. The c-Cbl linker region interacts with
-helix 1 of UBE2L3 [134]. Currently, there are 9 E2-E3 complex
structures deposited in PDB; UBE2D2/CNOT4 complex (pdb code:
1ur6:AB), Baculoviral IAP repeat-containing protein 3/ UBE2D2
(pdb code: 3eb6:AB), Signal transduction protein C-cbl/UBE2L3
Kar et al.
complex (pdb code: 1fbv:AC), HECT(NEDD4L)/ UBE2D2 complex (pdb codes: 3jw0:AC and 3jvz:AC), TNF receptor-associated
factor 6 (TRAF6)/UBE2N complex (pdb code: 3hct:AB), STIP1
homology and U-Box containing protein 1 (CHIP)/UBE2D1 complex (pdb code: 2oxq:AC), Ubiquitin-protein ligase Ube3a (E6AP)/
UBE2L3 complex (pdb code: 1c4z:AD) and U-box domain of human E4B ubiquitin ligase/UBE2D3 complex (pdb code: 3l1z:AB).
Comparison of UBE2L3 binding to RING type c-Cbl and HECT
type E6-AP revealed that there are common structural elements of
this E2 recognized by both E3s: most extensive contacts are provided by F63, P97 and A98 residues of UBE2L3 binding to both
RING type c-Cbl and HECT type E6-AP [134]. Comparison of all
known E2-E3 structures through identifying the structurally conserved contacts implied that loop L1 second position residue contacts are only employed when interacting with HECT E3s [135].
Combining such structural details with protein interactions networks of UPS, obviously, improves drug design concept as outlined
in the following section.
Structural Interaction Network of UPS Proteins
Network-based approaches have gained importance in drug
discovery with the realization that diseases are complex as they are
often consequences of multiple molecular abnormalities rather than
being the result of a single defect or phenomena [136]. To go a step
further in the mechanistic understanding one has to combine interaction networks information with molecular details of protein interfaces in the effort of identifying all target proteins that are influenced by a drug, either positively or negatively.
The number of distinct binding motifs is limited in nature [137]
implying that structurally different proteins can share similar interface architectures [138]. Following this notion, an algorithm called
Prism has been developed that predicts potential protein associations based on known protein interface motifs [139, 140]. Exploiting this algorithm and the available structural proteome, recently,
we have constructed a human E2-E3 structural interaction network
[135]. Considering the all human genome, protein structures for 24
Fig. (3). Interactions of c-Cbl E3 ubiquitin ligase with two E2 proteins, UBE2L3 and UBE2D1. (A) c-Cbl interacts with UBE2L3 (pdb code: 1fbv:AC). helix 1, loop L1 and loop L2 regions of UBE2L3 are displayed in yellow, green and orange color, respectively. Two loop regions, L1 and L2 of UBE2L3 are
in contact with the -helix and zinc-chelating loops of the RING domain. The central F63 residue in loop L1, P97 and A98 in loop L2 of UBE2L3 mediate its
binding to c-Cbl. The c-Cbl linker region interacts with -helix 1 of UBE2L3 [134]. (B) Modeled c-Cbl-UBE2D1 complex. Although the interaction was reported earlier [157], binding details and the structure of the complex was not available. c-Cbl residues labeled in red color was observed to be critical: C384,
C404, R420 were mutated in patients with acute myelogenous leukemia [158]. Additionally, Ile383 and Trp408 of c-Cbl were shown to have a critical role in
E2 binding [134]. These residues correspond to computational hotspots predicted by Hotpoint server indicating the usefulness of structural modeling of E2-E3
associations.
Emerging Role of the Ubiquitin-proteasome System
E2s are available (out of ~40) while the structural coverage is relatively low for E3 proteins; 32 of them (out of ~500) have resolved
structures in PDB. Among these, we predict 107 interactions between 22 E2s and 16 E3s. In our network, the number of all possible E2-E3 pairs is 352 (i.e. 22x16) and 51 of these were already
reported in earlier studies. We recover 76% (39/51) of the known
pairs through 107 predicted interactions verifying 36% (39/107) of
the network. On the basis of known E2-E3 interaction data, the
accuracy of the predictions is 76% indicating that the predicted E2E3 interactions are in agreement with validated functional E2-E3
pairs. Our method depends on the coverage of known interface
architectures and there are only 9 E2-E3 known complex structures
in the PDB (as aforementioned above) which are used as a basis for
modeling. Because the size and coverage of the PDB increases
exponentially, we expect the prediction efficiency of our method to
increase.
We observed that E3 proteins could interact with multiple E2s
and likewise E2 proteins could interact with multiple E3s, as expected. To reveal which residues are conserved among E2s interacting with a common E3 and which residues differ, hence, to provide
clues to specificity, we analyzed the E2-E3 interfaces and predicted
hotspot residues at interfaces using the Hotpoint server [141]. The
Hotpoint server determines computational hot spot residues based
on conservation, solvent accessibility and statistical pairwise residue potentials of the interface residues. The predicted hot spots are
observed to match with the experimental hot spots with an accuracy
of 70% [141]. Considering E2-E3 interface regions, some residues
are structurally conserved among E2 proteins and appear to be essential for all E2-E3 interactions, whereas others, particularly in
loop L1, appear to play important roles in E3 selectivity. The E2
proteins which have been characterized so far are known to recognize E3s through the L1 and L2 loops and the N-terminal -helix 1
on the E2 surface [142]. In particular, loop L1 has been shown to be
critical in E3 binding. In our network, E2 interface residues also
mostly correspond to the -helix 1, loop L1 and L2 regions. The
residue in the sixth position in loop L1 is widely utilized as an interface hot spot and appears indispensible for E2 interactions. Other
loop L1 residues also confer specificity on the E2-E3 interactions:
HECT E3s are in contact with the residue in the second position in
loop L1 of E2s; but this is not the case for the RING finger type E3s
[135].
Knowledge of such structural details and all interacting partners
of E3s at a proteome-level are crucial in drug design. This effort
will aid in explaining: i) which E3s interact with which E2s and
which substrates; ii) the molecular determinants of these interactions; iii) what the corresponding functions and outcomes are. In the
drug design field, to date, researchers have focused primarily on
blocking E3-substrate interfaces (as aforementioned above). However, targeting and disrupting E2-E3 interactions may be relatively
easy since these interactions are usually relatively weak [142]. Such
inhibitors can block the interaction by binding to the E2, the E3 or
the E2-E3 interface. Structural examples showing the binding of the
inhibitors are provided as case studies in the next section.
Flexibility and Molecular Dynamics of UPS Proteins
It is more and more recognized that increment in protein flexibility is paralleled by an increment in binding promiscuity of the
flexible region. This is particularly true for those region that undergo post-translational modifications such as phosphorylation
[143, 144] or ubiquitination [145, 146] and for their partners [147,
148]. These flexible changes are mostly studied by NMR and Molecular Dynamics (MD) [149]. Studies using NMR residual dipolar
couplings has shown neatly that free ubiquitin samples conformations globally similar to those in the bound state [150, 151]. Lange
and colleagues compared a number of X-ray structures of ubiquitin,
bound to different partners, with NMR structures of the ubiquitin
protein free in solution. In this pioneering study the authors were
Current Pharmaceutical Design, 2013, Vol. 19, No. 00
9
able to demonstrate, by analyzing the dynamic behavior of ubiquitin from the the picosecond to microsecond time scale, that for
each bound ubiquitin structure there is a member of the unbound
sampled ensemble that is structurally similar to it [151]. The work
strongly supported an underlying conformational selection mechanism for these multibinding promiscuous proteins. Lately, by Molecular Dynamics investigations of the same data by Wlodarski and
Zagrovic suggested that during the binding event the conformation
structurally most similar to the bound structure is the dominant one
via the conformational selection model. After this selection, reoptimization of the binding interface occurs via a ‘residual induced
fit’ mechanism, therefore calling in cause both mechanisms of binding for these enzymes [152]. Long and Brüschweiler performed
microsecond time scale all-atom MD simulations of ubiquitin and
its binding partners using the latest generation of molecular mechanics force field [153]. In this study, the effect of ligand molecules on the flexibility and plasticity of ubiquitin binding properties
is analyzed. The study focuses on the ubiquitin interacting motif
(UIM), a short alpha-helical conserved motif for ubiquitin recognition. The energy landscape and dynamics of ubiquitin in response to
the approach of the UIM ligand is analyzed in details. By the use of
Boltzmann sampling of molecular dynamics snapshots, the authors
show a statistical reweighing of the populations of ubiquitin conformers in the presence of its ligand molecule at intermediate distance range (1–2 nm) and are able to examine the population redistribution mechanisms.
Nussinov and collaborators focused on cullin-RING E3 ubiquitin ligases and their flexible role in binding to many partners
[145, 146]. They performed structural analysis and MD simulations
starting from Cul1, Cul4A, and Cul5 crystal structures. The MD
work clearly shows that at least three cullins do not act as merely
rigid scaffolds but are flexible with conserved hinges in the Nterminal domain. In another study, dynamical properties of the E2
enzymes of family 3R are characterized, showing a structural role
of Loop 7 and Loop 8 in stabilizing closed conformations [154].
Taken together, all these evidences highlight the importance of
flexibility in the recognition and functional activity of these E3
ligase enzymes and pave the way for more studies in which Molecular Dynamics techniques are used for the conformational characterization and identification of flexible regions to be targeted in
drug design.
4. CASE STUDIES
Here we present two case studies of UPS proteins relevant to
drug design. The first example illustrates how structural modeling
of E2-E3 associations may be beneficial to drug discovery. In the
second example, an interesting approach of designing a smallmolecule antagonist to target an E3 protein in cancer is explained.
Modeling E2-E3 Complexes: UBE2D1-c-Cbl Ligase Example
c-Cbl is an E3 ubiquitin ligase that attenuates signaling through
poly- or monoubiquitination of several activated receptor tyrosine
kinases (such as Flt-3, c-kit, and M-CSF) and other tyrosine kinases
of the Scr family [155]. The RING domain has a central role in cCbl function because its deletion or disruption abolishes the function of c-Cbl [156]. From its RING domain, c-Cbl binds to an E2
and mediates ubiquitin transfer from E2 to the target substrates.
Structure of c-Cbl interacting with one of its E2 partners, UBE2L3,
is deposited in Protein Data Bank (PDB) (pdb code: 1fbv) [134].
Two loop regions, L1 and L2 of UBE2L3 are in contact with the helix and zinc-chelating loops of the RING domain. The central
F63 residue in loop L1, P97 and A98 in loop L2 of UBE2L3 mediate its binding to c-Cbl. The c-Cbl linker region interacts with helix 1 of UBE2L3 [134]. c-Cbl ligase-UBE2L3 complex showing
the critical binding regions (-helix 1, loop L1 and L2) of UBE2L3
is visualized in (Fig. 3A).
10 Current Pharmaceutical Design, 2013, Vol. 19, No. 00
c-Cbl is also reported to interact with another E2, UBE2D1, and
ubiquitinate epidermal growth factor receptor [157], although the
structure of the complex is not available as yet. Using Prism algorithm, we modeled c-Cbl-UBE2D1 complex based on a known
interface of another E2-E3 complex; UBE2D2-CNOT4 (pdb code:
1ur6:AB) [135]. We observe that UBE2D1 (pdb code: 2c4p:A)
binds to c-Cbl (pdb code: 1fbv:A) mainly through its -helix 1, loop
L1 and L2 regions. Earlier work suggested that mutations in the cCbl RING domain are associated with acute myelogenous leukemia
and myeloproliferative neoplasms and the impairing the degradation of tyrosine kinases is an important mechanism in cancer [158].
In particular, on the RING domain of c-Cbl, the substitution of
cysteine or arginine residues at position 384 (C384Y), 404 (C404S),
and 420 (R420Q) are observed in patients with acute myelogenous
leukemia [158]. Additionally, Ile383 and Trp408 of c-Cbl were
shown to have a critical role in E2 binding [134] and the mutation
of Trp408 to alanine reduces c-Cbl’s affinity for the E2 and eliminates its ubiquitin-ligase activity in vitro [159]. Interface analysis of
our c-Cbl-UBE2D1 model indicates that these critical residues;
I383, C384, C404 and W408, correspond to computational hotspots
predicted by Hotpoint server [141], implying their importance in
binding and function of c-Cbl. Such structural modeling of E2-E3
associations could help in understanding E2-E3 selectivity and
discovering drug candidates targeting E3s. (Fig. 3B) illustrates the
modeled c-Cbl-UBE2D1 complex and the critical residues in binding.
A Potent Small Molecule Antagonist of IAP Proteins: GDC0152
Inhibitors of apoptosis (IAP) family of proteins function in the
regulation of signal transduction pathways associated with malignancy [160, 161] and are frequently overexpressed in cancer cells
[162, 163]. Cellular IAP (cIAP) proteins have been observed to
regulate TNF-mediated NF-B activation via its RING domain
and ubiquitinate receptor interacting protein RIP-1 and NF-B inducing kinase, NIK [164].
Viability of cancer cells depend on the aberrations in the apoptosis signaling pathways. Therefore, drugs that can restore apoptosis in tumor cells might be efficient in treating cancer [165]. In
cancer, one approach to target the IAP proteins is to design of small
Kar et al.
molecules that mimic the binding of IAP antagonist, second mitochondria-derived activator of caspases/direct IAP-binding protein
with low pI (SMAC/DIABLO) [166, 167], to baculoviral IAP repeat (BIR) domains [168]. The IAP BIR domains are necessary for
the antiapoptotic function of the IAP proteins [165]. One such antagonist is GDC-0152, which has entered Phase I clinical trials, can
bind to BIR3 domain of cIAP1 [165]. Binding of GDC-0152 to
cIAP1 promotes degradation of cIAP1, induces activation of
caspase-3/7, and leads to decreased viability of breast cancer cells
without affecting normal mammary epithelial cells [165]. (Fig. 4A)
illustrates N-terminal peptide from SMAC/DIABLO binding to
BIR3 domain of cIAP1 (pdb code: 3d9u). GDC-0152 antagonist
(pdb code: 3uw4), mimicking SMAC/DIABLO binding to cIAP1,
is visualized in (Fig. 4B). With the complete knowledge of structural and binding details of UPS proteins, it will become undoubtedly easier to design such small-molecule compounds.
5. CONCLUSIONS
Since the approval of the proteasome inhibitor Bortezomib in
2003, UPS components have emerged as crucial drug targets in
treatment of cancer, neurodegenenerative diseases, immunological
disorders and microbial infection. Small-molecule inhibitors of
ubiquitin-conjugating and –deconjugating enzymes are continuously being developed and evaluated for their potential use as
therapeutics. The major difficulty is, however, to design targetspecific inhibitors since UPS has diverse roles affecting hundreds of
different processes in the cell. Although attracted little attention so
far, developing selective compounds targeting E2-E3 interactions
would be a crucial potential therapeutic intervention: inhibitors can
be designed to bind to E3 ligases proximal to E2 binding region,
thus blocking E2-E3 interface or disrupt E2-E3 interactions allosterically by inducing long-range conformational changes. At this
point, knowledge of structural details of the interactions in UPS,
especially at a proteome-scale, appears to be essential.
In addition to targeting UPS with a single small-molecule compound, combinatorial drug therapy in UPS can be also efficient
since drug combinations currently provide a route to overcome the
complexity of human cancers [169]. Novel approaches including
large-scale genomics, systems and structural biology will set the
basis for future potential therapeutic interventions in UPS-
Fig. (4). A potent small molecule antagonist targeting an E3, cIAP1 in cancer treatment. (A) cIAP1 interacting with N-terminal peptide from
SMAC/DIABLO (pdb code: 3d9u). (B) GDC-0152 antagonist mimicks SMAC/DIABLO binding to cIAP1 (pdb code: 3uw4). Binding of GDC-0152 to cIAP1
promotes degradation of cIAP1, induces activation of caspase-3/7, and leads to decreased viability of breast cancer cells without affecting normal mammary
epithelial cells [165].
Emerging Role of the Ubiquitin-proteasome System
associated diseases and guide the identification of best possible
drug combinations targeting UPS.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflicts of
interest.
ACKNOWLEDGEMENTS
Declared none.
Current Pharmaceutical Design, 2013, Vol. 19, No. 00
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[26]
[27]
[28]
[29]
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Received: October 2, 2012
Accepted: November 1, 2012
Current Pharmaceutical Design, 2013, Vol. 19, No. 00
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