Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

Article abstract of DOI:10.1074/jbc.RA118.002649

Constitutive NF-B signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-B pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys⁶³-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6 -Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NF-B activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6 -Ubc13 interaction and thereby counteract NF-B signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6 -Ubc13 interaction that reduces TRAF6 -Ubc13 activity both in vitro and in cells. We found that this compound, C25-140, impedes NF-B activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical in vivo mouse models. Hence, the first-in-class TRAF6 -Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases.

Full text of DOI:10.1074/jbc.RA118.002649

JBC Papers in Press. Published on June 27, 2018 as Manuscript RA118.002649
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.002649
Targeting TRAF6 E3 ligase activity with a small molecule inhibitor combats autoimmunity
Jara K. Brenke1, Grzegorz M. Popowicz2,3, Kenji Schorpp1, Ina Rothenaigner1, Manfred Roesner4,
Isabel Meininger5#, Cédric Kalinski6, Larissa Ringelstetter1, Omar R’kyek7,8, Gerrit Jürjens7,8,
Michelle Vincendeau5,9, Oliver Plettenburg7,8, Michael Sattler2,3, Daniel Krappmann5 and Kamyar
Hadian1*
1Assay Development and Screening Platform, Institute of Molecular Toxicology and Pharmacology,
2
Helmholtz Zentrum München, Neuherberg, Germany; Institute of Structural Biology, Helmholtz
3
Zentrum München, Neuherberg, Germany; Center for Integrated Protein Science Munich at Chair of
Biomolecular NMR, Department Chemistry, Technische Universität München, Garching, Germany;
5
4mroe-consulting, Eppstein, Germany; Research Unit Cellular Signal Integration, Institute of Molecular
6
Toxicology and Pharmacology, Helmholtz Zentrum München, Neuherberg, Germany; Innovation
7
Management, Helmholtz Zentrum München, Neuherberg, Germany; Institute of Medicinal Chemistry,
Helmholtz Zentrum München, Neuherberg, Germany; 8Institute of Organic Chemistry, Leibnitz
9
Universität Hannover, Hannover, Germany; Institute of Virology, Helmholtz Zentrum München,
Neuherberg, Germany
Running title: Inhibition of TRAF6 activity counteracts autoimmunity
#Present address: AbbVie Deutschland GmbH, Wiesbaden, Germany
*To whom correspondence should be addressed: Kamyar Hadian: Assay Development and Screening
Platform, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München GmbH,
Neuherberg, Germany; kamyar.hadian@helmholtz muenchen.de; Tel. +49 89 3187 2664
Keywords: TRAF6, Ubc13, NF-κB, small molecule, protein-protein-interaction, inflammation,
autoimmunity, rheumatoid arthritis, psoriasis, drug discovery
_____________________________________________________________________________________
ABSTRACT
Constitutive NF-κB signaling represents
hallmark of chronic inflammation and
autoimmune diseases. The E3 ligase TNF
receptor-associated factor 6 (TRAF6) acts as a
key regulator bridging innate immunity, pro-
inflammatory cytokines, and antigen receptors to
the canonical NF-κB pathway. Structural
analysis and point mutations have unraveled the
essential role of TRAF6 binding to the E2
consequently, NF-κB activation. However, to
date no small molecule modulator is available to
inhibit the TRAF6-Ubc13 interaction and
thereby counteract NF-κB signaling and
a
associated diseases. Here, using
a
high-
throughput small-molecule screening approach,
we discovered an inhibitor of the TRAF6-Ubc13
interaction that reduces TRAF6-Ubc13 activity
both in vitro and in cells. We found that this
compound, C25-140, impedes NF-κB activation
in various immune and inflammatory signaling
pathways also in primary human and murine
conjugating
enzyme
Ubiquitin-conjugating
enzyme E2 N (Ubc13 or UBE2N) to generate
Lys63-linked ubiquitin chains for inflammatory
and immune signal propagation. Genetic
mutations disrupting TRAF6-Ubc13 binding
have been shown to reduce TRAF6 activity and
cells.
Importantly,
C25-140
ameliorated
inflammation and improved disease outcomes of
autoimmune psoriasis and rheumatoid arthritis in
preclinical in vivo mouse models. Hence, the
1

first-in-class TRAF6-Ubc13 inhibitor C25-140
expands the toolbox for studying the impact of
the ubiquitin system on immune signaling and
underscores the importance of TRAF6 E3 ligase
activity in psoriasis and rheumatoid arthritis. We
propose that inhibition of TRAF6 activity by
small molecules represents a promising novel
strategy for targeting autoimmune and chronic
inflammatory diseases.
influencing protein homeostasis. In contrast,
regulators of the ubiquitination cascade might
serve for more specific drug targets. Especially
E3 ligases are final regulators of the
ubiquitination reaction with more than 600
representatives and therefore, drugging E3
ligases might give rise to more specificity
compared to the 26S proteasome (7).
The TRAF protein family comprises
_______________________________________
seven E3 ligase members including TRAF6.
Different members of this class are involved in
distinct receptor signaling pathways such as IL-1
receptor family, T-cell receptor (TCR), IL-17
receptor, TNF receptor and more (8,9). TRAF-
dependent signaling pathways contribute to the
control of diverse cellular processes including
survival, proliferation, differentiation and
cytokine production (8). The TRAF6 protein
belongs to the RING-type of E3 ligases, which
interacts with the heterodimeric E2 enzyme
complex Ubc13/Uev1a to attach Lys63-linked
ubiquitin chains to its substrate proteins (10-12).
These Lys63-linked ubiquitin chains are
necessary to activate innate and adaptive
immune responses mainly through the NF-κB
axis (13,14). Of important note, the TRAF6
ligase activity is strictly determined by its
protein-protein-interaction with a specific set of
E2 enzymes including Ubc13. Importantly
ubiquitin is directly transferred from ubiquitin-
charged Ubc13 (E2) to the substrate, both
proteins bound by the E3 ligase TRAF6 (15).
Thus, targeting TRAF6 E3 activity requires the
identification of protein-protein-interaction
inhibitors, which disrupt the TRAF6-Ubc13
interaction, because TRAF6 has no intrinsic
catalytic activity (7).
Ubiquitination of proteins is a cellular process
that either drives protein degradation by the
proteasome in order to sustain protein
homeostasis or regulates distinct cellular
signaling pathways. Three enzymes, namely E1,
E2 and E3 are needed to covalently attach
ubiquitin to the substrate proteins. E1 is the
activating enzyme initially binding the ubiquitin,
E2 is the conjugating enzyme thereby
determining the linkage type and the E3 ligase is
involved in substrate specificity and transfer of
ubiquitin (1). Depending on the type of linkage,
polyubiquitination can have various functions
(1-3). While, e.g. Lys48-linked chains are
known to target the substrate protein for
proteasomal
degradation
via
the
26S
proteasome, Lys63-linked chains are associated
with a variety of non-proteolytic functions such
as trafficking, DNA damage response as well as
NF-κB signaling in innate and adaptive
immunity.
The eukaryotic transcription factor NF-
κB plays a critical role in regulating the
expression of a large variety of genes that are
involved in several cellular processes including
innate and adaptive immune response, cell
growth and survival as well as cell development
(4). Several signals including cytokines,
pathogens, injuries and other stress conditions
induce activation of the NF-κB transcription
factors that are tightly regulated via the
formation of poly-ubiquitin chains. Deregulation
of NF-κB signaling contributes to the
Overexpression of TRAF6 as well as
enhanced TRAF6 activity have been shown to
promote chronic immune stimulation in a variety
of
disorders
including
autoimmunity,
inflammation and cancer (8). For example, it has
previously been demonstrated that TRAF6
expression is elevated in rheumatoid arthritis
(RA) patients (16,17) and in Lupus cohorts (18).
On a molecular level, disruption of TRAF6-
Ubc13 binding by the cellular protein A20 (19)
or by genetic mutations (20,21) counteracts
TRAF6 activity and NF-κB activation. To date,
efforts on targeting TRAF6 activity have
pathogenesis
inflammation and various cancers (5,6). Within
NF-κB signaling, approved ubiquitin-
of
autoimmunity,
chronic
proteasome inhibitors mainly focus on the
inhibition of the 26S proteasome (e.g.
Bortezomib and Carfilzomib), thereby broadly
focused
on
disrupting
TRAF6-substrate
2

interaction (22), whereas inhibition of the
TRAF6 activity by interfering with the TRAF6-
Ubc13 association has not been attempted so far
and represents an attractive novel strategy to
combat TRAF6-dependent disease formation.
In this report, we demonstrate the
identification of the first TRAF6-Ubc13 protein-
protein-interaction inhibitor that ameliorates
disease outcome of two preclinical murine
vitro (Fig. 1D). These data demonstrate that
C25-140 directly binds to TRAF6, thereby
blocks the interaction of TRAF6 with Ubc13 and
as a consequence lowers TRAF6 activity.
Selectivity profiling of C25-140 against other
E3 ligases and E2 enzymes
To better understand the selectivity of
C25-140 towards other E3 ligases, we analyzed
the activity of various E3 ligases upon C25-140
treatment. These studies revealed that only the
activity of cIAP1, an E3 ligase generating
Lys63-linked poly-ubiquitin chains (26) similar
to TRAF6, was affected by C25-140 (Fig. 2A).
Notably, several other E3 ligases (MDM2,
TRIM63, ITCH, E6AP and RNF4) building
other types of poly-ubiquitin chains were not
influenced by C25-140 (Fig. 2B-D). In addition
to these studies on a set of E3 ligases, we tested
whether C25-140 inhibits E1/E2 reactions. To
this end, we treated different E1/E2 pairs
employing nine different E2 enzymes with
compound C25-140. All reactions including
UbcH13/Uev1a were not affected by C25-140
(Fig. 3), confirming that C25-140 acts on the E3
ligase side.
models
of autoimmunity
and
chronic
inflammation.
Results
Identification of TRAF6-Ubc13 inhibitors
In order to identify small molecule
inhibitors of the TRAF6-Ubc13 interaction, we
established an AlphaScreen® binding assay with
robust performance (Figure S1A) and carried out
a High-Throughput-Screening (HTS) campaign
using 25,000 compounds (Figure S1B). After
removing AlphaScreen® frequent hitters using
our own developed chemoinformatics filters
(23,24) and by employing strict selection criteria
(dose-response and cell-based NF-κB activation
assays), we were able to select the small
molecule C25 as the most promising scaffold for
further
evaluation
(Figure
S1C-E).
Effects of compound C25-140 on pro-
inflammatory signaling
Subsequently, we performed a small Structure
Activity Relationship (SAR) analysis by testing
few commercially available analogs (Fig. S2A).
Three analogs showed similar inhibitory effects
on TRAF6-Ubc13 interaction when compared to
C25 (Fig. S2B). Compound C25-140, however,
had the best inhibitory potential on NF-κB
activation after IL-1β stimulation (Fig. S2C),
which led us choose this compound for
additional investigations. C25-140 (Fig. 1A and
Fig. S2A) disrupted the TRAF6-Ubc13 binding
in vitro (Fig. S2D), without affecting the
interaction of Ubc13 and the deubiquitinase
OTUB1(25) (Fig. S2D), which competes with
TRAF6 for Ubc13 binding (25). Furthermore,
the binding of Ubc13 to Uev1a remained
unimpaired upon compound treatment (Fig.
S2E). Importantly, treatment of cells with C25-
140 dose-dependently impeded TRAF6-Ubc13
Before moving into cell-based validation
of C25-140, we analyzed whether C25-140
affects cell viability and cell cycle phases. C25-
140 neither substantially influenced cell viability
in MEF cells and Jurkat T-cells at concentration
up to 50 µM (Fig. S3A) nor did it influence cell
cycle phases of Jurkat T-cells (Fig. S3B).
We first tested the inhibitory potential of
C25-140 in MEF cells that can be stimulated
with pro-inflammatory cytokines IL-1β and
TNFα to trigger NF-κB activation. Stimulation
of these cells with IL-1β initially induces
TRAF6 auto-ubiquitination that is required for
signal progression through the IKK complex
towards NF-κB activation and expression of NF-
κB-dependent target genes (8). C25-140
treatment of MEF cells led to a dose-dependent
reduction of TRAF6 auto-ubiquitination (Fig.
4A) followed by decreased levels of
phosphorylated IκBα (Fig. 4B), which translated
into diminished target gene expression (Fig. 4C).
interaction
in
co-immunoprecipitation
experiments (Fig. 1B) and NMR studies proved
that C25-140 directly binds to TRAF6 (Fig. 1C).
Furthermore, C25-140 effectively reduced
TRAF6-mediated ubiquitin chain formation in
3

These data illustrate that inhibition of TRAF6
activity early during IL-1 receptor signaling
hampers the signaling cascade towards NF-κB
primary human origin. In addition to primary
human cells, we verified the inhibitory potential
of C25-140 in ex vivo primary murine T-cells
and observed dose-dependent reduction of IL-2
mRNA and protein levels after CD3/CD28
stimulation (Fig. 5D).
activation.
Additionally,
TNFα
induced
phosphorylation of IκBα (Fig. S4A) as well as
NF-κB induced target gene expression (Fig.
S4B) were affected by C25-140 treatment.
Taken together, C25-140 efficiently inhibits IL-
1β and TNFα mediated receptor signaling in the
context of cytokine activation.
Absorption/Distribution/Metabolism/Excretion
(ADME)
and
pharmacokinetics
(PK)
assessment of C25-140
Before moving into in vivo mouse
studies, we analyzed the compounds suitability
for in vivo application. To this end, we
performed ADME studies assessing key
parameters (Fig. S5A). Here, C25-140 was
stable in plasma as well as in microsomes,
showed 96.8% plasma protein binding and
exhibited low binding to hERG. Only in terms of
Cytochrome P450, the compound showed some
inhibitory potential on distinct CYPs (Fig. S5A).
Next, we tested the pharmacokinetics
(PK) of C25-140 after intravenous (IV), peroral
(PO) and intraperitoneal (IP) injection of mice
with 10 mg/kg C25-140 each. In general, a rapid
initial distribution phase was observed and C25-
140 had good calculated oral bioavailability
(Fig. S5B and C).
Effects of C25-140 on T-cell activation
In addition to IL-1β receptor signaling,
we tested C25-140 on T-cell activation as TCR
mediated NF-κB activation relies on TRAF6
activity (27,28). In the human Jurkat T-cell line,
T-cell activation by PMA/Ionomycin (P/I)
stimulation led to TRAF6 auto-ubiquitination
that was diminished by C25-140 in a dose-
dependent manner (Fig. 4D). Again, decreased
TRAF6 auto-ubiquitination affected downstream
signaling as evident from reduced IκBα
phosphorylation and cytokine secretion (Fig. 4E
and F). Moreover, P/I induced JNK
phosphorylation in MAPK signaling was
diminished by C25-140 (Fig. S4C). All these
data emphasize that C25-140 also affects TCR
mediated immune response in a human derived
cell line.
Altogether, results from ADME and
pharmacokinetics measurements underscored
that compound C25-140 had appropriate
properties to be applied in mouse studies for in
vivo efficacy testing.
C25-140 is effective in cytokine signaling of
primary human and murine cells
Next, we used primary human peripheral
C25-140 efficacy in a psoriasis mouse model
In a first in vivo mouse model we aimed
blood mononuclear cells (PBMCs) from three
healthy individuals to investigate whether C25-
140 is also competing TRAF6-dependent
receptor signaling in primary human cells. In
line with our data acquired in cell lines (Fig. 4),
C25-140 reduced secretion of NF-κB-driven
inflammatory cytokines such as TNFα and IL-6
after IL-1β stimulation (Fig. 5A) or IL-1β and
TNFα upon LPS stimulation (Fig. 5B), clearly
indicating that C25-140 also reduces cytokine
and innate immune responses in primary human
blood cells. Similar inhibitory effects by C25-
140 could be proven in human PBMCs after
stimulating the adaptive immune response with
anti-CD3/CD28 antibodies for TCR signaling
(Fig. 5C). Therefore, C25-140 is capable of
inhibiting cytokine signaling also in cells of
at investigating the effects of C25-140 on
psoriasis, an autoimmune disease. In this model,
psoriasis is induced by imiquimod (IMQ), which
activates TLR7 signaling (29), a pathway
employing TRAF6 for signal progression. IMQ
was topically applied to the shaved back as well
as the right ear of mice. On the same regions
C25-140 was topically applied twice daily at a
final dose of approx. 1.5 mg/kg per application.
Parameters describing disease outcome were
scored every day and samples for IL-17 cytokine
measurement were collected at day 6 (Fig. 6A).
C25-140 was able to significantly reduce the
“cumulative score” (Figure 6B), the overall
“thickness score” (Fig. 6C), “scaling” (Figure
6D) and “erythema” (Fig. 6E). Moreover,
4

analysis of IL-17 cytokine expression in the
right ear tissue proved its downregulation as a
consequence of C25-140 treatment (Fig. 6F).
This first efficacy study demonstrates that C25-
140, an inhibitor of TRAF6 activity, is able to
ameliorate symptoms of autoimmune psoriasis.
damage and several more (Fig. 7D and E and
Fig. S8). These data also underlined the dose-
dependent improvement of RA disease outcome.
Taken together, we present
a
successful
preclinical proof of concept study for C25-140
in a RA efficacy model.
C25-140 efficacy in a preclinical mouse model
for rheumatoid arthritis (RA)
Discussion
In this report, we present a high-
As pointed out earlier, it has previously
throughput screening campaign that successfully
discovered a novel protein-protein-interaction
inhibitor, namely C25-140, which is capable of
reducing TRAF6 E3 ligase activity by
interfering with the TRAF6-Ubc13 interaction.
Moreover, C25-140 exhibits inhibitory effects
on various immune and inflammatory signaling
pathways in cell lines as well as primary human
and murine cells. Notably, inhibition of these
pathways should be beneficial for treating a
highly inflamed immune system. In line with
this notion, C25-140 ameliorated symptoms of
autoimmune diseases in in vivo preclinical
efficacy models of psoriasis and rheumatoid
arthritis (RA). Hence, our reverse chemical
genetics approach presented in this study
underlines the importance of TRAF6 activity for
the development and persistence of autoimmune
diseases such as psoriasis and RA.
been reported that levels of TRAF6 are elevated
in RA patients (16,17). Therefore, we
investigated whether C25-140 is effective in a
preclinical mouse model of autoimmune
rheumatoid arthritis. We chose the collagen-
induced arthritis (CIA) mouse model (30) in
which TRAF6 is upregulated during disease
progression and RA symptoms are reversed by
the application of siRNA-based TRAF6 knock-
down (17), stressing the biological relevance of
this in vivo model for the TRAF6 E3 ligase
functions. In our study, RA was induced by
injection of collagen at day 0, followed by a
collagen boost injection at day 21 to promote the
development of arthritic symptoms. At day 28,
compound C25-140 was intraperitoneally
administered twice daily at three doses (6mg/kg,
10mg/kg and 14mg/kg) over a period of 14 days.
In RA, patients usually receive treatment when
symptoms become apparent. Thus, we started
dosing C25-140 in the CIA model when mice
already had developed symptoms of RA
(arthritic index of approx. 3), thereby reflecting
the diseased patient. Prednisolone served as a
positive control. Mice were daily scored for
arthritic index (AI) and euthanized on day 42 in
order to collect limbs for histopathology (Fig.
7A). Throughout the entire study the body
weight of all mice was monitored and did not
show any reduction, indicating that there were
no signs of obvious toxicity (Fig. S6).
Intriguingly, C25-140 ameliorated the arthritic
index to almost baseline levels in this efficacy
model at the doses of 10mg/kg and 14mg/kg
(Fig. 7B). Tissue sections of the limbs (Fig. 7C
and Fig. S7) evidently verified that C25-140
dose-dependently improved symptoms of RA
including inflammation and structural damages.
In addition, all sections were quantified based on
parameters of disease appearance such as
summed scores, inflammation, pannus, cartilage
TRAF6 belongs to the RING type of E3
ligases, where the RING domain makes a
protein-protein-interaction with the ubiquitin-
charged Ubc13 protein. Here, the ubiquitin is
transferred from Ubc13 (E2) to the substrate and
TRAF6 acts as a scaffold bridging these two
proteins (15). Therefore, targeting the E3
activity of TRAF6 requires protein-protein-
interaction inhibitors, which interfere with the
binding of TRAF6 to Ubc13 (7). Protein-
protein-interaction inhibitors (PPIs) have been
the very difficult to target in drug discovery as
the interaction interfaces are usually large and
contain many hydrophobic residues (31-33).
However, PPIs comprise hotspot regions and
genetic point mutation can lead to complete
disruption of binding, thereby suggesting that
small molecules have the chance to interfere
with these interfaces (33). Indeed, crystal
structures of TRAF6-Ubc13 binding clearly
imply that distinct amino acids of TRAF6 (e.g.
C70 or D57) are crucial for its interaction with
Ubc13 (10). Single point mutations of these
5

amino acids entirely abrogated TRAF6-Ubc13
interaction (Figure S1A and (10)), emphasizing
that there is a good chance to impede the
TRAF6-Ubc13 PPI.
with target-based function against RA is
Tofacitinib, a JAK inhibitor (41-43), which yet
comprises serious side effects. Thus, the field
relies on the discovery of additional approaches
that use small molecules against novel disease-
related targets. Accordingly, C25-140 is a novel
small molecule inhibitor that interferes with a
distinct target, i.e. TRAF6 activity, which is
critical for activation of various NF-κB signaling
pathways. This novel strategy effectively leads
to reduction of pro-inflammatory signaling to
improve RA disease outcome.
The goal of this study was to identify a
selective inhibitor of TRAF6 activity that can be
applied to chemical biology as well as drug
discovery studies. Our selectivity profiling on
other E3 ligases including RING (e.g. cIAP1 and
MDM2) and HECT (e.g. ITCH and E6AP) type
of E3 ligases revealed that C25-140 comprises
good selectivity for TRAF6 over other E3
ligases with the exception that it also inhibits
cIAP1, a RING E3 ligase generating Lys63
ubiquitin chains, similar to TRAF6. Importantly,
C25-140 did not interfere with Lys48-linked
poly-ubiquitin chain formation, which is critical
to maintain cellular protein homeostasis. The
compound also did not inhibit several other
E1/E2 reactions including UbcH13, underlining
that it acts on the E3 ligase side of the PPI.
Hence, C25-140 is fairly selective and does not
act as a broadband inhibitor of E3 ligases.
In the past, some inhibitors of MYD88-
mediated receptor signaling have been described
including IRAK4 activity (44), Ubc13-Uev1a
interaction (45), MYD88 dimerization (46) and
Ubc13 (47), but targeting the E3 ligase activity
of TRAF6 has never been accomplished as its
ligase activity is determined by the E2-E3
protein-protein-interaction. In general, inhibition
of E2-E3 interactions have been challenging and
only inhibitors of an acetylated-E2 together with
a Cullin type E3 ligase have been reported (48).
In contrast, our study describes the first-in-class
protein-protein-interaction inhibitor of a RING-
E3 ligase (TRAF6) / E2 enzyme (Ubc13)
binding and presents a novel strategy to treat
autoimmune and chronic inflammatory diseases.
The inhibition of TNFα signaling by
C25-140 was unexpected, as this particular
receptor activation does not employ TRAF6 for
signal progression towards NF-κB (34). This
finding, however, can be explained by the fact
that C25-140 also inhibits cIAP1 E3 ligase
activity, which is a central regulator of TNFα
receptor signaling (26). In fact, this C25-140 off-
target activity may eventually be beneficial for
the treatment of autoimmune and inflammatory
diseases as uncontrolled function of TNFα
signaling has been shown to highly contribute to
the development of these diseases (35).
Therefore, our study provides a novel small
molecule that counteracts TRAF6-Ubc13
interaction as a target to reduce NF-κB signaling
by various receptors (IL-1β receptor, TLR4, T-
cell receptor and TNFα receptor), thereby
encompassing cumulative effects on signaling
processes that drive chronic inflammation.
Experimental procedure
Plasmids, cell lines, antibodies and compounds
Cell lines:
HEK293T and MEF were grown in DMEM
(Gibco) supplemented with 10% Fetal bovine
serum (FBS) (Gibco) and 100 U/ml Penicillin/
Streptomycin (Gibco). Jurkat cells were grown
in RPMI 1640 (Gibco) supplemented with 10%
FBS
(Gibco)
and
100
U/ml
Penicillin/Streptomycin (Gibco). Cells were re-
thawed every three to four weeks. Human
PBMCs were cultured in RPMI supplemented
with
10%
FBS,
100U/ml
Penicillin/
Streptomycin and 50 µM b-Mercaptoethanol
(Gibco). Primary mouse CD4+ T-cells were
cultured as previously described (49).
Current therapeutics for RA are mostly
“disease modifying anti-rheumatic drugs”
(DMARDs) and glucocorticoids with broad and
diffuse immunosuppressive effects in the early
stage (36-38) with addition of antibody/biologics
therapy against specific surface targets (39,40) at
later stages. The only approved small molecule
Plasmids:
cDNA of TRAF6 RZ 12 (residues 50-187) was
cloned into pEF-HA backbone vector (50).
cDNA of TRAF6 RZ1 (residues 50-159) or the
TRAF6 D57K mutant was cloned into pASK
6

IBA 3+ (IBA Lifesciences) and pGEX4T1 (GE
Healthcare). Full length Ubc13 was cloned into
the pGEX4T1 vector with and without C-
terminal Flag-His-tag. OTUB1 full-length
cDNA was cloned into pGEX4T1 and Uev1a
was cloned into pET28a vector.
inhibitor Complete mini (Roche Diagnostics).
The suspension was sonified (8 x 30 seconds)
and subsequently centrifuged at 53,000 xg for 2
x 30 minutes. For the purification of the StrepII-
tagged proteins, the protein solution was applied
to Strep-Tactin columns using the ÄKTA
purifier (GE Healthcare). After washing, the
StrepII-protein was eluted from the column
Antibodies:
These antibodies were used for Western
Blotting: β-Actin I-19 (Santa Cruz sc-1616,
1/1000); p-IκBα Ser32/36 5A5 (Cell signaling
#9246, 1/1000); IκBα C21 (Santa Cruz sc-371,
1/1000) for MEF cells; IκBα L35A5 (Cell
Signaling #4814, 1/1000) for Jurkat T-cells;
RNF4 (Abnova A01, 1/2000); TRAF6 (Abcam
EP591Y, 1/1000); Ubc13 (Invitrogen 37-1100,
1/1000); Ubiquitin P4D1 (Santa Cruz sc-8017,
1:1000).
using
the
Strep-elution
buffer
(IBA
Lifesciences) containing desthiobiotin. The
eluted protein was concentrated using Amicons
(cut off 3 kDa) down to 1 ml. Using the ÄKTA
purifier and a HiTrap Desalting 5 ml column
(GE Healthcare), the protein was desalted and
exchanged to storage buffer (20 mM Tris-HCL
pH 8.0; 20 mM NaCl, 1 mM 1,4 Dithiothreitol
and 100 µM ZnCl2). The protein was
concentrated in Amicons up to 2 µg/µl and
stored in small aliquots at -80°C.
Anti-HA Affinity Matrix (Roche Diagnostics,
1/40) was used for immunoprecipitation.
Purification of untagged TRAF6 RZ1 protein
The cDNA sequence of wildtype TRAF6 RZ1
domain was cloned into the pGEX4T1 vector
(GE Healthcare Life Sciences). For the
purification of untagged TRAF6 RZ1, the
protein was expressed in BL21 codon plus RIPL
cells in M9 minimal medium. For 15N-labeled
Compounds:
All compounds were purchased from ChemDiv:
C25 (Cat. No.: G827-0171)
C25-140 (Cat. No.: G827-0140)
C25-167 (Cat. No.: G827-0167)
C25-189 (Cat. No.: G827-0189)
C25-031 (Cat. No.: G827-0031)
C25-166 (Cat. No.: G827-0166)
C25-054 (Cat. No.: G827-0054)
C25-024 (Cat. No.: G827-0024)
For all experiments, C25-140 was re-synthesized
to ensure quality control for working with the
correct molecule (synthesis procedure below).
15
protein, the regular NH4 was replaced by NH4.
For protein production, 1 mM IPTG and 100 µM
ZnCl2 was added at OD
= 0.6-0.7. After 16
600
hours at 18°C, cells were centrifuged and the
pellet resuspended in 10 ml TRAF6 washbuffer
(20 mM Tris pH 8.0, 20 mM NaCl) including 1
Complete mini pill (Roche Diagnostics). After
sonification (8 x 30 seconds) and centrifugation
(2 x 30 minutes at 53,000 xg), the protein
suspension was incubated with 1 ml pre-washed
Glutathione Sepharose 4 fast flow beads (GE
Healthcare) for 2 hours at 4°C. Subsequently,
beads were washed with 100 ml TRAF6 wash
buffer and then incubated with 50 Units
Thrombin for 3 hours at room temperature to
cleave off the GST-tag. The TRAF6 protein was
eluted in 5 ml wash buffer and cleared for
remaining GST with 1 ml Glutathione Sepharose
4 fast flow beads. Afterwards, the solution was
centrifugated for 5 minutes at 200 xg and the
supernatant transferred into an Amicon for
concentration followed by centrifugation at
20,000 xg for 30minutes. The supernatant was
then applied to a HiLoad Superdex75 column
Recombinant
purification
protein
expression
and
Purification of Strep-tagged TRAF6 RZ1 protein
The plasmid was transformed into BL21 codon
plus RIPL cells (Agilent Technologies) and
cultured in Luria/Miller medium containing
Ampicillin and Chloramphenicol. Protein
production was induced at an OD
= 0.6-0.8
600
by adding 200 µg anhydrotetracycline, 0.5 mM
Isopropyl-β-D-thiogalactopyranosid (IPTG) and
100 µM ZnCl2. Cells were incubated at 18°C for
16 hours. Afterwards, cells were centrifuged at
2,600 xg for 20 minutes at 4°C. The pellet was
resuspended in 10 ml Strep-wash buffer (IBA
Lifesciences) including 1 pill of protease
7

(GE Healthcare) using the ÄKTA purifier for
separating the untagged TRAF6 protein from
uncleaved protein as well as buffer exchange in
desalting buffer (2 mM Tris pH 8.0, 20 mM
NaCl, 1 mM DTT and 100 µM ZnCl2). These
proteins were used for 2D-NMR and in vitro
ubiquitination experiments.
included on every plate of the screening.
Compounds that inhibited TRAF6WTStrepII–
Ubc13FH by more than 25 % were considered as
actives. After elimination of AlphaScreen®
frequent hitter compounds and His-tag frequent
hitters(23), 178 compounds were defined as
primary hits and were subsequently tested in
five-point serial dilution assays (2.5 – 40 µM) in
Flag-His-tagged Ubc13 protein (Ubc13-FH)
and GST proteins (including GST-OTUB1 and
GST-Ubc13) were produced as previously
described (51).
TRAF6WTStrepII–Ubc13FH
experiments. Only compounds with dose-
dependent effects on TRAF6WTStrepII–
AlphaScreen
Ubc13FH (n = 27) were further taken.
His-tagged Uev1a protein was purified
according to GST-OTUB1 protocol with the
following buffer: lysis buffer (20 mM NaH2PO4,
20 mM NaCl, 50 mM Imidazol), elution buffer
(20 mM NaH2PO4, 20 mM NaCl, 500 mM
Imidazol) and desalting buffer (20 mM
NaH2PO4, 20 mM NaCl). Ni-Sepharose 6 Fast
Flow (GE Healthcare) was used instead.
AlphaScreen® counter assays involved the
interactions of GST-OTUB1/Ubc13FH and
GST-Ubc13/Uev1a-His. For each assay, the
GST-tagged protein was applied at a final
concentration of 30 nM (30 µl), wherein the His-
tagged protein was used at a concentration of 20
nM (10 µl). Glutathione-Donor beads and
Nickel-Chelate Acceptor beads (Perkin Elmer)
were used at a final concentration of 3 µg/ml (10
µl each).
AlphaScreen® assay for compound screening of
TRAF6-Ubc13 PPI inhibitors
For performance of an automated AlphaScreen®
assay, all following components were pre-
diluted in AlphaScreen® buffer (1x PBS, 0.5 %
BSA, 0.01 % Tween-20). First, the TRAF6
protein (end concentration 100 nM, 30 µl
volume) was added to 384-well Opti-plates
(PerkinElmer) involving the MultiFlo dispensing
system (BioTek) followed by transfer of the
compounds via the Sciclone G3 transfer station
(PerkinElmer). After addition of Ubc13FH (end
concentration 75 nM, 10 µl volume) with the
MultiFlo system and incubation for one hour at
room temperature, both beads (Strep-Tactin
Alpha Donor beads and Nickel-Chelate acceptor
beads; end concentrations 4 µg each, 10 µl
volume each, PerkinElmer) were added using
the MultiFlo system in subdued light. Read-out
of the plates occurred after another hour of
incubation at room temperature. Statistical
parameters including the coefficient of variation
(%CV), Z´ factor and signal window (SW) were
calculated to determine the quality of the assay.
To evaluate the efficacy of the compounds,
AlphaScreen® units of compound-treated
samples were referred to DMSO control treated
samples. Thereby, the TRAF6D57K mutant served
as the control for minimum signal and was
Two-dimensional-Nuclear-Magnetic-Resonance
(2D-NMR)
To determine whether the inhibitory compounds
directly bind to TRAF6 rather than to Ubc13,
2D-NMR
experiments
were
conducted.
Untagged 15N-labeled TRAF6 RZ1 protein (c =
120 µM) and compound (c = 20 mM stock) were
incubated at a ratio of 1:5 in a 3 mm NMR tube
for 10 minutes before spectra acquisition. To
observe chemical shift perturbations upon
compound addition, two-dimensional SOFAST-
HMQC
spectra
were
acquired.
All
measurements were performed using the Bruker
Avance 600 MHz spectrometer.
In vitro Ubiquitination assays (Western Blot-
based)
The ability of TRAF6 to form K63-linked poly-
ubiquitin chains in conjunction with Ubc13 was
analyzed after compound treatment. Therefore,
untagged TRAF6WT as well as TRAF6D57K were
recombinantly purified. 0.125 µM of TRAF6
protein was pre-incubated with DMSO or
compound in a total volume of 100 µl in K63
assay buffer (25 nM Tris-HCl pH 7.6; 250 nM
MgCL2; 500 nM creatine phosphate; 0.3 U/mL
inorganic pyrophosphatase; 0.3 U/mL creatine
8

phosphokinase) for 30 minutes at room
temperature. Small aliquots for input samples
were taken. A master mix containing 0.01 µM
Co-Immunoprecipitation studies in HEK 293T
cells
To study the interaction of TRAF6 and Ubc13 in
cells, 1 x 106 293T cells were seeded and after
18 hours cells were transfected with 1 µg pEF-
HA empty or pEF-HA-TRAF6 RZ1 plasmid
using the X-tremeGENE HP DNA transfection
reagent (Roche Diagnostics). 6 hours upon
transfection, cells were treated with compound
or DMSO. After 42 hours of further incubation,
cells were harvested in Co-IP lysis buffer (150
mM NaCl; 25 mM Hepes (pH 7.5); 0.2% NP-40;
1 mM Glycerol; 10 mM NaF; 8 mM β-
Glycerophosphat; 1 mM DTT; 300 µM Sodium-
vanadate; Complete protease inhibitor) and HA-
TRAF6 was immunoprecipitated with anti-HA
Affinity Matrix (Roche Diagnostics). Binding of
Ubc13 to TRAF6 was examined by Western
Blot.
E1-activating
enzyme
(UBE1)
(Boston
Biochem), 0.2 µM E2-conjugating enzyme
complex (Ubc13/Uev1a) (Boston Biochem), 1
mM ZnCl2, 2 mM ATP and 4 µM mono-
ubiquitin (Boston Biochem) was added to the
TRAF6 protein and the reaction mixture was
incubated for 120 minutes at 37°C. The reaction
was stopped by adding 4x SDS loading buffer
and boiling at 95°C for 5 minutes. The input
samples were analyzed using the Pierce Silver
Stain
Kit
(Thermo
Fisher
Scientific).
Ubiquitination was detected in Western Blot
analysis using an ubiquitin antibody (52).
For analysis of cIAP1, MDM2, TRIM63, RNF4,
ITCH and E6AP mediated poly-ubiquitination,
the respective ubiquitin ligase kit from Boston
Biochem (K-102, K-260, K-200B, K-220, K-240
and K-270) was used. Here, reaction buffer, E1
activating enzyme, E2 conjugating enzyme and
E3 ligase and substrate (p53 for MDM2, S5A for
TRIM63 and S5A for E6AP) were mixed and
pre-incubated with compound or DMSO for 5
minutes at room temperature before adding
Mg2+-ATP and mono-ubiquitin for reaction start.
Ubiquitination was visualized by Western Blot
as described before (52).
Immunoprecipitation of TRAF6 for detection of
endogenous ubiquitination
2.5 x 106 MEF cells were seeded, treated with
compound for 6 hours and stimulated with IL-1β
(3.5 ng/ml) for 10 minutes. For Jurkats T-cells,
2.5 x 106 cells were seeded, treated with
compound for 6 hours and stimulated with PMA
(400 ng/ml) and Ionomycin (600 ng/ml) (P/I) for
10 minutes. Cells were lysed in Co-IP lysis
buffer containing 1 % SDS to eliminate all
cellular interactions. After lysis, centrifugation
and collection of input samples, the samples
were diluted to 0.1 % SDS and incubated with 5
µl TRAF6 antibody EP591Y (Abcam) and
immunoprecipitated with Protein-G-Sepharose
beads (GE Healthcare). After washing of beads
in Co-IP wash buffer (150 mM NaCl; 25 mM
Hepes (pH 7.5); 0.2% NP-40; 1 mM Glycerol),
TRAF6 protein was eluted and levels of TRAF6
ubiquitination were detected by Western Blot.
E1/E2 reactions
UbcH (E2) enzyme kit (K-980B) and E1 enzyme
(UBE1, e-305) were purchased from Boston
Biochem. Ubiquitination assays were set as
described above for cIAP1 but only involving
E1/E2 enzymes. After 30 min of incubation at
37°C, the reaction was stopped and analyzed in
Western Blot staining for Ubiquitin P4D1.
Cell culture methods
AlphaSurefire
In order to detect protein levels of p-IκBα
(Ser32/36) as well as total IκBα, 1x104 MEF
cells were seeded in 96-well plates, treated with
compound for 6 hours and stimulated with 1
ng/ml IL-1β for 7 minutes. The p-IκBα and total
IκBα protein levels were analyzed using the
AlphaSurefire kits (PerkinElmer) following the
manufacturer´s instruction. The general 2-plate
protocol was conducted.
Densitometric quantification of Western Blot
bands
Western blots were densitometricaly quantified
using the LabImage1D software provided by
Kapelan Bio-Imaging. Here, background-
subtracted values of the protein of interest were
calculated relative to the background-subtracted
values of the control protein.
9

5´ TGC TGA TTA AGT CCC
TGG GTC 3´(SEQ ID NO:18)
Human TNFα 5´ CCC AGG GAC CTC TCT
for CTA ATC 3´(SEQ ID NO:19)
Human TNFα 5´ GCT ACA GGC TTG TCA
Human IL-2 rev
Analysis of mRNA expression
For MEF cells, 1.2 x 105 cells per sample were
seeded, treated with compound for 6 hours and
stimulated with either IL-1β (1 ng/ml) or TNFα
(10 ng/ml) for 60 minutes. Jurkat T-cells and
primary mouse CD4+ T-cells were seeded at 2 x
105 cells per sample, also treated with compound
for 6 hours and stimulated with P/I for 3 hours.
RNA was isolated according to the
manufacturer´s protocol of the Qiagen RNeasy
Kit (MEF cells) or the STRATEC INVITRAP®
SPIN UNIVERSAL RNA MINI KIT (Jurkat T-
cells). To clear isolated RNA from genomic
DNA, samples (up to 5µg RNA) were treated
with RQ1 RNase-Free DNaseI (Promega) as
described in the technical manual. For the
reverse transcription of RNA into cDNA, the
DNaseI digested RNA samples were processed
using the SuperScript III First Strand cDNA
Synthesis System (Invitrogen/ThermoFisher
Scientific) following the manufacturer´s
protocol. Here, random hexamers provided with
the kit were used for reverse transcription.
cDNA samples were stored at -20°C. For
quantification of cDNA in the samples, the
LightCylcer480 (Roche) and the KAPA SYBR
FAST qPCR Kit were used as described
earlier(52). For relative quantification, the ΔΔCp
method first described by Pfaffl (53) was used.
The following primer were used for qRT- PCR:
rev
CTC GG 3´(SEQ ID NO:20)
RNA 5´ GCA CCA CGT CCA ATG
ACA 3´(SEQ ID NO:21)
RNA 5´ GTG CGG CTG CTT CCA
TAA 3´(SEQ ID NO:22)
Human
Pol II for
Human
Pol II rev
Enzyme-Linked ImmunoSorbent Assay (ELISA)
For measurement of cytokine secretion, 2 x 105
Jurkat T-cells or primary mouse CD4+ T-cells or
human PBMCs were seeded per sample,
subsequently treated with compound for 6 hours
and stimulated as indicated. 20 hours after
stimulation, supernatants were harvested and
analyzed with the IL-2, IL-1β, IL-6 and TNFα
Ready-Set-Go! ELISA (2nd Generation) kits
provided by Affymatrix eBioscience.
Viability, cell cycle, ADME, PK and safety
studies
Viability assay
For cell viability experiments the CellTiter-
Glo® 2.0 system (Promega) was used. MEF
cells (1.2 x 103 cells/well) and Jurkat T-cells (5 x
103 cells/well) were seeded in 384 well plates
and 18 hours later, cells were treated with
compounds using the Sciclone G3 automation
system. After 24 hours incubation, the luciferase
assay was performed according to the
manufacturer’s manual. Signals were measured
on an Envision plate reader (PerkinElmer).
5´ GCT CAA CTG GTG TCG
TGA AG 3´(SEQ ID NO:5)
5´ ATG AGG CAG TTT CCA
TCA CC 3´(SEQ ID NO:6)
Mouse A20 for
Mouse A20 rev
Mouse β-Actin
for
Mouse β-Actin
rev
5′ CCT CTA TGC CAA CAC
AGT GC 3′(SEQ ID NO:7)
5′ GTA CTC CTG CTT GCT
GAT CC 3′(SEQ ID NO:8)
Cell cycle analyses
For cell cycle staining, cells were seeded at 2 x
105 cells per sample and treated with compound
or DMSO. After the indicated incubation time
(24 or 72 hours), cells were fixed in 100 %
Ethanol and subsequently stained with the
Propidium Iodide staining solution (0.1 % Triton
X-100; 20 µg/ml Propidium Iodide; 200 µg/ml
RNAse A in PBS) for 40 min in the dark. The
Propidium Iodide fluorescence was collected
using the Attune Flow Cytometer (Thermo
Fisher Scientific) and analysis of cell cycle
phases was performed using the Single Cell
analysis software FlowJo as previously
published (54).
Mouse ICAM-1 5´ CGC TCA GAA GAA CCA
for CCT TC 3´(SEQ ID NO:9)
Mouse ICAM-1 5´ GGA GAC GCA GAG GAC
rev
CTT AAC 3´(SEQ ID NO:10)
5′ GAG TGC CAA TTC GAT
GAT GAG 3′(SEQ ID NO:13)
5′ AGG GCT TGT TGA GAT
GAT GC 3′(SEQ ID NO:14)
Mouse IL-2 for
Mouse IL-2 rev
Human IL-2 for
5´ CAC AGC TAC AAC TGG
AGC ATT TAC 3´(SEQ ID
NO:17)
10

ADME and pharmacokinetic studies
from 0 to 4: 0=none; 1=slight; 2=moderate;
3=marked and 4=very marked. Ear thickness
was measured by electronic calipers as an
indicator of edema. IL-17 cytokine secretion of
the right ear tissue was measured by ELISA.
Bioethical permission of the IMQ-induced
psoriasis study was granted under the IACUC
number 17-063 to WB.
The Bienta Enamine Biology Services of the
CRO Bienta/Enamine Ltd. conducted all ADME
(plasma stability, plasma protein binding,
microsomal stability, LogD, Caco-2 assay,
CYP450 inhibition and hERG predictor assay)
as well as pharmacokinetic studies (Tmax, Cmax
,
AUC0-240min, Mean Residence Time, Elimination
half life, Elimination rate constant, Volume of
Distribution and Clearance) of C25-140 by using
their standard protocols. Pharmacokinetic
measurements were done by intraperitoneal (IP),
intravenous (IV) or peroral (PO) application of
10 mg/kg C25-140. Each time point was
investigated with n = 4 mice. Bioethical
permission of pharmacokinetic measurements in
mice was granted under the numbers: IACUC
number №1 from 19.05.2015 and IACUC
number №6 from 08.12.2015 to Bienta.
Collagen-Induced Arthritis Model (in vivo)
In order to test the potential of C25-140 to
ameliorate RA symptoms, a Collagen-Induced
Arthritis (CIA) Model in DBA1/J mice was
performed
by
the
CRO
Washington
Biotechnology, Inc. (Baltimore, USA). Each
group of 10 mice was subjected to a single
subcutaneous injection of collagen/complete
Freund´s Adjuvant emulsion (0.05 ml/mouse;
100 µg collagen/CFA). After 20 days (day 21),
mice were booster injected with collagen (0.05
ml/mouse; 100 µg/mouse collagen in Incomplete
Freund´s Adjuvant). On day 28, mice were
scored for their Arthritic Index (AI) and 14 days
intraperitoneally dosed twice daily with vehicle;
6 mg/kg C25-140, 10 mg/kg C25-140 and 14
mg/kg C25-140. 10 mg/kg prednisolone was
applied once daily. Mice were daily scored for
macroscopic signs of arthritis. Thereby, the
following scoring index was applied to each
individual paw: 0 = no visible effects of arthritis;
1 = edema and/or erythema of 1 digit; 2 = edema
and/or erythema of 2 digits; 3 = edema and/or
erythema of more than 2 digits; 4 = severe
arthritis of entire paw and digits. Single paw
scores were added and recorded. On day 42,
animals were euthanized, limbs were collected
and histopathology was performed using
Toluidine blue staining with scoring (0-5) of
Mouse studies
Isolation of primary mouse CD4 positive T-cells
(ex vivo)
CD4+ T-cells from the peripheral lymph nodes
of Balb/C mice were isolated by negative
magnetic-activation cell sorting (MACS)
selection using the CD4+ T-cell isolation kit II
(Miltenyi). Per sample, 2 x 105 cells were seeded
and treated with compound for 6 hours. Cells
were stimulated with anti-CD3 (0.5 mg/ml) and
anti-CD28 (1 mg/ml) on plates that were pre-
coated with rabbit anti-hamster IgG. Samples for
qRT-PCR were harvested after 4 hours whereas
supernatants for analysis of cytokine secretion
were harvested 20 hours after stimulation.
Psoriasis mouse study (in vivo)
To evaluate the anti-inflammatory activity of the
compound C25-140, an Imiquimod (IMQ)-
induced psoriasis mouse model was carried out
by the CRO Washington Biotechnology, Inc.
(Baltimore, USA). Per group (vehicle and C25-
140 treated), 8 male Balb/C mice were shaved in
the back (1.5 cm x 2 cm) and IMQ and C25-140
were applied topically to the shaved back and
the right ear every day for 6 days. Here, C25-
140 was dissolved in acetone and applied at a
total dosage of 500 µg per day (2 x 250 µg).
This refers to a total of approx. 1.5 mg/kg per
application. Mice were monitored and daily
scores were independently collected on a scale
several
parameters
(summed
score,
inflammation, pannus, bone resorption, cartilage
damage, periosteal bone formation and bone
width). Bioethical permission of the collagen-
induced arthritis study was granted under the
IACUC number 17-059 to WB.
Experiments with human primary PBMCs
For isolation of peripheral blood mononuclear
cells (PBMCs) from 50 ml human blood of three
different donors, blood was mixed with 800
units of Heparin (Sigma-Aldrich) and
centrifuged at 300 xg for 10 minutes without
11

brake. After removal of the plasma fraction,
buffy coat was diluted in 2 volumes PBS and
added on top of 15 ml Lymphoprep
(STEMCELL technologies). After centrifugation
at 160 xg for 20 minutes at room temperature
without brake, platelets were removed and the
cell suspension was centrifuged again at 350 xg
for 20 minutes without brake. Mononuclear cells
from the intermediate layer were transferred to a
new falcon and washed 3 times in PBS
supplemented with 0.1 % BSA and 2 mM
EDTA. In the end, cells were resuspended in
RPMI medium containing 10% FCS, 1%
Penicillin/Streptomycin and 50 µM β-
mercaptoethanol. Per sample, 2 x 105 cells were
seeded and treated with compound for 6 hours.
The cells were stimulated with either 1 µg/ml
Lipopolysaccharide (LPS) (Sigma-Aldrich); 20
ng/ml IL-1b (R&D Systems) or CD3/CD28 (1
µg hCD3 (IgG2A), 4 µg hCD28 (IgG1), 2 µg ant-
IgG2A and 2 µg ant-IgG2A; all BD Pharmingen).
Supernatants for analysis of cytokine secretion
were harvested 20 hours after stimulation. The
permission to isolate, treat and analyze human
peripheral white blood cells ex vivo was granted
by the ethics commission of the Technical
University Munich (TUM) (project number
358/15).
Synthesis procedure
C25-140
General Methods:
All solvents were purchased from
Sigma-Aldrich and were used as received;
anhydrous solvents were used for reactions, and
HPLC grade solvents were used for aqueous
work
ups,
recrystallizations
and
chromatography. Other reagents were purchased
from various vendors and were used as received.
Glassware was dried by keeping it in an oven at
70 °C for 12 h prior to use. The pH of aqueous
solutions was estimated using pH paper.
Vacuum filtrations were carried out using a
house vacuum line. In the individual procedures,
the phrase “concentrated in vacuo” means that
solvent was removed on a rotary evaporator
using a diaphragm pump (with an automatic
vacuum regulator) and then remaining traces of
volatiles were removed on a high-vacuum (<1
torr) oil pump.
Reactions were monitored by TLC using
EMD silica gel 60 F254 (250 µm) glass-backed
plates (visualized by UV fluorescence) and by
LC-MS Waters Acquity H UPLC CLASS
system, with Aquity UPLC BEH C18 column
(1.7 µm, 2.1x50 mm), eluting at 0.8 ml/min,
using a 3 min linear gradient method with a
mobile phase consisting of water/acetonitrile
(0.05 % v/v TFA added to each): 95:5→5:95 (0-
2.25 min), 95:5 (2.27-3 min). Sample runs were
monitored using alternating positive/negative
electrospray ionization (50-1000 amu) and UV
detection at 254 nm. Automated preparative
normal-phase chromatography was carried out
on a Büchi Reveleris Prep. Purification system
with 3 channel viable UV-Vis and ELS detection
(runs were monitored at 220-400 nm) Pre-
packed silica gel cartridges (12, 25 and 40 g)
Ethical statement
Mouse studies:
• Bioethical permission of pharmacokinetic
measurements in mice was granted to
Bienta/Enamine under the numbers:
IACUC number №1 from 19.05.2015 and
IACUC number №6 from 08.12.2015.
• Bioethical permission of the IMQ-induced
psoriasis study was granted to Washington
Biotechnology, Inc. under the IACUC
number 17-063.
• Bioethical permission of the CIA-induced
RA study was granted to Washington
Biotechechnology, Inc. under the IACUC
number 17-059.
were
employed
for
normal-phase
1
chromatography, eluting at 20-30 ml/min. H
NMR spectra were recorded at 400 MHz on a
Bruker spectrometer and are reported in ppm
using
the
residual
solvent
signal
(dimethylsulfoxide-d6 = 2.50 ppm) as an internal
standard. Data are reported as: {(δ shift),
[(s=singlet, d=doublet, dd=doublet of doublets,
br=broad, m=multiplet), (J=coupling constant in
Hz) and (integration)]}.
Human ex vivo studies (PBMCs):
The permission to isolate, treat and analyze
peripheral white blood cells from healthy donors
ex vivo was granted by the ethics commission of
the Technical University Munich (TUM)
(project number 358/15). The studies abide by
the Declaration of Helsinki principles.
12

315.40, tR = 0.98 min.
1-(3-Methyl-[1,2,4]-triazolo-[4,3-b]-pyridazin-
6-yl)-hydrazine. A round-bottomed flask was
charged with 6-chloro-3-methyl-[1,2,4]-triazolo-
[4,3-b]-pyridazine (2.5 g, 0.015 mol) and ethanol
(35 mL). To the resulting solution was added the
hydrazine hydrate (2.6 mL, 0.053 mol). The
reaction mixture was then heated at 80°C for 3h.
After cooling to room temperature, the formed
product slurry was filtered, washed with little
ethanol and air dried to afford 2.43g (98%) of
3-(3,5-Dimethyl-1-(3-methyl-[1,2,4]-triazolo-
[4,3-b]-pyridazin-6-yl)-1H-pyrazol-4-yl)-
propanoic acid. To a solution of methyl 3-(3,5-
dimethyl-1-(3-methyl-[1,2,4]triazolo[4,3-
b]pyridazin-6-yl)-1H-pyrazol-4-yl)propanoate
(175 mg, 0.557 mmol) in THF (1.75 ml) and
water (0.7 ml) was added LiOH.H2O (70.16 mg,
1.671 mmol). The reaction mixture was stirred at
room temperature for 20 h. A 3N HCl solution
(8 ml) was added and the mixture was extracted
with a mixture of 20 % isopropanol in
dichloromethane (3x). The combined organic
layers were dried over magnesium sulfate,
filtered and concentrated in vacuo. The white
solid obtained (140 mg, 84%) was used without
further purification. 1H NMR (400MHz, DMSO-
d6): δ 12.19 (s, 1H), 8.36 (d, J = 10 Hz; 1H),
7.85 (d, J = 10 Hz; 1H), 2.76-2.66 (br m, 5H),
2.6 (s, 3H), 2.55-2.48 (br m, 2H), 2.24 (s, 3H);
LC-MS (ESI+) m/z: [M+H]+ Calcd. for
C14H17N6O2 301.32 found 301.40, tR = 0.84 min.
1
the desired product as a white solid. H NMR
(400MHz, DMSO-d6): δ 8.46 (br s, 1H), 7.86 (d,
J = 10 Hz; 1H), 6.78 (d, J = 10 Hz ; 1H), 4.28
(br s, 2H), 2.55 (s, 3H); LC-MS (ESI+) m/z:
[M+H]+ Calcd. for C6H9N6 165.17 found 165.20,
tR = 0.17 min.
Methyl
3-(3,5-dimethyl-1-(3-methyl-[1,2,4]-
triazolo-[4,3-b]-pyridazin-6-yl)-1H-pyrazol-4-
yl)-propanoate. A round-bottomed flask filled
with
1-(3-methyl-[1,2,4]-triazolo-[4,3-b]-
pyridazin-6-yl)-hydrazine (190 mg, 1.066 mmol)
and glacial acetic acid (1.90 ml) was added
methyl 4-acetyl-5-oxohexanoate (0.22 mg, 1.17
mmol). The reaction mixture was stirred at
room temperature for 15 min, and then the acetic
acid was removed under vacuo. An aqueous
solution of 5 % sodium bicarbonate solution was
added and the mixture was extracted with ethyl
acetate (3x). The combined organic layers were
dried over magnesium sulfate, filtered and
concentrated in vacuo. The residue obtained was
purified by flash chromatography eluting with a
1-(4-Benzylpiperidin-1-yl)-3-(3,5-dimethyl-1-
(3-methyl-[1,2,4]-triazolo-[4,3-b]-pyridazin-6-
yl)-1H-pyrazol-4-yl)-propan-1-one. A round-
bottomed flask filled with a solution of 3-(3,5-
Dimethyl-1-(3-methyl-[1,2,4]-triazolo-[4,3-b]-
pyridazin-6-yl)-1H-pyrazol-4-yl)-propanoic acid
(50 mg, 0.17 mmol), in dimethylformamide (3
ml), were added 4-benzylpiperidine (0.06 ml,
0.33 mmol), HATU (68.52 mg, 0.18 mmol)) and
triethyl amine (0.04 ml, 0.306 mmol). The
resulting reaction mixture was stirred at room
temperature until complete consumption of the
starting material (17h) was observed. It was then
diluted with water (15 ml) and extracted with
ethyl acetate (3x10mL). The combined organic
layers were dried over MgSO4, filtered and
concentrated in vacuo. The product was purified
by flash chromatography eluting with a linear
gradient of ethyl acetate
/
methanol
100:00→90:10 as eluent. The combination of
the appropriate fractions yielded 243mg (66%)
1
of the title compound as a light yellow solid. H
NMR (400MHz, DMSO-d6): δ 8.37 (d, J =10
Hz; 1H), 7.85 (d, J = 10 Hz; 1H), 3.6 (s, 3H),
2.73-2.66 (br m, 5H), 2.6 (s, 3H) , 2.55-2.48 (br
m, 2H), 2.24 (s, 3H); LC-MS (ESI+) m/z:
[M+H]+ Calcd. for C15H19N6O2 315.35 found
13

gradient of ethyl acetate
100:00→50:50 as eluent. The combination of
/
methanol
2.71-2.61 (br m, 6H), 2.59 (s, 3H), 2.47-2.14 (br
m, 4H), 2.24 (s, 3H), 1.77-1.63 (br m, 1H), 1.59-
1.43 (br m, 2H), 0.99-0.73 (br m, 2H); LC-MS
(ESI+) m/z: [M+H]+ Calcd. for C26H32N7O
458.58 found 458.61, tR = 1.16 min.
the appropriate fractions yielded 55 mg (70%) of
1
the title product: H NMR (400MHz, DMSO-
d6): δ 8.4 (d, J = 10Hz, 1H), 7.88 (d, J = 10Hz,
1H), 7.33-7.09 (br m, 3H), 7.10-6.99 (br m, 2H),
4.39 (br d, 1H), 3.76 (br d, 1H), 2.86 (t, 1H),
Acknowledgements: We thank Martin Göttlicher for helpful discussions. We further thank Scarlett
Dornauer and Stefanie Brandner for excellent technical assistance and Sabrina Schreiner for gifting the
RNF4 antibody. This work has been supported by the Life Science Foundation to K.H. and the SFB 1054
project A04 to D.K.. A patent for the clinical use of C25-140 has been submitted to the European patent
office.
Conflict of interest: The authors declare that they have no conflict of interest with the content of this
article.
14

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18

Figure 1. C25-140 binds TRAF6, inhibits TRAF6-Ubc13 interaction and TRAF6 activity. A,
Chemical structure of C25-140 is indicated. B, Ectopically expressed HA-TRAF6 RZ12 in HEK-293T
cells co-immunoprecipitates less endogenous Ubc13 after C25-140 treatment. Ubc13 levels in the IP
fractions were densitometrically quantified in relation to precipitated TRAF6. Error bars indicate mean
+/- S.D.; n = 4 biological replicates were quantified; unpaired t-test (two-tailed); ****P<0.0001; D =
DMSO. C, Heteronuclear correlation NMR experiments using 15N-labeled wild-type TRAF6 RZ1 either
alone (blue) or in the presence of C25-140 (red; molar ratio TRAF6:C25-140 = 1:5). Several NMR
signals experience intensity reduction or broadening beyond detection (arrows) indicating binding in so
called “intermediate exchange regime”. This suggests that protein-compound complex lifetime is in
millisecond time scales and confirms direct binding of the compound to TRAF6. D, C25-140 dose-
dependently counteracts in vitro ubiquitination by untagged Wild-type TRAF6 RZ1 together with the E2
complex Ubc13/Uev1a.
19

Figure 2. Effect of C25-140 on various E3 ligases/E2 enzyme reactions. A, cIAP1 generating K63-
ubiquitin chains was also inhibited by C25-140. B, MDM2 and TRIM63 generating K48-ubiquitin chains
were not impaired by C25-140. C, RNF4, a SUMO targeted ubiquitin ligase, was also not affected by
C25-140. D, Activity of the HECT E3 ligases ITCH and E6AP was not inhibited by C25-140. The
substrates and the compound concentrations are indicated; assays were purchased from Boston Biochem.
20

Figure 3. Effect of C25-140 on E1 and E2 enzymes. Various E1/E2 ubiquitination reactions including
UbcH13/Uev1a were carried out in the absence or presence of C25-140 (30 µM). None of the reactions is
affected by the compound. C = control without ATP; D = DMSO; 140 = compound C25-140
21

Figure 4. C25-140 effect on proinflammatory signaling and T-cell activation. A, Endogenous TRAF6
auto-ubiquitination in MEF cells upon IL-1β stimulation is reduced after C25-140 treatment. B, C25-140
impairs IL-1β-induced IκBα phosphorylation. pIκBα levels were densitometrically quantified in relation
to β-Actin. Error bars indicate mean +/- S.D.; n = 3 biological replicates were quantified; unpaired t-test
(two-tailed); ****P<0.0001. C, Target gene (ICAM-1 and A20) expression is diminished after IL-1β
stimulation and C25-140 treatment; error bars indicate mean +/- S.D.; n ≥ 3 biological replicates;
unpaired t-test (two-tailed). *P<0.05, **P<0.01, ****P<0.0001. D, Endogenous TRAF6 auto-
ubiquitination after PMA/Ionomycin (P/I) stimulation is reduced after C25-140 treatment. E, C25-140
decreases IκBα phosphorylation after P/I stimulation in Jurkat T-cells. pIκBα levels were
densitometrically quantified in relation to β-Actin. Error bars indicate mean +/- S.D.; n = 3 biological
replicates were quantified; unpaired t-test (two-tailed); *P<0.05, **P<0.01. F, Upon P/I stimulation, IL-2
and TNFα cytokine secretion is attenuated after C25-140 treatment. Error bars indicate mean +/- S.D.; n =
3 biological replicates; unpaired t-test (two-tailed); *P<0.05, **P<0.01; D = DMSO.
22

Figure 5. C25-140 impedes immune receptor signaling in primary human PBMC and primary
murine T-cells. A-C, PBMCs from healthy individuals were isolated and treated with C25-140 prior to
stimulation with A, IL-1β, B, LPS and C, CD3/CD28. Secretion of various NF-κB driven cytokines was
measured by ELISA and showed reduction of distinct cytokine secretion by C25-140 in all donors. D,
Primary mouse CD4+ T-cells were isolated and treated with C25-140 and subsequently stimulated with
CD3/CD28. IL-2 mRNA expression and protein secretion were measured using qRT-PCR and ELISA,
respectively. C25-140 dose-dependently reduced IL-2 expression; error bars indicate mean +/- S.D.; n = 4
biological replicates; signals were normalized to DMSO; unpaired t-test (two-tailed); **P<0.01,
***P<0.001, ****P<0.0001; D = DMSO.
23

Figure 6. Topical application of the inhibitor C25-140 reduces in vivo outcome of imiquimod-
induced psoriasis. A, Preclinical mouse model for imiquimod (IMQ)-induced psoriasis. Mice (n = 8 per
group) were topically treated with IMQ to the back and ear once a day. C25-140 was topically applied
twice daily. Mice were scored throughout the study and samples were collected at day 6 for cytokine
measurement. (B-E) Mice were scored for B, cumulative score, C, thickness score, D, scaling and E,
erythema. All symptoms were improved by C25-140 treatment; error bars indicate mean +/- S.D.; 2-way
ANOVA test (Sidak’s multiple comparison test). F, IL-17 cytokine levels were evaluated by ELISA and
show a significant decrease after treatment; Unpaired t-test (two-tailed); error bars indicate mean +/- S.D.;
*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
24

Figure 7. C25-140 ameliorates symptoms of rheumatoid arthritis (RA) in a preclinical mouse
model. A, Study design of a collagen-induced arthritis (CIA) preclinical mouse model for RA (n = 10 per
group). RA was induced by collagen application at day 0 and a booster injection at day 21. At day 28,
C25-140 was intraperitoneally applied twice daily for 14 days. Mice were scored throughout the study
and limbs were collected at day 14 of treatment for histopathology. B, The average arthritic index was
determined for each group to analyze efficacy of treatment. C25-140 and the control prednisolone
significantly reduced the arthritic index; error bars indicate mean +/- S.E.M.; 2-way ANOVA test
(Sidak’s multiple comparison test). In histopathology analyses, mice were scored for C, H&E staining of
sections for histopathology (ankle and hindpaw). C25-140 at 14 mg/kg ameliorates disease outcome;
more histology images in Figure S7. D, Summed histology score, E, inflammation, pannus, and cartilage
damage. All quantified parameters demonstrate a dose-dependent improvement of RA symptoms after
C25-140 treatment; error bars indicate mean +/- S.E.M.; One-way ANOVA test (Sidak’s multiple
comparison test); more quantification of histology data are in Figure S8; ns = not significant; ***P<0.001,
****P<0.0001; BID = twice daily.
25

Targeting TRAF6 E3 ligase activity with a small molecule inhibitor combats
autoimmunity
Jara Kerstin Brenke, Grzegorz Maria Popowicz, Kenji Schorpp, Ina Rothenaigner, Manfred
Roesner, Isabel Meininger, Cédric Kalinski, Larissa Ringelstetter, Omar R'kyek, Gerrit
Jürjens, Michelle Vincendeau, Oliver Plettenburg, Michael Sattler, Daniel Krappmann and
Kamyar Hadian
J. Biol. Chem. published online June 27, 2018
Access the most updated version of this article at doi: 10.1074/jbc.RA118.002649
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