A Non-Competitive Inhibitor of VCP/p97 and VPS4 Reveals Conserved Allosteric Circuits in Type I and II AAA ATPases

Article abstract of DOI:10.1002/anie.201711429

AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases.

Full text of DOI:10.1002/anie.201711429

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Accepted Article
Title: A non-competitive inhibitor of VCP/p97 and VPS4 reveals
conserved allosteric circuits in type I and II AAA ATPases
Authors: Robert Pöhler, Jan H. Krahn, Johannes van den Boom,
Grzegorz Dobrynin, Farnusch Kaschani, Hans-Michael
Eggenweiler, Frank T. Zenke, Markus Kaiser, and Hemmo
Meyer
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201711429
Angew. Chem. 10.1002/ange.201711429
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A non-competitive inhibitor of VCP/p97 and VPS4 reveals
conserved allosteric circuits in type I and II AAA ATPases
[
a]
[b]
[a]
[a]
Robert Pöhler, Jan H. Krahn, Johannes van den Boom, Grzegorz Dobrynin, Farnusch
[b]
[c]
[c]
[b]
[a]
Kaschani, Hans-Michael Eggenweiler, Frank T. Zenke, Markus Kaiser,* and Hemmo Meyer*
Abstract: AAA ATPases have pivotal functions in diverse cellular
processes essential for survival and proliferation. Revealing
strategies for chemical inhibition of this class of enzymes is therefore
of great interest for the development of novel chemotherapies or
chemical tools. Here, we characterize the compound MSC1094308
as a reversible, allosteric inhibitor of the type II AAA ATPase human
ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase
VPS4B. Subsequent proteomic, genetic and biochemical studies
indicate that MSC1094308 binds to a previously characterized
drugable hotspot of p97, thereby inhibiting the D2 ATPase activity.
Our results furthermore indicate that a similar allosteric site exists in
VPS4B, suggesting conserved allosteric circuits and drugable sites
in both type I and II AAA ATPases. Our results may thus guide future
chemical tool and drug discovery efforts for the biomedically relevant
AAA ATPases.
VPS4
p97
A
NTD
D1
D1
D2
B
C
Proteins of the AAA (ATPases associated with diverse cellular
activities) family are involved in fundamental processes ranging
from DNA replication to membrane traffic and ubiquitin-
dependent degradation via ATP-driven conformational
[
1]
p97:
IC50 = 7.2 ± 1.1 µM
rearrangements in substrate molecules. In humans, more than
00 different AAA ATPases are known that are characterized by
VPS4B: IC50 = 0.71 ± 0.10 µM
NSF
1
a highly conserved ATPase module harboring the Walker A and
B motifs of the P-loop NTPases.[ Most AAA ATPases form
single (type I) or double-ring-shaped (type II) homohexamers as
D
2]
[
3]
active units. Representative examples of type I are the
endolysosomal sorting ATPase VPS4A/B or the proteasomal
[
4]
base complex, while archetypical examples of type II (in which
the two ATPase domains are denoted as D1 and D2) are the
ubiquitin-directed
unfoldase
Valosin-containing
protein
(
(
VCP)/p97 or the NEM-sensitive membrane fusion factor (NSF)
Fig. 1A). In addition, many AAA ATPases also feature
[
5]
Figure 1. MSC1094308 is a non-competitive p97 and VPS4B inhibitor. A)
[
4a]
Cryo EM structure of the type I AAA ATPase VPS4 (pdb 5uie) from yeast
regulatory N-terminal domains (NTDs) that are involved in the
regulation of ATPase activity [5b, 6]
[5b]
and the type II AAA ATPase p97 (without the NTD, pdb 5ftk) from human
and a corresponding schematic of the general domain structure. B) Chemical
structure of MSC1094308. C) Malachite green-based ATPase assay reveals
inhibition of VPS4, p97 and NSF with different specificity. Shown are mean
and error (s.d.) of three replicates. D) Kinetic measurements of the ATPase
activity of p97 and VPS4B. Using the NADH coupled assay, the activity was
measured in the presence of different concentrations of MSC1094308. Data
were fitted to the Hill equation, shown are mean and error (s.d.) of three
replicates.
[
a]
Dr. R. Pöhler, Dr. J. van den Boom, Dr. G. Dobrynin, Prof. Dr. H
Meyer*
Molecular Biology I, Centre for Medical Biotechnology, University of
Duisburg-Essen, 45117 Essen, Germany
E-mail: hemmo.meyer@uni-due.de
[
b]
c]
Dr. J. H. Krahn, Dr. F. Kaschani, Prof. Dr. M. Kaiser*
Chemical Biology, Centre for Medical Biotechnology, University of
Duisburg-Essen, 45117 Essen, Germany
Chemical inhibitors may represent promising tools for
deciphering allosteric circuits in AAA ATPases. So far, most
efforts have focused on the development of p97 inhibitors due to
p97’s important role in the ubiquitin proteasome system (UPS)
E-mail: markus.kaiser@uni-due.de
[
Dr. H.-M. Eggenweiler, Dr. F. T. Zenke,
Medicinal Chemistry DA and Translational Innovation Platform
Oncology, Global R&D, Healthcare, Merck KGaA, Frankfurter Str.
[7]
and their potential biomedical application in cancer therapy.
These efforts have led to the development of competitive
ATPase inhibitors such as DBeQ and its ‘optimized’ analogue
CB-5083 which has entered clinical trials for treatment of certain
250, 64293 Darmstadt, Germany
Supporting information for this article is given via a link at the end of
the document.
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cancer types.[7b, 8] In addition, two allosteric inhibitors of p97,
This is in contrast to the published compound NMS-873 which
we confirmed is highly selective for p97 (Supporting Fig. 2).
MSC1094308 inhibition was reversible, as expected due to
[
9]
NMS-873 and UPCDC30245, have recently been reported
9]
(
Supporting Fig. 1).[5b,
Although both compounds are
structurally different, they target the same allosteric pocket in D2. absence of bioreactive groups in the compound and confirmed
For most other AAA ATPases, chemical inhibitors or structural
information on allosteric circuits are however not yet available.
with an initial treatment at 20 µM followed by a dilution series
that progressively relieved the inhibitory effect (Supporting Fig.
3).
Here, we report
a
chemical compound class that
allosterically inhibits both the type I ATPase VPS4 and the type
II ATPase p97, thereby demonstrating conserved allosteric
circuits in both types of AAA ATPases.
Enzyme kinetic studies for the AAA ATPases p97 and
VPS4B revealed that MSC1094308 did not affect the K or
M
cooperativity; however, Vmax was progressively reduced with
increasing inhibitor concentrations, indicating a non-competitive
inhibitory mechanism for both ATPases (Fig. 1D and Supporting
Table 1). This was corroborated by binding studies with
fluorescent ATP to p97, which was not affected by the presence
or absence of MSC1094308 (Supporting Fig. 4). Together with
our findings that MSC1094308 inhibition is reversible, these
experiments thus strongly suggest an allosteric inhibition of both
AAA ATPases.
We next performed structure-activity relationship (SAR)
studies, aiming to identify the impact of the different structural
units of MSC1094308.
A set of focused analogues was
synthesized and tested for p97 inhibition. The influence of the
bisfluorophenyl moiety was evaluated with derivatives 2 – 4 (Fig.
2
A and Table 1). While removal of the fluoride residues in 2 led
to an only three-fold decrease in activity (IC50 of 22.6 ± 5.6 µM),
further displacement of one (3) or both phenyl residues (4)
abolished their inhibitory potential. In contrast, modifications at
the tetrahydrocarbazole system were better tolerated (Fig. 2B
and Table 1). Removal of the fluoride residue (5) again had an
only weak effect (IC50 of 14.1 ± 3.8 µM). Deconstruction of the
ring system led to tetralin derivative 6 or cyclohexane derivative
7
that inhibited with IC50s of 25.2 ± 4.6 µM and 46.1 ± 6.1 µM,
respectively. Incorporation of a benzyl residue (8) instead of the
tetrahydrocarbazole moiety also resulted in derivatives with
remaining, nevertheless weak inhibitory potential (IC50 of 62.3 ±
7.2 µM). For VPS4B, a different picture was however obtained.
Here, the structural reductions leading to derivatives 2 – 8 had a
much lower impact on inhibition (Table 1). Indeed, all
Figure 2. Overview on synthesized chemical compounds for structure-activity-
relationship (SAR) studies. A) Chemical structures of fluoro-bisphenyl
analogues. B) Chemical structures of derivatives for probing the inhibitory
impact of the tetrahydrocarbazole moiety. C) Chemical synthesis of
MSC1094308 analogues. a) i) BnO(CH
2
)
3
PPh
, Pd/C, EtOH, rt, o/n, 46%; b) i) MsCl (2 eq.), NEt
h, ii) 1,4-dioxaspiro[4.5]decane-8-carboxamide (2 eq.), MeCN, 80 °C, rt,
2%; c) Ar-NHNH (2 eq.), TFA/DCM (1:3), 2 h, rt, 13-83%.
3
Br (1 eq.), KOtBu (1.1 eq.), THF,
‘tetrahydrocarbazole’ derivatives 5 – 8 were almost equipotent to
7
3
8
0 °C, 6 h, ii) H
2
3
(2.5 eq.), rt,
the parent inhibitor MSC1094308. Removal of residues on the
bisphenyl system were also much better tolerated, although
derivatives 3 and 4 no longer displayed a similar, classical
sigmoidal inhibition curve as observed for MSC1094308, thus
indicating that these compounds may exhibit an alternative
inhibition mode. Altogether, our SAR therefore indicates that the
fluorobisphenyl moiety is most important for effective inhibition
and that allosteric inhibitors for targeting both the type I ATPase
VPS4B and the type II ATPase p97 should feature the whole
bisfluorophenyl tetrahydrocarbazole scaffold. We therefore next
determined the impact of different substitutions at the
tetrahydrocarbzole moiety on p97 and VPS4B inhibition and
found that most substitutions were well tolerated (Fig. 2C and
Table 1). Intriguingly, we however also noticed a correlation
between p97 and VPS4B inhibition, i.e. more potent p97
inhibitors are also more potent VPS4B inhibitors and vice versa;
indeed, most tetrahydrocarbazole VPS4B inhibitors are about
2
Our studies started from the identification of MSC1094308 (1) in
a biochemical NADH-coupled high throughput screen for p97
ATPase inhibitors (Fig. 1B). We validated the inhibitory effect vs.
p97 by a malachite green assay and observed a sigmoidal dose-
activity relationship and an IC50 of 7.2 ± 1.1 µM, comparable to
the published activity of the frequently used p97 inhibitor DBeQ
8a]
(
Fig. 1C and Table 1).[
To deduce the specificity of
MSC1094308, we tested its inhibitory potential vs. two other
AAA ATPases (Fig. 1C and Table 1). For the type II AAA
ATPase NSF, we observed dose-dependent inhibition only at
higher concentrations, which prevented reliable determination of
an IC50. For the type I AAA ATPase VPS4B, ATPase activity was
determined in presence of an activating fragment of its substrate
CHMP1B.[10] MSC1094308 inhibited VPS4B about ten times
more potently than p97 with an IC50 of 0.71 ± 0.10 µM.
10-fold more potent than against p97, thereby indicating that the
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generated compounds target a structurally homologous binding
site.
it suggests that this allosteric mechanism is also conserved in
VPS4B even though it contains only one ATPase domain.
To assess the functional relevance of this binding region for
VPS4B, we generated a set of VPS4B mutants based on
structural information of the predicted binding site and tested
their ATPase activity.[ All generated mutants from this binding
site (V260A, Q261A, N269Y, G271Q, G271F) were catalytically
inactive. Proper protein folding was confirmed by ATP binding
assays and CD spectra for the Q261A and G271F mutants
Table 1. Inhibition potencies of synthesized compounds.
4a]
Compound
p97 IC50 (µM)
VPS4B IC50 (µM)
MSC1094308 (1)
7.2 ± 1.1
22.6 ± 5.6
> 100
0.71 ± 0.10
6.6 ± 3.4
3.0 ± 0.7
17.3 ± 5.9
1.7 ± 0.7
1.9 ± 0.8
1.2 ± 0.3
1.4 ± 0.5
0.63 ± 0.08
1.6 ± 0.5
1.4 ± 0.7
0.76 ± 0.22
7.9 ± 1.2
8.1 ± 4.4
2.3 ± 1.1
2
3
4
5
6
7
8
(Supporting Fig. 8). In contrast, mutants with changes outside
the binding region maintained their activity (e.g. Q221A, N218A,
K217A), thereby indicating that the allosteric binding site has
indeed functional relevance for VPS4B.
> 100
14.1 ± 3.8
25.2 ± 4.6
46.1 ± 6.1
62.3 ± 7.2
9.0 ± 2.2
16.7 ± 10.6
15.5 ± 4.1
8.6 ± 1.2
23.6 ± 4.9
46.7 ± 5.6
80.2 ± 22.6
To clarify which of the two ATPase domains in p97 is
targeted by MSC1094308, we introduced Walker B mutations
(
that allow ATP binding but impair ATP hydrolysis) in either the
D1 or D2 ATPase domain and measured the effect of the
inhibitor on the residual activity in our ATPase assays (Fig.
3
B).[11] Incubation with 20 µM MSC1094308 effectively inhibited
12a
12b
12c
12d
12e
D2 ATPase activity by roughly 80% in the mutant deficient in D1
hydrolysis and thus to the same extent as wild-type p97. In
contrast, MSC1094308 inhibited the residual D1 ATPase activity
in the D2 hydrolysis mutant only by 30%, suggesting that
MSC1094308 targets predominantly the D2 domain, as
expected for an allosteric inhibitor targeting the NMS-873
binding site. In support of this notion, MSC80829 also stabilized
the D2 domain in limited proteolysis experiments to a similar
degree as NMS-873 (Supporting Fig. 9). [9]
12f
13
Next, we tested the effect of mutations in the allosteric site
on MSC1094308-mediated inhibition. Previous studies revealed
that the N616F mutation which is located in the allosteric binding
site reduced the sensitivity of allosteric p97 inhibition by NMS-
To narrow down the position of the allosteric binding site, we
made use of our findings that benzyl derivative 8 still retained
8
73 and may thus be used as a molecular ‘sensor’ for binding at
VPS4B inhibitory potential. Accordingly, we synthesized
a
[9]
this site. We therefore explored the effect of the N616F
mutation on the activity of MSC1094308 (Fig. 3C). As observed
for NMS-873, the mutation almost completely abolished
inhibition of p97 ATPase activity by MSC1094308. These results
thus strongly suggest that the two allosteric inhibitors NMS-873
and MSC1094308 act via a similar molecular mechanism.
phenyl azido derivative 13 for chemical crosslinking studies that
inhibited VPS4B with an IC50 of 2.3 ± 1.1 µM while p97 inhibition,
as expected, was much lower (Supporting Fig. 5 and Table 1).
We then performed photoaffinity labeling experiments with
VPS4B (in the presence of CHMP1B 106-199), using either 13
or DMSO as a control. After UV crosslink, VPS4B was digested
with LysC and trypsin and the abundance of the resulting
unmodified tryptic peptides was measured via LC-MS/MS. This
analysis revealed two groups of VPS4B tryptic peptides that
were less abundant in the 13- than DMSO-treated samples: a
first set of peptides corresponding to the N-terminal MIT domain
Finally, we tested whether our allosteric p97 inhibitor is also
an efficient p97 inhibitor in living cells. Consistent with p97
inhibition, MSC1094308, like the proteasome inhibitor MG132,
led to accumulation of polyubiquitin as
a biomarker for
degradation inhibition (Fig. 3D). To discriminate between
proteasome and p97 inhibition, we used a cell line that
expresses two proteasomal model substrates, ODD-luciferase,
which is degraded independently of p97, and an Ubiquitin-GFP
fusion (Ub-GFP) that requires p97 for degradation.[ Levels of
both proteins were monitored by Western blot after they were
accumulated by proteasome inhibition and then released for
degradation. While ODD-luciferase degradation was inhibited by
MG132 during the release (Supporting Fig. 10A), 10 µM
MSC1094308 did not affect the degradation (Fig. 3E). In
contrast, degradation of Ub-GFP was sensitive to both inhibitors
at the same concentrations (Fig. 3E). These results demonstrate
that MSC1094308 is active in cells, and specifically inhibits p97,
(
(
residues 24 – 46) and a second set from the ATPase domain
residues 256 – 289) (Supporting Fig. 6 and 7 and Supporting
Table 2). The MIT domain-derived peptides are not homologous
to elements of p97. The other peptide however stems from a
highly conserved region in the large ATPase subdomain of the
AAA module and is therefore homologous to equivalent regions
in the p97 D1 and D2 ATPase domains. Intriguingly, this region
corresponds to the previously described binding area of the
allosteric inhibitors NMS-873 and UPCDC30245 in the p97 D2
8a]
Fig. 3A).[5b, 9] This indicates that MSC1094308 may modulate
p97 by binding to this known inhibitory allosteric site. Moreover,
(
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but not the proteasome. As a second cellular assay, we also
followed IB degradation that is p97 and proteasome
dependent and occurs after stimulation of the NFB pathway.[12]
Accordingly, we monitored IB levels by Western blot analysis
after TNF stimulation and observed a significantly delayed
IB degradation after 10 µM MSC1094308 application,
comparable to inhibition by the equipotent inhibitor DBeQ
(Supporting Fig. 10B).
Figure 3. MSC1094308 targets a conserved allosteric site on p97 and VPS4. A) D2 AAA domain of p97 (left panel, pdb 5ftj) in complex with the allosteric
regulator UPCDC30245 and AAA domain of VPS4 (right panel, pdb 5uie) reveals conserved structural features. The 13-labelled peptide and its corresponding
homologue in p97 is colored yellow, the allosteric gatekeeper is colored red and brown labels indicate residues involved in UPCDC30245 binding (in p97) or the
corresponding homologous residues in VPS4. Note that for NMS-873, a slightly different binding mode (that involves stronger interactions with the yellow labelled
helix) has been proposed. B) Inhibition of p97 Walker mutants by MSC1094308. ATPase activity was measured in the presence of 20 µM MSC1094308 and
normalized to uninhibited wild type (wt) activity. B1: Walker B in D1 (E305Q); B2: Walker B in D2 (E578Q). Shown are mean and error (s.d.) of three replicates. C)
The p97 N616F mutation in the proposed binding site reduces inhibition by MSC1094308 and NMS-873. Shown are mean and error (s.d.) of three replicates. D)
MSC1094308 induces accumulation of polyubiquitinated proteins. HCT116 cells were treated with MSC1094308 or the proteasome inhibitor MG132 (10 µM each)
for 8 h. Cells were lysed and ubiquitin (Ub) was detected by Western Blotting. Tub, tubulin control. E) MSC1094308 inhibits p97 but not the proteasome in a
reporter cell line. Specificity of the inhibitor was shown by a cycloheximide chase experiment in a double reporter HeLa cell line, expressing Ub-GFP and ODD-
Luciferase. Both constructs are degraded in the proteasome, but only Ub-GFP in a p97-dependent manner. 10 µM MSC1094308 stabilized Ub-GFP, but not
ODD-Luciferase.
Together, we have shown that MSC1094308 is an allosteric
reversible inhibitor of p97 and VPS4B. Subsequent genetic and
biochemical studies indicate that MSC1094308 binds to an
Acknowledgements
allosteric binding site that is shared by two previously described,
This work was funded by the DFG SFB1093 to H.M. and M.K.
structurally unrelated allosteric p97 inhibitors, suggesting that
this site is a promising hotspot for chemical modulation. This
binding site is located at the D2 domain in the interface area
between the D1 and D2 (often denoted as the D1/D2 interface)
of p97. Intriguingly, a similar allosteric site seems to be present
in VPS4B, thereby indicating conserved allosteric circuits and
drugable binding sites in both type I and II AAA ATPases. This is
unexpected as type I AAA ATPases lack the D1/D2 interface,
and display a structurally different NTD-D1 interface. Our
findings thus indicate that allosteric circuitry is a generic feature
of AAA ATPases and not limited to type II AAA ATPases. We
anticipate that these findings may guide future drug discovery
efforts, e.g. to achieve domain or cofactor-specific regulation of
the biomedically highly relevant AAA ATPases.[13]
Keywords: AAA ATPase • p97 • VPS4 • allostery • drug
discovery
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
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One class of allosteric inhibitors
for different AAA ATPases. An
allosteric inhibitor of the type I AAA
ATPase VPS4B and the type II AAA
ATPase p97 reveals unexpected
conserved allosteric features in both
architectural diverse AAA ATPases.
R. Pöhler, J. H. Krahn, J. van den
Boom, G. Dobrynin, F. Kaschani, H.-
M. Eggenweiler, F. T. Zenke, M.
Kaiser*, H. Meyer*
Page No. – Page No.
A non-competitive inhibitor of
VCP/p97 and VPS4 reveals
conserved allosteric circuits in type
I and II AAA ATPases
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