RESULTS
Gossypol Acetate (GA) was identified as a VCP inhibitor which
binds to interface between N and D1 domain
To identify novel modulator of VCP, we established a high-throughput
screening (HTS) biochemical assay for VCP ATPase activity measurement.
Upon screening our in-house natural small molecular library of 700
compounds (at 10 µM each) (Supplementary Fig. 1A), 9 hits were
identified with each inhibiting VCP ATPase activity by ≥50% at 10 µM.
The most potent hit was gossypol acetate (Fig. 1A), an acetic acid
mixture with gossypol, which almost completely inhibited the ATPase
activity of VCP at 10 µM (Supplementary Fig. 1A). Gossypol is an active
ingredient from the seeds or root bark of malvaceae or other plants.
Gossypol acetate has better absorption, longer serum half-life and less
side effects than gossypol itself and has been widely used in the clinic
(27 ). We thus focused on gossypol acetate for further study.
Gossypol acetate inhibited VCP enzymatic activity with an
IC50 of 6.53 ± 0.6 µM (Fig. 1B). To confirm the binding
of gossypol acetate to VCP, we performed partial trypsin digestion assay
using purified 6xHis-VCP protein in the presence of gossypol, gossypol
acetate or the allosteric VCP inhibitor NMS873 (28 ) (Fig. 1C). We
observed significant difference between each inhibitor-treated group and
the DMSO control group in the cleavage profile. Gossypol acetate as well
as gossypol reduced VCP’s sensitivity to trypsin digestion compared to
DMSO (Fig. 1C, red arrow), confirming their direct binding to VCP.
Notably, the digestion patterns of gossypol and gossypol acetate group
that were quite identical as expected, were quite different from that of
NMS873, suggesting that the influence of NMS873 on VCP conformation is
different from gossypol or gossypol acetate. Gossypol acetate inhibited
ATPase enzymatic activity of both VCP-FL(full length) and VCP-N+D1
domain, but not the one of VCP-D1+D2 domain (Fig. 1D), suggesting that
inhibition of VCP enzymatic activity by gossypol acetate required both N
and D1 domain of VCP.
To verify the binding of gossypol acetate to VCP, we carried out thermal
shift assay based on ligand-induced thermal stabilization of target
proteins. Thermostability of 6XHis-VCP protein was gradually increased
with increased concentration of gossypol acetate when temperature was
fixed at 70℃(Supplementary Fig. 1B). At 5 μM concentration of gossypol
acetate, VCP was significantly more stabilized compared to the DMSO
control group. Next, VCP protein melting temperature
(Tm) was determined upon incubating with 5 μM gossypol
acetate or DMSO control. Gossypol acetate significantly increased VCP’s
Tm from 64.2 ± 0.1℃ to 71.1 ± 0.7℃ (Fig. 1E, 1F),
confirming the direct binding of gossypol acetate to VCP. Consistent
with the result that VCP enzymatic inhibition by gossypol acetate
required the presence of both N and D1 domains, thermal shift assay also
confirmed that gossypol acetate bound to N+D1 domain. Incubation with 5
μM gossypol acetate significantly increased thermal stability of
VCP-N+D1 domain (Tm increased from 56.9±0.1℃ to 62.3±1.6℃) (Fig. 1G,
1H). We then used Isothermal Titration Calorimetry (ITC) to quantify the
interaction between VCP and gossypol acetate. The calorimetry
experiments showed that KD value is about 6.9 μM for the
binding between gossypol acetate and VCP (Supplementary Fig. 1C).
Next, we attempted to gain additional insight into the interaction by
docking gossypol to the crystal structure of VCP (PDB code 5DYG) in a
simulation experiment. The result of molecular docking, which suggested
that gossypol bound at the interface between VCP N domain and D1 domain
(Supplementary Fig. 1D), was consistent with the experimental results
that inhibition of VCP ATPase activity by gossypol acetate requires both
N and D1 domain. Given that the function of VCP relies on the formation
of hexamers, we next used native gel electrophoresis assay to determine
whether gossypol acetate affects VCP hexamer formation. Native gel
electrophoresis of the product of VCP incubated with gossypol acetate
showed that the oligomeric state of VCP complex was unchanged
(Supplementary Fig. 1E). Together, these results demonstrated that
gossypol acetate is bound to the interface between N and D1 domains of
VCP, thereby inhibiting the VCP ATPase activity in vitro without
affecting its oligomeric state.
Gossypol acetate reduced the mHTT protein level and neuronal
toxicity
As a crucial regulator of proteostasis, VCP regulate degradation of
damaged protein in proteasome and autophagy pathway. It has been
implicated in the pathology of HD as well as other neurodegenerative
disease, thus molecular modulator targeting VCP might be of therapeutic
benefits for treatment of neurodegenerative diseases including
Huntington’s disease. We tested whether gossypol acetate could
facilitate degradation of the toxic, aggregation-prone mHTT and improve
neuron survival in HD cellular models.
We first investigated whether gossypol acetate could promote degradation
of autophagy substrate-mHTT proteins in HD patient iPS-derived Q47
striatal neurons generated as previous described (29). The
well-established HTRF assay was used for measurement of mHTT level
(30 ) and assessment of mHTT clearance by gossypol acetate
treatment. Endogenous mHTT protein level was decreased dose-dependently
by gossypol acetate treatment (Fig. 2A). The observed reduction is not
due to cell loss, because the total protein concentration for mHTT
measurement was controlled. Meanwhile, compound treatment slightly
increased neuron cell number and improved cell morphology as shown in
Fig. 2D, excluding compound toxicity at the dose used. Western-blot
analysis was used to detect the level of mHTT and wtHTT protein. The 2B7
and MW1 antibodies were used, with MW1 specifically detecting the polyQ
mHTT and 2B7 detecting both mHTT and wtHTT. As shown in Fig 2B, gossypol
acetate induced obvious reduction of mHTT, while the level of total HTT
were only slightly decreased (Fig. 2B). Cotreatment with the autophagy
inhibitor CQ almost completely blocked mHTT degradation induced by
gossypol acetate (Fig 2C), implying that the observed mHTT lowering
mainly depends on autophagy pathway.
Since gossypol acetate induced mHTT degradation, we next examined
whether it could improve neuronal survival. Withdraw of brain-derived
neurophic factor (BDNF) induced neuronal apoptosis, shrinkage and loss,
which are mHTT-dependent (Figure 2D). These phenotypes under stress
condition were previously used for assaying huntington’s
disease-associated cytotoxicity. Treatment of gossypol acetate
significantly reduced caspase3 activation (Figure 2D-E) as well as
rescued stress-induced neuronal shrinkage and loss (Figure 2D, 2F). The
neuroprotective effect of gossypol acetate was further confirmed in the
mouse striatal cell line STHdhQ7/Q111 (31 ). In
this cell model, gossypol acetate remarkably induced mHTT degradation in
a dose-dependent manner as shown in HTRF assay and Western-blot analysis
(Supplementary Fig. 2A,B). Consistently, gossypol acetate reduced
apoptosis (detected by caspase3 activation) induced by serum starvation
(Supplementary Fig. 2C), indicating suppression of mHTT-induced
cytotoxicity. In summary, gossypol acetate treatment induced degradation
of mHTT proteins and significantly suppressed mHTT toxicity, which
relies on autophagy.
Gossypol acetate increased the formation of mHTT-VCP-LC3
ternary complex
We next investigated the mechanism by which gossypol acetate induced
mHTT degradation by harnessing the autophagic machinary. Previous
studies showed that VCP colocalized with expanded polyQ in HD mouse
brains and in postmortem patient brains (14, 32 ). We used
pull-down experiment to confirm the VCP-mHTT interaction and further
revealed that VCP’s N-terminal domain is the binding site for mHTT
(Supplementary fig 3A). Meanwhile, it has been reported that VCP
interacts with LC3 through LIR motif in VCP’s N terminal domain
(26 ). However, it remains unclear whether VCP is in a complex
with mHTT and LC3 and play a role in autophagy-mediated mHTT
degradation.
We carried out in vitro pull-down experiments to determine
whether VCP formed a complex with mHTT and LC3 and examined the effects
of gossypol acetate on VCP interaction with LC3 or mHTT. First, gossypol
acetate enhanced interaction between GST-LC3 and 6XHis-VCP in a
dose-dependent manner (Fig. 3A). In contrast, the VCP enzymatic
inhibitor MNS873 and DBEQ did not affect VCP’s interaction with LC3
(Supplementary Fig. 3B), in accordance with the observation that they
have different binding sites on VCP as well as different trypsin
digestion pattern Second, we examined whether gossypol acetate affected
VCP’s interaction with mHTT or with wtHTT. The pull-down assay revealed
that VCP’s interaction with mHTT is much stronger compared with its
interaction with wtHTT. Gossypol acetate significantly increased VCP’s
interaction with mHTT (Fig 3B), with little influence on VCP-wtHTT
interaction. Next, we determined whether VCP was in a complex with mHTT
and LC3 and the effects of gossypol acetate on complex formation.
Pull-down experiment showed VCP did form a complex with mHTT and LC3
(Fig.3C). Consistent with the enhanced interaction of VCP-LC3 and
VCP-mHTT respectively, gossypol acetate could also increase the complex
formation of 6XHis-VCP, GST-LC3 and mHTT proteins (Fig.3C). However, in
the absence of VCP, gossypol acetate did not obviously affect the
binding between GST-LC3 and mHTT-Q72 (Supplementary Fig. 3C), suggesting
that enhanced LC3-VCP- mHTT complex formation depends on gossypol
acetate’s effect on VCP. VCP-FL, VCP-N domain and VCP-N+D1 domain
interacted with LC3, respectively (Fig.3D), indicating that VCP
interacts with LC3 through the N domain of VCP. Gossypol acetate
increased LC3’s interaction with VCP-FL, VCP-N+D1 domain, but not VCP-N
alone. This further suggested the enhanced VCP-LC3 interaction relies on
gossypol acetate’s binding to VCP, which depends on the presence of both
N and D1 domain (Fig.1). These data revealed that gossypol acetate
enhanced formation of mHTT-VCP-LC3 complex, which may facilitate
autophagy-mediated degradation of mHTT by increasing the efficiency of
substrate recognition by autophagosomes.