DISCUSSION
VCP plays a critical role in governing proteostasis and protecting cell
from cytotoxic effects caused by damaged proteins. Its dysfunction is
related to pathogenesis of neurodegenerative diseases, thus VCP is
believed to be a potential therapeutic target for treatment of
neurodegenerative disease. In the present study, we identified gossypol
acetate, a drug from traditional Chinese medicine for treatment of
uterine leiomyoma and endometriosis, as a novel inhibitor of the ATPase
of VCP. We showed that gossypol acetate induced autophagy-mediated
degradation of mHTT and rescued HD-relevant phenotypes.
We found that gossypol has a unique mechanism of action through
enhancing formation of the ternary VCP-LC3-mHTT complex, thus
facilitating autophagy-mediated mHTT degradation. This unique mechanism
distinguished it from previous reported VCP inhibitors as well as other
autophagy inducers. Moreover, although VCP has been shown to co-localize
with mHTT and involved in HD pathogenesis (14 ), whether VCP plays
a crucial role in autophagy-mediated mHTT degradation remains unclear.
In our study, we revealed N-terminal domain of VCP mediated the
formation of the ternary complex of VCP-LC3-mHTT and facilitate
autophagy-mediated degradation of mHTT, which was significantly enhanced
upon gossypol acetate treatment. Gossypol acetate binding to VCP
increased the interactionof VCP with LC3 and mHTT respectively, thus
leading to enhanced complex formation. In contrast, the formation of
wtHTT-VCP-LC3 complex is largely unaffected by gossypol acetate
treatment and VCP interaction with wtHTT is much weaker compared to
interaction between VCP and mHTT, suggesting that gossypol selectively
recruit mHTT to the ternary complex for autophagic degradation.
Furthermore, enhanced interaction between mHTT and LC3 complex requires
VCP, since gossypol acetate did not affect binding between LC3 and mHTT
in the absent of VCP. Based on these observations, we proposed a model
where gossypol acetate bound to VCP, acted in a gain-of-function manner
to promote the formation of mHTT-VCP-LC3 ternary complex, thus
selectively accelerating autophagy-mediated degradation of mHTT. This
mechanism of action of gossypol acetate is completely different from
previously reported VCP inhibitors and is reminiscent of the mode of
action of rapamycin and FK506 which act through induction of the
formation of mTOR-rapamycin-FKBP12 and Calcineurin-FK506-FKBP12
complexes respectively (34, 35 ). This is the unique merit of
small molecules, revealing a mechanism that cannot be captured by
genetic deletion of knock-down experiment.
Different from previously reported VCP inhibitors which target VCP D2
enzymatic domain, gossypol acetate binds to interface between VCP’s N
and D2 domain and enhanced VCP-N domain interaction with LC3 and mHTT.
The N terminal domain of VCP is involved in cofactor binding and
substrate recognition (36,37 ). The interface between N-domain and
D1 domain was considered to be important for N-D1 communication, as well
as D1-controlled conformational changes of the N domain, which in turn
might regulate co-factor interactions. In our study, we demonstrated the
N terminal domain is responsible for VCP’s interaction with LC3 as well
as mHTT. Gossypol acetate bound to the interface between N and D1
domain, and its inhibition of VCP enzymatic activity as well as its
interaction with VCP required the presence of both N and D1 domain. The
binding of gossypol acetate to VCP N-D1 interface increased VCP
interaction with both mHTT and LC3 without affecting VCP
oligomerization. Thus, binding of gossypol acetate to N-D1 interface
might regulate N-terminal domain conformation and thus increased
LC3-VCP-mHTT complex formation. Two other known inhibitors of VCP,
NMS-873 and DBEQ are binding to different positions of VCP, and our
trypsin digestion pattern of VCP also implicated that gossypol acetate’s
binding site is quite different from that of NMS-873. As an interaction
hub, VCP has multiple cofactor binding sites and diverse mechanism to
regulate distinct aspect of VCP-mediated process. It has been reported
that various disease-associated mutations might affect the association
of VCP with certain cofactors, leading to impairment of distinct subsets
of VCP/p97 functions and different effects (10 ). It is reasonable
that inhibitors binding to different site of VCP might regulate distinct
aspect of VCP and have different regulatory effects (36 ).
We showed in our study that gossypol acetate had beneficial effects in
HD treatment through targeting VCP and harnessing the autophagy
machinery. Although gossypol has been used for the treatments of various
diseases through targeting different proteins, this is the first time
that its neuroprotective role has been identified and its unique
underlying mechanism is unraveled. The improvement of neuronal survival
by gossypol acetate treatment was comparable to the effects of mHTT
knock-down in human iPS-derived HD neurons. Its neuronal protective
effect was potent and specific to mHTT-caused neuronal toxicity in vivo
in drosophila HD models. In our HD cell and mouse model, gossypol
acetate lowered the level of mHTT about 20-40% and rescued Huntington’s
disease-associated phenotypes. It is reasonable since previous study in
human ESC-derived neuronal model suggested that 10-20% reduction of
mHTT alone is sufficient to show a significant reduction of toxicity,
whereas reducing wtHTT by up to ~90% seemed to be safe
(38 ). Reducing mHTT protein has been proved to be an effective
therapeutic approach in animal model and the recent clinical trial (39).
Gossypol has been reported to be able to penetrate blood–brain barrier
(BBB) (40 ). In our study, gossypol acetate improved HD-relevant
motor function deficits in both Drosophila and mouse HD knock-in models.
Thus, gossypol acetate is a promising lead for developing new treatment
of HD.
Binding of gossypol to VCP enhance mHTT-VCP-LC3 ternary complex
formation and ensuing degradation of the disease-causing mHTT protein.
This novel mechanism of action that bears similarity to rapamycin and
FK506 revealed a novel strategy to use small molecules targeting VCP to
facilitate autophagy-mediated clearance of disease-causing mHTT.
Recently, a tethering compound that interacted with both LC3 and mHTT
has been reported to be able to target mHTT for autophagic degradation,
reduce mHTT level and rescue disease-relevant phenotypes (41 ).
Herein, gossypol similarly acted in a gain-of-function way to induce
mHTT degradation by autophagy, yet these effects depends on the
regulation of VCP by gossypol. In summary, our study showed that small
molecules targeting VCP could have therapeutic potential for treatment
of HD disease and we have identified gossypol as the first small
molecule modulator of VCP in selective autophagic degradation of mutant
HTT. As gossypol has already been used as a drug in China for many
years, our results also suggest that it can be directly repurposed for
clinical evaluation as a new therapy for HD.