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.