Discovery of a potent SCAP degrader that ameliorates HFD-induced obesity, hyperlipidemia and insulin resistance via an autophagy-independent lysosomal pathway.

SCAP (SREBF chaperone) regulates SREBFs (sterol regulatory element binding transcription factors) processing and stability, and, thus, becomes an emerging drug target to treat dyslipidemia and fatty liver disease. However, the current known SCAP inhibitors, such as oxysterols, induce endoplasmic reticulum (ER) stress and NR1H3/LXRα (nuclear receptor subfamily 1 group H member 3)-SREBF1/SREBP-1 c-mediated hepatic steatosis, which severely limited the clinical application of this inhibitor. In this study, we identified a small molecule, lycorine, which binds to SCAP, which suppressed the SREBF pathway without inducing ER stress or activating NR1H3. Mechanistically, lycorine promotes SCAP lysosomal degradation in a macroautophagy/autophagy-independent pathway, a mechanism completely distinct from current SCAP inhibitors. Furthermore, we determined that SQSTM1 captured SCAP after its exit from the ER. The interaction of SCAP and SQSTM1 requires the WD40 domain of SCAP and the TB domain of SQSTM1. Interestingly, lycorine triggers the lysosome translocation of SCAP independent of autophagy. We termed this novel protein degradation pathway as the SQSTM1-mediated autophagy-independent lysosomal degradation (SMAILD) pathway. In vivo, lycorine ameliorates high-fat diet-induced hyperlipidemia, hepatic steatosis, and insulin resistance in mice. Our study demonstrated that the inhibition of SCAP through the SMAILD pathway could be employed as a useful therapeutic strategy for treating metabolic diseases.Abbreviation: 25-OHD: 25-hydroxyvitamin D; 3-MA: 3-methyladenine; ABCG5: ATP binding cassette subfamily G member 5; ABCG8: ATP binding cassette subfamily G member 8; ACACA: acetyl-CoA carboxylase alpha; AEBSF: 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; AHI: anhydroicaritin; AKT/protein kinase B: AKT serine/threonine kinase; APOE: apolipoprotein E; ATF6: activating transcription factor 6; ATG: autophagy-related; BAT: brown adipose tissue; CD274/PD-L1: CD274 molecule; CETSA: cellular thermal shift assay; CMA: chaperone-mediated autophagy; COPII: cytoplasmic coat protein complex-II; CQ: chloroquine; DDIT3/CHOP: DNA damage inducible transcript 3; DNL: de novo lipogenesis; EE: energy expenditure; EGFR: epithelial growth factor receptor; eMI: endosomal microautophagy; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FADS2: fatty acid desaturase 2; FASN: fatty acid synthase; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic-pyruvate transaminase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; HMGCS1: 3-hydroxy-3-methylglutaryl-CoA synthase 1; HSP90B1/GRP94: heat shock protein 90 beta family member 1; HSPA5/GRP78: heat hock protein family A (Hsp70) member 5; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; INSIG1: insulin induced gene 1; LAMP2A: lysosomal associated membrane protein 2A; LDLR: low density lipoprotein receptor; LyTACs: lysosome targeting chimeras; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MBTPS1: membrane bound transcription factor peptidase, site 1; MEF: mouse embryonic fibroblast; MST: microscale thermophoresis; MTOR: mechanistic target of rapamycin kinase; MVK: mevalonate kinase; PROTAC: proteolysis targeting chimera; RQ: respiratory quotient; SCAP: SREBF chaperone; SCD1: stearoyl-coenzemy A desaturase 1; SMAILD: sequestosome 1 mediated autophagy-independent lysosomal degradation; SQSTM1: sequestosome 1; SREBF: sterol regulatory element binding transcription factor; TNFRSF10B/DR5: TNF receptor superfamily member 10b; TRAF6: TNF receptor associated factor 6; UPR: unfolded protein response; WAT: white adipose tissue; XBP1: X-box binding protein 1.

Sodium cantharidinate, a novel anti-pancreatic cancer agent that activates functional p53.

Despite the use of many types of chemotherapies for pancreatic cancer, optimal efficacy has not been obtained so far. Pancreatic cancer shows a high incidence of TP53 mutations, inactivating its tumor suppressor activity. In this study, we identified sodium cantharidinate as a novel, potential anti-pancreatic cancer agent that activates p53 function. Sodium cantharidinate reduced the viability of pancreatic cancer cells, including the human primary pancreatic cancer cells, PANC-1, AsPC-1, SW1990 and BXPC-3, in a dose-dependent manner. Sodium cantharidinate induced apoptosis and DNA damage of pancreatic cancer cells. Furthermore, proteome-wide sequencing analysis detected a marked perturbation in p53 signaling pathway on PANC-1 cells upon sodium cantharidinate. Consistent with the previous results, sodium cantharidinate treatment decreased Bcl-2 and mitochondrial cytochrome-c protein expression, as well as phosphorylation of MDM2; meanwhile, it increased the levels of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, Bax, and phosphorylated p53, thus inducing the apoptosis of pancreatic cancer cells. The p53-activating effect of sodium cantharidinate was strongly abrogated by treatment with TP53-targeting shRNA. Moreover, sodium cantharidinate inhibited neoplasm growth via the JAK2-STAT3 pathway, which was inhibited by shRNA-TP53 and triggered by combination with gemcitabine. Combination therapy indicated that sodium cantharidinate and gemcitabine synergistically reduced ex vivo and in vivo growth of pancreatic neoplasm. Further docking studies revealed the different binding fates of sodium cantharidinate to activate wild-type p53 function. Thus, sodium cantharidinate could be a potential agent with promising anti-pancreatic cancer efficacy.

Dual targeting of SREBP2 and ERRα by carnosic acid suppresses RANKL-mediated osteoclastogenesis and prevents ovariectomy-induced bone loss.

Osteoporosis develops because of impaired bone formation and/or excessive bone resorption. Several pharmacological treatment of osteoporosis has been developed; however, new treatments are still necessary. Cholesterol and estrogen receptor-related receptor alpha (ERRα) promote osteoclasts formation, survival, and cellular fusion and thus become high risk factors of osteoporosis. In this study, we identified that carnosic acid (CA) suppressed bone loss by dual-targeting of sterol regulatory element-binding protein 2 (SREBP2, a major regulator that regulates cholesterol synthesis) and ERRα. Mechanistically, CA reduced nuclear localization of mature SREBP2 and suppressed de novo biogenesis of cholesterol. CA subsequently decreased the interaction between ERRα and peroxisome proliferator-activated receptor gamma coactivator 1-beta (PGC1ß), resulting in decreased the transcription activity of ERRα and its target genes expression. Meanwhile, CA directly bound to the ligand-binding domain of ERRα and significantly promoted its ubiquitination and proteasomal degradation. Subsequently, STUB1 was identified as the E3 ligase of ERRα. The lysine residues (K51 and K68) are essential for ubiquitination and proteasomal degradation of ERRα by CA. In conclusion, CA dually targets SREBP2 and ERRα, thus inhibits the RANKL-induced osteoclast formation and improves OVX-induced bone loss. CA may serve as a lead compound for pharmacological control of osteoporosis.

Inhibition of HSP90ß Improves Lipid Disorders by Promoting Mature SREBPs Degradation via the Ubiquitin-proteasome System.

Rationale: Heat shock protein 9 (HSP90) are a family of the most highly expressed cellular proteins and attractive drug targets against cancer, neurodegeneration diseases, etc. HSP90 proteins have also been suggested to be linked to lipid metabolism. However, the specific function of HSP90 paralogs, as well as the underlying molecular cascades remains largely unknown. This study aims to unravel the paralog-specific role of HSP90 in lipid metabolism and try to discover paralog-specific HSP90 inhibitors. Methods: In non-alcohol fatty liver disease (NAFLD) patients, as well as in diet induced obese (DIO) mice, expression of HSP90 paralogs were analyzed by immunohistochemistry and western blot. In hepatocytes and in DIO mice, HSP90 proteins were knockdown by siRNAs/shRNAs, metabolic parameters, as well as downstream signaling were then investigated. By virtue screening, corylin was found to bind specifically to HSP90ß. Using photo-affinity labeling and mass spectrum, corylin binding proteins were identified. After oral administration of corylin, its lipid lowering effects in different metabolic disease mice models were evaluated. Results: We showed that hepatic HSP90ß, rather than HSP90α, was overexpressed in NAFLD patients and obese mice. Hepatic HSP90ß was also clinical relevant to serum lipid level. Depletion of HSP90ß promoted mature sterol regulatory element-binding proteins (mSREBPs) degradation through Akt-GSK3ß-FBW7 pathway, thereby dramatically decreased the content of neutral lipids and cholesterol. We discovered an HSP90ß-selective inhibitor (corylin) that only bound to its middle domain. We found that corylin treatment partially suppressed Akt activity only at Thr308 site and specifically promoted mSREBPs ubiquitination and proteasomal degradation. Corylin treatment significantly reduced lipid content in both liver cell lines and human primary hepatocytes. In animal studies, we showed that corylin ameliorated obesity-induced fatty liver disease, type 2 diabetes and atherosclerosis. Principle conclusions: HSP90ß plays a parolog-specific role in regulating lipid homeostasis. Compound that selectively inhibits HSP90ß could be useful in the clinic for the treatment for metabolic diseases.

Phellodendrine chloride suppresses proliferation of KRAS mutated pancreatic cancer cells through inhibition of nutrients uptake via macropinocytosis.

Despite the massive efforts to develop the treatment of pancreatic cancers, no effective application exhibits satisfactory clinical outcome. Macropinocytosis plays a critical role for continuous proliferation of pancreatic ductal adenocarcinoma (PDAC). In this study, we generated a screening method and identified phellodendrine chloride (PC) as a potential macropinocytosis inhibitor. PC significantly inhibited the viability of KRAS mutant pancreatic cancer cells (PANC-1 and MiaPaCa-2) in a dose-dependent manner; however, it did not affect the wild type KRAS pancreatic cancer cells (BxPC-3). Further experiments indicated that PC reduced the growth of PANC-1 cells through inhibition of macropinocytosis and diminishing the intracellular glutamine level. Disruption of glutamine metabolism led to enhance the reactive oxygen species level and induce mitochondrial membrane potential depolarization in PANC-1 cells. PC treatment caused increased Bax and decreased Bcl-2 expression, along with the activation of cleaved caspase-3, 7, 9 and cleaved-PARP, thus induced mitochondrial apoptosis. Moreover, PC inhibited macropinocytosis in vivo and effectively reduced the growth of PANC-1 xenograft tumors. All together, we demonstrated that inhibition of macropinocytosis might be an effective strategy to treat pancreatic cancers. Thus, PC could be a potential compound with improved therapeutic efficacy in patients with pancreatic cancers.

Correction: Praeruptorin B improves diet-induced hyperlipidemia and alleviates insulin resistance via regulating SREBP signaling pathway.

[This corrects the article DOI: 10.1039/C7RA11797C.].

Jujuboside A promotes Aß clearance and ameliorates cognitive deficiency in Alzheimer's disease through activating Axl/HSP90/PPARγ pathway.

Rationale: It has been reported that peroxisome proliferator activated receptor γ (PPARγ) level decreases significantly in the brains of Alzheimer's disease (AD) patients and mice models, while the mechanism is unclear. This study aims to unravel the mechanism that amyloid ß (Aß) decreases PPARγ and attempted to discover lead compound that preserves PPARγ. Methods: In APP/PS1 transgenic mice and Aß treated microglia, the interaction between HSP90 and PPARγ were analyzed by western blot. Using a PPRE (PPARγ responsive element) containing reporter cell line, compounds that activate PPARγ activity were identified. After genetic ablation or pharmacological inhibition of potential target pathways, the target of jujuboside A (JuA) was discovered through Axl/HSP90ß. After oral administration or intrathecal injection, the anti-AD activity of JuA was evaluated by Morris water maze (MWM) test and object recognition test. Soluble Aß42 levels and plaque numbers after JuA treatment were detected by thioflavin S staining, and the activation of microglia was assayed by immunofluorescence staining against Iba-1. Results: We found that Aß stress decreased heat shock protein 90 ß (HSP90ß), subsequently reduced the abundance of PPARγ, and down-regulated Aß clearance-related genes in BV2 cells and primary microglia. We identified that JuA stimulated the expression of HSP90ß, strengthened the interaction between HSP90ß and PPARγ, preserved PPARγ levels, and thus effectively promoted the clearance of Aß42. We demonstrated that JuA increased HSP90ß expression through Axl/ERK pathway. JuA significantly ameliorated cognitive deficiency in APP/PS1 transgenic mice, meanwhile, JuA significantly reduced the soluble Aß42 levels and plaque numbers in the brain. Notably, the therapeutic effects of JuA were dampened by R428, an Axl inhibitor. Conclusions: This study suggests that the up-regulation of HSP90ß by JuA through Axl is a potential therapeutic strategy to facilitate Aß42 clearance and ameliorate cognitive deficiency in AD.

Anhydroicaritin, a SREBPs inhibitor, inhibits RANKL-induced osteoclastic differentiation and improves diabetic osteoporosis in STZ-induced mice.

Nowadays, more and more attention has been paid to osteoporosis caused by diabetes mellitus. Elevated levels of pro-inflammatory cytokines in diabetic patients activate the activity of osteoclasts through the RANKL/OPG pathway. The nuclear transcription factor SREBP2, a master regulator of cholesterol metabolism, has been found involved in osteoclastogenesis. In our previous study, we have identified anhydroicaritin as a potent inhibitor of transcription factor SREBPs, which improves dyslipidemia and insulin resistance. In this study, we demonstrated that anhydroicaritin could also decrease the level of SREBP2 and its target genes in osteoclasts induced by RANKL without significant cytotoxicity. Moreover, anhydroicaritin suppressed RANKL-induced osteoclasts differentiation. In STZ-induced diabetic mice model, we found that the osteoclasts were largely increased accompanied with deterioration of bone structure. Anhydroicaritin decreased the level of blood glucose and alleviated insulin resistance. More importantly, anhydroicaritin inhibited osteoclast differentiation and rescued diabetes-induced bone loss in vivo. In conclusion, anhydroicaritin, a potent SREBP2 inhibitor, inhibits the osteoclasts formation and improves diabetes-induced bone loss.

Anhydroicaritin improves diet-induced obesity and hyperlipidemia and alleviates insulin resistance by suppressing SREBPs activation.

SREBPs play important roles in the regulation of lipid metabolism, and are closely related to the occurrence and development of many metabolic diseases. Small molecular inhibitors of SERBPs are important tools in developing efficient treatment of metabolic diseases. However, there are no listing drug targeting SREBPs. Therefore, there is an urgent need to develop highly specific small molecules that inhibit SREBPs. In this study, using a hepatocyte-based high-throughput screening, we identified anhydroicaritin (AHI) as a novel inhibitor of SREBPs. HepG2, HL-7702, and human primary hepatocytes were used to verify the effects of AHI. We explored the mechanism by which AHI blocks the binding of SCAP/SREBPs complex with Sec23α/24D via regulating LKB1/AMPK/mTOR pathway. AHI reduced liver cell lipid level by preventing de novo lipogenesis. In diet induced obese mice, AHI ameliorated obesity, insulin resistance, fatty accumulation in liver and hyperlipemia. In conclusion, AHI improves diet-induced obesity and alleviates insulin resistance by suppressing SREBPs maturation which is dependent on LKB1/AMPK/mTOR pathway. Thus, AHI can serve as a leading compound for pharmacological control of metabolic diseases.