Study Of A New Nur77-selective Celastrol Analog For Its Anti-Obesity Activity And The Underlying Mechanism Of Action

Celastrol represents one of the very few natural products with proven clinical efficacy and potential drug development most.Emerging evidence indicates that celastrol exerts broad therapeutic effects on anti-inflammatory,anti-tumor and antiobesity activities.However,its clinical application is limited largely because of its severe toxicity likely attributed to its multiple intracellular targets.Our recent discovery that orphan nuclear receptor Nur77 serves as an important intracellular target of celastrol allows a rational structure-activity relationship analysis of celastrol and its modification with the hope to identify new celastrol derivatives with improved therapeutic profiles for clinical application.The C-6 site of celastrol is known to form covalent bond with cysteine through Michael addition reaction.In studying how celastrol binds to Nur77,we found that celastrol binds to Nur77 via both non-covalent bond and reversible covalent bond.These exciting discoveries provide an opportunity to identify Nur77-selective celastrol analogs with improved efficacy and reduced toxicity.Thus,we propose that Nur77-selective celastrol analogs could be rationally designed to reduce its toxicity and side effects.To address this hypothesis,we modified celastrol to eliminate its covalent binding ability.The modified celastrol analog,XS0503,was then evaluated for its binding to Nur77 and other known targets,its efficacy and toxicity,and its molecular mechanism of action.Through blocking C-6 and covalent binding,the ability of XS0503 binding to Nur77 decreases a little,but almost lost in binding other targets,which reveals an effective method to improve the selectivity of Nur77 binding.The Nur77-selective celastrol analog that we designed and synthesized,XS0503,was evaluated in cells,mouse model,and zebrafish model for its toxicity,and our results showed that the toxicity of XS0503 was significantly reduced as compared to celastrol.By contrast,XS0503 showed a similar anti-obesity effect in a high-fat induced obesity mouse model.Moreover,by using the db/db mouse model with leptin receptor deletion,the ob/ob mouse model with leptin deletion,the Lean mouse model with leptin low expression and the combined model of leptin and XS0503,we found that the anti-obesity effect of XS0503 was due to its ability to sensitize the effect of leptin.Obesity is a systemic chronic inflammation that can lead to hypothalamic neuroinflammation and thus leptin resistance.In studying the molecular mechanism of XS0503 action,we found that XS0503 could enhance leptin sensitivity by inducing autophagy of dysfunctional mitochondria(mitophagy)to alleviate neuronal inflammation,thus inhibiting leptin resistance.Further in vitro experiments demonstrated that the mitophagic effect of XS0503 was not mediated by the classical PINK1-Parkin pathway,but by our newly discovered Nur77-p62 mitophagic pathway.These results further revealed the significance to the new Nur77-p62 mitophagic pathway in modulating inflammation and obesity.In conclusion,through reversible covalent binding theory,our ability to design a new Nur77-selective celastrol analog XS0503 with potent anti-obesity activity but lack toxicity and reveal the mechanism of hypothalamus inflammation clear and thus leptin sensitivity improvement by Nur77-p62 mitophagic pathway,provides not only a promising drug candicate but also a valuable tool to dissect the Nur77-dependent mitophagic pathway,whose theoretical significance and clinical application value are of much concern.