Characterization, SAR Analysis, And Structure Optimization Of Celastrol And Derivatives For Their Binding To Orphan Nuclear Receptor Nur77

Nur77,an orphan member of the nuclear receptor superfamily,plays a vital role in diverse biological processes,including survival,proliferation,apoptosis,inflammation and immunity.Identification of small molecule modulators of Nur77 is therefore of significant in developing new therapeutics for inflammation-related diseases and cancer.Previous studies from our laboratory identified celastrol as a direct binder of Nur77,inducing its translocation from the nucleus to damaged mitochondria,a process that results in elimination of damaged mitochondria through autophagy to alleviate deleterious inflammatory responses.This discovery provides a new strategy to develop Nur77-based therapeutics for inflammatoryrelated diseases and cancer.However,celastrol has several limitations including poor target selectivity,toxicity,and poor water-solubility.Understanding how celastrol binds Nur77 is therefore important for developing improved celastrol analogs for therapy.The goal of this study was to determine the binding mode of celastrol to Nur77 and to study S AR between celastrol and Nur77 with the aim of identifying improved celastrol analogs for drug development.First,we demonstrated that celastrol can covalently bind to Cys551 in Nur77 in a reversible manner.Our results also revealed that non-covalent interactions mediated by Asp499 and Gln547 in Nur77 are required to bring celastrol close to Cys551,making the C-6 position of celastrol form covalent bond with Cys551.Thus,celastrol binds Nur77 through the specific non-covalent interactions followed by covalent interaction.These results not only establish a unique binding mechanism of celastrol to Nur77 but also identify a new binding site on the surface of Nur77 for small molecule interaction,providing a new strategy for synthesizing new Nur77 ligands.Next,we synthesized 24 derivatives of celastrol based on the above binding mechanism of celastrol to Nur77.Our structure-activity relationship(SAR)study between celastrol and Nur77 suggested a new direction to improve the Nur77 selectivity of celastrol analogs through modification of ring A while keeping the ring B in native state.By combining our SAR study and the binding mode between celastrol and Nur77,we designed and synthesized a new celastrol derivative XS0557.Using various methods such as nuclear magnetic resonance,mass spectrometry,quantum calculation and X-Ray single crystal diffraction,we confirmed that XS0557 has a novel structure with the Kd of 310 nM for Nur77,which is comparable to celastrol.Importantly,the ability of celastrol to covalently bind to proteins was greatly impaired in XS0557,avoiding the multi-covalent targets like celastrol.Thus,celastrol has improved selectivity toward Nur77 as compared to celastrol.The biological evaluation of XS0557 indicated that the compound potent anticancer activity with much reduced toxicity in vitro and in animals.Thus,our studies identify XS0557 as promising Nur77-selective compound that warrants further investigation.To further develop XS0557,it is necessary to investigate the structure-activity relationship between XS0557 and Nur77,so we preliminarily designed and synthesized 15 derivatives of XS0557,to contribute the further optimization of XS0557.