Design,Synthesis And Target Identification Of Thiazolo-pyrones Via Photo-affinity Labeling (PAL) Protocol

Inflammation is the basis of many physiological and pathological processes.Although inflammation is a normal body reaction,uncontrolled and misleading inflammation can lead to inflammatory diseases and even cancer.For the treatment of inflammation,non-steroidal anti-inflammatory drugs(NSAIDs)like aspirin,ibuprofen,naproxen,and celecoxib are the most widely used drugs to relieve pain,but these drugs often have adverse side effects or even have fatal side effects.Therefore,the industrial and academic research of anti-inflammatory drugs has become the focus.According to the basic framework of COX-2 inhibitors,a new kind of chiral thiazolopyranones was designed and synthesized.Previous studies found that these compounds not only have significant anti-inflammatory activities,but also have some therapeutic effects in the animal model of acute liver injury and liver fibrosis.In order to further explore the specific mechanism of thiazolopyranone,we designed a kind of photoaffinity probe derived from thiazolopyranone.Firstly,the photo sensitive azide heterocyclic group can help the compound to covalently couple with its binding protein.Secondly,biotin modification of terminal alkynes was performed by the classic Click reaction to facilitate the separation of target proteins by streptavidin magnetic beads.Finally,the possible target proteins were determined by protein mass spectrometry and omics analysis.Using chemical biological methods to directly prove the target of thiazolopyrone compounds and related mechanisms,verify anti-inflammatory and anti-fibrotic activities,and deliberately elaborate the specific relationship between inflammation and fibrosis.Based on the method developed by the research group,the photoaffinity probe 3c was successfully constructed via 2c and alkenyl thiazolone as the original materials.It was shown that the 3c compound has the same chiral structure as the 3a compound from NMR and HPLC.After obtaining the photocrosslinking probe 3c,the equivalence of activity was evaluated by the NO inhibition test of RAW 264.7 and the PGE2 level release inhibition test,indicating that the probe molecule and the parent compound had equivalent biological activity,and the probe compound was used for the target protein Omics analysis.For the exploration of target molecules,first use Click reaction to carry out specificfluorescent labeling,firstly qualitatively analyze the target molecules by SDS-PAGE,and secondly confirm the analysis by LC-MS.In qualitative analysis,specific bands were clearly found in RAW 264.7,HEK 293 a,L02,GMC cells,and the target bands in different cells were different.In the confirmation analysis,first,Ptgs2(COX-2),Alox5ap(5-LOX),Pla2g4a(cPLA2),Lta4h(LTA4H),Ptges3(PTGES3),Cyp2d12(CYP450)and other target molecules are involved in arachidonic acid metabolism and the release of prostaglandins and interleukins.Secondly,ANXA2,ENO1,RAC1,MAPK3 and other target molecules participate in the p38 MAPK signaling pathway,On the one hand,it mediates the inflammatory response and promotes the release of inflammation-related rate-limiting enzymes and inflammatory mediators;on the other hand,the increased expression of nuclear α-SMA(ACTA2)and Collagen(COL1A1)via the p38 MAPK pathway is associated with the ITGA / B-RHOA pathway activated various actins form focal adhesions and cause fibrosis.At the same time,CUL3,CUL5,SMURF1,RPS27 A,NEDD4 and other related target molecules are also involved in the ubiquitination modification of proteins in various signaling processes in signaling cells.In summary,in this paper,a photo-affinity group modified thiazolopyrone probe compound was used to conduct a systematic proteomics analysis of its target molecules by chemical biological methods.Through the network correlation analysis of proteins with different functions,the anti-inflammatory and anti-fibrotic functions of thiazolopyrone are elucidated.These studies help us to understand the anti-inflammatory and anti-fibrotic mechanism of thiazolopyrone compounds,and provide an experimental basis for further drug modification.This study also helps to reveal the mechanism of inflammation and fibrosis.The design and development of new drugs in this field have certain guiding significance.