Discovery Of Novel PPARγ-Selective Modulators On The Basis Of High-Throughput Virtual Screening And Ser273 Phosphorylation Mechanism

Research Background and Objectives:Diabetes mellitus（DM）,a metabolic disorder characterized by hyperglycemia,is the result of the deficient secretion of insulin in islets or insulin resistance on peripheral target organs.According to the statistical data obtained from the International Diabetes Federation（IDF）,the morbidity of diabetes is predicted to increase to>700 million people by 2045.This epidemic is predominantly caused by a rise in the prevalence of T2DM,a complex disorder that is mainly characterized by insulin resistance in the liver and peripheral tissues.In view of the continuing increase in the global incidence of diabetes,the prevention and treatment of this disease have become global public health issues.Hence,overcoming insulin resistance is a key therapeutic index for the treatment of T2DM.Numerous studies revealed that peroxisome proliferator activated receptorγ（PPARγ）is a clinically validated target for the treatment of insulin resistance and and plays an important role in the regulation of tissue insulin sensitivity and the uptake and storage of glucose and lipids,and up to date,several PPARγfull agonists Thiazolidinedione class of drugs,including Rosiglitazone and Pioglitazone,have been developed as the major frontline insulin-sensitizing drugs for clinical treatment of T2DM through an activation function 2（AF-2）involved“lock”mechanism.Despite their therapeutic efficiency,such action of mechanism also led to the risk of adverse effects such as increased body weight with long-term use of these compounds.Based on the fact,the use of these drugs has been restricted in the clinical.In 2010,Spiegelman initially described a new antidiabetic mechanism of PPARγ-targeted drugs.This novel mechanism depends upon the inhibition of PPARγSer 273 phosphorylation by cyclin dependent kinase 5（CDK5）,which represses transcription of brown-fat-like genes and induces expression of diabetogenic genes involved in insulin resistance.On basis of the mechanism,a number of PPARγpartial agonists such as MRL 24 and SR1664 has been developed thus far.However,there is no such agents have advanced to the clinic use to date.In recent years,virtual screening techniques such as ligand-based virtual screening（LBVS）and structure-based virtual screening（SBVS）have emerged as powerful drug discovery tools for identifying the potent ligands with the corresponding biological targets from a large database of chemical structures.Obviously,they can improve the probability of drugs discovery and reduce the time and cost of drug development.In order to rapidly discover lead compound/approved drug for clinical use,we initiated a program of Structure-based Virtual High-throughput Screening from the Diversity Skeleton Compound Library,Approved Drug Library and Medicine Food Homology Compound Library to seek for novel PPARγ-selective modulators which specifically target the area includes the helix 3,theβ-sheet and the H2-H2’link region（Arm 3,in relation to the CDK5-mediated PPARγSer 273 phosphorylation）.Methods:（1）A program of SB-VHTS from the Diversity Skeleton Compound Library,Approved Drug Library and Medicine Food Homology Compound Library to seek for novel PPARγ-selective modulators/lead compounds which specifically target the Arm3 pocket of the PPARγLBD has been employed.Then,a series of target hits base on the lead compounds were design and synthesized by the molecular docking.（2）TR-FRET assay and Surface Plasmon Resonance（SPR）were established to evaluate the binding affinities of compounds/drugs.Then,the ability of transactivation of PPARγinduced by the target compounds was evaluated by plasmid transfection and dual luciferase reporter gene assay in COS-7 cell line.（3）Murine 3T3-L1 preadipocytes were treated with the test compounds（10μM）and the differentiation was visualized using Oil Red O staining.The PPARγfull agonist Rosi（10μM）was employed as the positive control.Then the expression levels of a P2,C/EBPα,Fasn,LPL,CD36,Adiponectin and Glut4 were detected by using reverse transcription and quantitative real time PCR.（4）db/db mice was used as the animal model to evaluate the hypoglycemic effect and toxic side effects of drugs in vivo,in which a PPARγpartial agonist FN（an marketed anti-fungal drug）reported recently by our team was used as the positive control.The blood glucose,glucose tolerance and blood insulin solubility of mice were tested after treating with our selected administration concentrations of potential compounds.The side effects of target compounds/drugs were further assessed by analyzing the body weight and morphological changes of main organs and adipose tissues such as liver and heart.To confirm its hypoglycemic mechanism,and finally,the western blotting experiment was carried out to detect the Ser273 phosphorylation expression of PPARγin liver and adipose tissues.Besides,quantitative real-time PCR was used to evaluate the expression of the adipogenic genes such as a P2,C/EBPα,PPARγ,Fasn,LPL and CD36 as well as sensitivity-related genes,including Adiponectin and Glut4 in the liver and adipose tissues of mice.Results:（1）In this study,Chrysin,Vagistat,Oxiconazole,Sertaconazole,Raloxifene,Dicumarol,Licorice chalcore A,β-Elemonic Acid and Bilobetin were identified through the structure-based virtual screening and TR-FRET as novel PPARγligands with IC₅₀values of 52.79μM,25.46μM,43.65μM,7.57μM,3.72μM,2.55μM,6.44μM,2.52μM and 0.39μM,respectively.The biological data from transactivation assays revealed that all of compounds exhibited weak PPARγagonistic activities.The results from adipocyte differentiation experiments showed that these developed PPARγLigands revealed stronger efficacy than Vehicle but were still less active in this setting than full agonist Rosi.（2）Based on the structural features of Chrysin,we further designed and synthesized a series of flavonoid derivatives.2-（（5-Hydroxy-4-oxygen-2-phenyl-4H-chromoene-7-group）oxygen）-N-（4-（trifluoromethyl）benzyl）acetamide（YGT-29）showed better binding affinity with an IC₅₀of 1.72μM compared with Chrysin.The structure-activity relationship studies（SARs）suggested that the electron-absorbing benzyl amine substituted analogue showed better binding activities than other compounds.On the other hand,the biological data from transactivation assays and quantitative real-time PCR revealed that YGT-29 had a weak PPARγagonistic activity without inducing the expression of key adipogenic genes encoding a P2,C/EBPα,Fasn,LPL,PPARγand CD36.（3）The treatment of the db/db mice with flavonoid derivative YGT-29（10mg/kg/day and 20 mg/kg/day）and Dicoumarin（5 mg/kg/day,10 mg/kg/day and 20mg/kg/day）3 weeks by intraperitoneal administration,and they significantly reduced blood glucose and improved the glucose tolerance,compared to FN（20 mg/kg/day）.Moreover,there were no obvious changes in body weight,important organs and fat mass of db/db mice after treating with YGT-29 and Dicoumarin.The results of western blotting experiments and RT-q PCR assays showed that YGT-29 and Dicoumarin inhibited CDK5-mediated Ser 273 phosphorylation of PPARγand selectively up-regulated the expression of insulin sensitivity-related genes including Glut4 and Adiponectin.（4）The binding modes of flavone derivative YGT-29 and Dicoumarin with PPARγwere evaluated by a docking study,and the results demonstrated that the interactions of these molecules bound to PPARγare different from that of the partial agonists FN and MRL 24.For example,the flavone derivative YGT-29 formed hydrogen bonds with Ser289,Ser342 and Glu343 with bond lengths of 2.7（?）,2.8（?）,2.5（?）and 2.7（?）,and Dicoumarin formed hydrogen bonds with Cys285 and Ser342with bond lengths of 2.4（?）,2.2（?）and 2.7（?）.In contrast to the binding patterns of Rosi,the two compounds have no obvious H-bonding interactions with His323,His449 and Tyr473.Subsequently,SPR analysis suggested that YGT-29 and Dicoumarin bound tightly to PPARγand the Ser342 is the key amino acid for targeting the inhibition of CDK5-mediated PPARγSer273 phosphorylation.Conclusion:Through structure-based high-throughput virtual screening,design,synthesis and comprehensive evaluation in vitro and in vivo,we draw the following conclusions:（1）Chrysin,Vagistat,Oxiconazole,Sertaconazole,Raloxifene,Dicumarol,Licorice chalcore A,β-Elemonic Acid and Bilobetin were identified through the structure-based virtual screening as a novel class of PPARγpartial agonists with IC₅₀values of 52.79μM,25.46μM,43.65μM,7.57μM,3.72μM,2.55μM,6.44μM,2.52μM and 0.39μM,respectively,and these ligands weakly induced adipocyte differe-ntiation as well as the expression of adipogenic genes.（2）We designed a partial PPARγagonist YGT-29 by using a structure-based strategy.The flavonoid derivatives YGT-29 and Dicoumarin acted as PPARγ-selective modulators with partial agonistic activities and strong affinities.As expected,these developed ligands also weakly induced the adipocyte differentiation as well as the expression of adipogenic genes.（3）The flavonoid derivatives YGT-29 and Dicoumarin exert an anti-diabetic effect through the efficient inhibition of PPARγ-Ser273 phosphorylation mediated by CDK5 without leading to the related side effects induced by PPARγfull agonists TZD drugs,thus not only providing the promising candidates for antidiabetic drug discovery,but also giving a new insight on the future design of novel PPARγmodulators.（5）The binding modes of flavone derivative YGT-29 and Dicoumarin with PPARγare different from that of the full agonist Rosi and partial agonists FN and MRL 24,and the Ser342 is the key amino acid for targeting the inhibition of CDK5-mediated PPARγSer 273 phosphorylation.