Structure-Activity Relationship Studies Of Dammarane-Type Natural Products As Potential Activators Of MM-Type Creatine Kinase

Objective:In previous research,we have found that the muscle-type creatine kinase?CK-MM?is the target of Ginsenoside in skeletal muscle tissue through the methods of affinity fishing",gel electrophoresis and protein mass spectrometry identification.The direct interaction between 20?S?-PPD and CK-MM was confirmed by biofilm interferometry?BLI?and isothermal titration calorimetry?ITC?,represented by 20?S?-proginseng glycol[20?S?-PPD].Moreover,the vivo and vitro experiments indicated that 20?S?-PPD could activate CK-MM activity by allosteric regulation,increase the content of creatine phosphate?PCr?in skeletal muscle tissue and enhance the function of CK/PCr system in skeletal muscle,which thus effectively delaying the accumulation of lactic acid induced by exercise training and exerting anti-fatigue effect.However,the maximum increase of CK-MM activity in vitro was about 10%or both mouse-derived and human-derived CK-MM which showed an unsatisfactory result of the activation of CK-MM.It is well known that 20?S?-PPD belongs to the natural products of Dammarane.This dissertation arms to explore the structure-activity relationship of the activation of CK-MM by natural products of Dammaranes,in order to provide preliminary support for finding better CK-MM activators from natural products or by structural modification.Method:Experiment 1:Preparation of Jujubogenin and 24-carboxyl-protopanaxadiol?24-COOH-PPD?High purity total saponins of juj ube kernel was prepared by macroporous adsorption resin D101 combined with anion-cation exchange resin D201 and D113.Then the crude jujubogenin was prepared by oxidation-cutting method according to the reported literature.Finally,high purity jujubogenin was prepared by silica gel column chromatography and methanol recrystallization,and purity was analyzed by TLC and HPLC.Rough 24-COOH-PPD was obtained by silica gel column chromatography and the purity of 24-COOH-PPD was analyzed by TLC and HPLC.Experimentation 2:The effect of Dammarane natural products on the activity of CK-MM in vitroTwelve different concentrations of Dammarane compounds,including ginsenoside Rgl,Rd and Rh,and Dammarane aglycones 20?S?-PPD,20?R?-PPD,20?S?-protopanaxatriol[20?S?-PPT],Panaxa-diol?PD?,Panaxatriol?PT?,Ocotillol,25-OH-protopanaxadiol?25-OH-PPD?,j uj ubogenin and 24-OH-PPD,were incubated with CK-MM,and those effects on CK-MM activity were detected by creatine kinase?CK?assay kit.Experimentation 3:Direct interaction between natural products of Dammaranes and CK-MMThe direct interaction strength between the above 12 Dammaranes and CK-MM was determined by the technique of BLI.Experiment 4:Molecular docking of protopanaxadiol with creatine kinaseThe docking of CK-MM and 20?S?-PPD was simulated by Schrodinger software.The docking morphology of 20?S?-PPD at each potential binding site was evaluated by the GlideScore tool,and the binding sites and modes of 20?S?-PPD and CK-MM were analyzed.Experiment 5:Preparation of Mutant and Human CK-MMThe plasmids pET11,pET12 and pET13 were constructed by site-directed mutagenesis using pET10 as template.The plasmids were transformed into Escherichia coli BL-21 cells to express the recombinant His tag CK-MM.Finally,the protein was purified by HisTrap HP column and the purity of protein was evaluated by SDS-PAGE on 10%olyacrylamide gel.Experiment6:Affinity determination of 20?S?-PPD with wild type and mutant human CK-MMForteBio Octet RED96 system was used to detect the binding of 20?S?-PPD with wild-type?WT?or three mutant human CK-MM.The binding constant?Kon?and dissociation constant?Kdis?were obtained by using ForteBio Data Analysis software.The affinity value?KD?was calculated according to the formula KD=Kdis/Kon.ResultExperiment 1:10 g purity total saponins of jujube kernel were obtained by macroporous resin adsorption method,and then 2 g crude Jujubogenin was prepared by oxidative cutting.100 mg Jujubogenin was obtained by repeated chromatography on silica gel column and the purity was 90.7%.150 mg crude 24-COOH-PPD was prepared by potassium permanganate oxidation method,and 30 mg refined 24-COOH-PPD were obtained by silica gel column chromatography and the purity of Panaxadiol was 90.2%.Experiment 2:The binding of 20?S?-PPD,20?R?-PPD,20?S?-PPT,24-COOH-PPD and PD with rabbit CK-MM can improve its enzyme activity;the binding of PT,Ocotillol with rabbit CK-MM has no direct effect;the binding of 25-OH-PPD and Jujubogenin with enzyme inhibits its activity.Experiment 3:?1?The effect of Dammarane aglycones on the activity of CK-MM.The activity of rabbit CK-MM was increased after incubated with 20?S?-PPD,20?R?-PPD,20?S?-PPT,24-COOH-PPD,25-OH-PPD and PD.The maximum activation degree was 20?S?-PPD>24-COOH-PPD>20?S?-PPT>20?R?-PPD>25-OH-PPD>PD.There was no significant effect on the activity of rabbit CK-MM after incubated PT and OcotillCK-MM,while the activity of rabbit CK-MM couid be inhibited after incubated with the Jubogenin and Ocotillgen.?2?The effects of ginsenosides and their aglycones on the activity of CK-MM.The activity of rat CK-MM was increased after incubated with 20?S?-PPT and Rh2.The maximum activation degree was 20?S?-PPD>20?S?-PPT>Rh2.There was no significant effect on the activity of CK-MM after incubated with Rd and Rg1.Experiment 4:FTMap and SiteMap software analysis showed that there were two active binding sites in human CK-MM crystal structure after analysised with FTMap and SiteMap softwares.There was also a proximity site(S2"could be found besides the ADP binding sites?S1?that all kinases possess.The conformation score of 20?S?-PPD on the sites connected to S2" ws higher than that of ADP binding pocket?S1"?.20?S?-PPD can interact with amino acid residues?Q318,R20 and S285?at the S2"site by forming three hydrogen bonds through two negative groups?3-OH and 12-OH?.Experiment 5:Three mutant human CK-MM,including mutant 2?R320T,320 arginine mutation to threonine?and mutant 3?S285T,285 serine mutation to threonine?mutants?Q318P,318 glutamine mutation to proline?.were prepared by site-directed mutagenesis and protein purification techniques based on the key amino acids?Q318,R320 and S285?predicted by molecular docking.Experiment 6:The KD values of wild type,mutant 1,mutant 2 and mutant 3 with 20?S?-PPD were y 2.37±0.11E-5M,3.09±0.11E-2M,5.14±0.13E-4M and 9₅6±0.10E-5M,respectivel.The affinity of wild type binding to 20?S?-ppd was 1300,20 and 4 times of that of mutant 1,mutant 2 and mutant 3 binding to 20?S?-ppd,respectively.Mutations in key amino acids?R320 and S285?at the S2"locus reduced the affinity between 20?S?-PPD and CK-MM,and the mutation at Q3 18 locus almost resulted in the loss of binding ability between 20?S?-PPD and CK-MM.Conclusion1.The position of C-3,C-6 and C-20 in the mother nucleus of Dammane natural products will weaken their ability to activate CK-MM and their interaction with CK-MM.2.Compared with the natural S-conformation,the R-conformation reduces its direct interaction with CK-MM.3.The formation of cyclic structures on the side chains of dammalane aglycones and the substituent groups at the C-6 position in the mother nucleus significantly affect their binding to CK-MM and their ability to activate CK-MM.4.The 3-OH and 12-OH on the mother nucleus of Dammarane natural products are important chemical groups for their direct interaction with CK-MM,and the importance of 3-OH is higher than that of 12-OH.