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.