Molecular Dynamics Simulation On Structure And Function Of Glycogen Synthesis Kinase 3 And Its Inhibitors Design

Glycogen synthase kinase 3(GSK3),a multifunctional serine/threonine kinase, is encoded by two highly homologous isoforms,αandβin mammalian.Now GSK3βis recognized as a well-established component of glycogen metabolism,Wnt and NF-κB pathways,and therefore has been implicated in numerous physiological processes by phosphorylation of different targets.The pathways in which GSK3βact as a key switch,when deregulated,have been linked with many human diseases,such as Alzheimer's disease(AD),cancer and diabetes,etc.GSK3βis also regarded as a therapeutic target and its pharmacological inhibitors draw more and more attentions. The thesis is about the theoretical calculation on the structural and functional effects of mutation on GSK3β-substrate complexes,as well as the binding mode of with its inhibitors.Axin is the scaffold protein of GSK3β-βcatenin complex in Wnt signaling pathway,while some single-point mutation destroys the stability of GSK3β-axin complex.In order to investigate the effects of V267G and L392P mutation on GSK3β-axin structure,as well as the unbinding mechanism for two mutant complexes, 2 ns molecular dynamics(MD)simulation studies were performed on the WT GSK3β-axin complex and two mutants(V267G and L392P).Besides,rough energy and residue-based energy decomposition were calculated by MM_GBSA(molecular mechanical Generalized_Born surface area)approach to illuminate the instability of the two mutants.The results reveal that hydrophobic interaction is the main factor to maintain stable WT complex.For both two mutants,the structure of GSK3βremains unchanged,while axin moves away from the interfacial hydrophobic pockets.Axin exhibits positional shift in V267G mutant,whereas,losing the hydrogen bonds that are indispensable for stabilizing the helix structure of wild type axin,the helix of axin is distorted in L392P mutant.To conclude,both two mutants destroy the hydrophobic interactions that are essential for the stability of GSK3β-axin complex.In addition, rough energy analysis explain complementarily the instability of two mutants, meanwhile the energy of key residues are unfavorable for binding,especially locating at the N-terminal of axin,which is agreement with axin behaviors as indicated from MD results.Many Ser/Thr protein kinases,to be fully activated,are obligated to introduce a phosphate Ser/Thr in their activation loop.Molecular dynamics simulations and energy analysis were employed to investigate the effect of a phosphorylated serine residue(pSer)on the structure of GSK3β-ATP-pSer complex,and the mechanisms of GSK3βselective deactivation on primed substrate due to R96K and R96A mutations. The results indicate that the introduction of pSer generates a slight lobe closure due to the electronic interaction between RD pocket and phophote ion,which may illuminate the experimental conclusion,whereas the conformations of GSK3βand ATP undergo significant changes in two mutants.As for GSK3β,the affected positions distribute over opened activation loop(A-loop),shifted or twisted C-loop and shifted glycine-rich loop(G-loop).Based on coupling among the mentioned positions,the allosteric mechanisms of hydrophobic interaction network for distorted ATP were proposed. Thus the phosphoryl transfer in-line manner is demolished.Additionally,energy calculation provides the qualitative complementary for instability of two mutants in contrast to WT.Energy decomposition reveals that Lys96 and Ala96 in two mutants show unfavorable energy for binding,while Arg180 and Lys205 possess similar contribution,which are all in agreement with MD results.And also the key residues of Arg96 and Arg180 were identified,which should be considered when designing non-ATP-competitive inhibitors with little possibility of cancer occurrence.Due to the highly conservation of the ATP-binding pocket between GSK3βand CDKs,many inhibitors of GSK3βalso affect CDKs.Hymenialdisine(HD)and Di- bromocatharelline(DT)are the integrendient of marines,the former shows similar inhibitory activity to GSK3β,CDK2 and CDKS,while the latter only inhibits GSK3βwith 3000nM.Based on the crystal structure of CDK2-HD complex,Autodock, FlexX and Gold three docking methods,as well as molecular dynamics simulation were performed to reveal the mechanism of DT selectivity and seek optimization clues.The results give similar binding modes of HD when docked into CDK5 and GSK3β,which is consistent with poor selectivity to both families.As for the docked GSK3β-DT complex,when superimposed with CDKS,smaller size pocket of CDK5 and dense eletronic interaction networks indicate steric conflict between the side chain of Asp144 and guanidine offing D,and therefore hardly inhibiting CDKs.MD similations reveal the unique binding modes of DT with GSK3β,such as weak hydrogen bond between the carbonl oxygen atom of ring A and side chain of Arg141, stable hydrogen bond involved N3 atom of guanidine and the carbonyl oxygen atom of Gln185,as well as salt bridge between N4 atom and side chains of Ash186.Additionally, the bromine and three rings form hydrophobic interaction with Leu132, Leu188,Val110 and Cys199.Utilizing the larger size residue Cys199 near to D ring GSK3β,compared to Ala of CDKs,a large substitution at D ring may be helpful for inhibitory activity.Indirubin analogues inhibit GSK3βand CDK2 with good and similar effects, and analogues with halogen substitution show good pharmacological property.In order to seek the rule in designing and optimizing indirubin analogues,two methods were used to build 3D-QSAR models for indirubin derivatives.Ligand-based(LB) studies were performed based on the lower energy conformations employing atom fit alignment rule.The receptor-based(RB)3D-QSAR models were also derived using bioactive conformations obtained by docking compounds to the active site of GSK3β. Some substitute shows different orientation between two methods,but these sub- stituents extend almost to the outward of binding pocket and also have little effects on activity,therefore conclusions of models based on two methods are similar and reliable. Contour maps of the receptor-based CoMSIA model including the steric,electronic and hydrophobic fields were employed to explain factors affecting activities of inhibitors.It is helpful that hydrophilic and small substituent is added to position 3' and 6' of A ring,which are surrounded by a polar residue chain.Negative substituent may be favored for activity when introduced in to A and B ring,which formπ-πinteraction with aromatic residue Tyr134.And big size and hydrophobic groups at position 5 and 6 of ring D is preferred to interact with hydrophobic residues such as Val70, Val110,Leu132 and Cys199.This study elucidates the structural and functional characters of mutant GSK3βcomplexed with substrates,which provides significant hints for other substrates.By comparing the binding pocket of GSK3βand CDKs,the rule for optimizing DT and indirubins inhibitors is proposed,which provide instructive information for the structure-based drug design.