Research On The Ristricted Intramolecular Rotation Of SAG Based On Nuclear Magnetic Resonance

The Smoothened receptor(Smo)is a key signal transducer in the hedgehog pathway.Smo can have its activity modulated directly by small molecules and plays an important role in embryonic development and cell carcinogenesis.Smo agonists have been proposed as desired therapeutic value of restoring tissue function in diseases associated with heart failure,traumatic and chronic degenerative conditions,wound repair,and retinal damage.Meanwhile,Smo antagonists have the therapeutic perspective of as anticancer drugs.Therefore,Smo receptor have a promising application in the research of regenerative medicine and anti-tumor drugs.Among several kinds of small molecules which were screened to bind with the Smo receptor,SAG agonists showed high affinity to Smo receptor and high specifity for Hedgehog signal pathway,thus SAG was widely used in the bio-chemical investigation of Smo receptor molecule functions,target significance of disease and physiologic function research of Hedgehog signal pathway.In the present work,the coexistence of two distinct conformations of SAG are revealed by solution NMR,the 3D structures of the two conformations are elucidated by ROESY spectroscopy and MMFF94 program.In deuterated water and methanol,there are two kinds of conformations with different proportions,the conformation with higher existing ratio is named M,and the one with lower existing ratio(non-advantage conformation)is named m;whereas in deuterated chloroform,the two conformations are present in a 1: 1 ratio.The two kinds of stable conformations were confirmed by NMR spectrum and molecular structural simulation,and the difference of the two conformations was proved to be caused by rotating of molecular around amide bond.The conformation m is more similar to the ligand conformation in the SAG-smo crystal complex(4QIN).Using the means of molecular docking,it is also confirmed that the conformation m is graded higher than that of the smo receptor binding marker with the conformation M.The thermodynamic parameters of transformation between the two conformations were calculated by dynamic NMR method,the activation energies of M to m in chloroform,methanol and heavy water were the lowest,the middle and the highest,respectively.The results indicated that the polarity and the hydrogen bond given ability of solvent can influence the transformation of two conformations.The experimental results are instructive for the rational design of better SAG analogues.By using conformational search method,we can design a drug structure that is more similar to m conformation,so as to improve the pharmacodynamics of this kind of drug.At the same time,it has instructive significance for the pharmacology and pharmacological effect of SAG in different solvents.