Molecular Basis Of Treating Perimenopausal Syndrome With Xiaoyaosan And Structural Studies And Drug Screening Of VBcl-2 Of Epstein-barr Virus

Xiaoyaosan(XYS)is a classical herbal formula for liver depression and spleen deficiency syndrome in traditional Chinese medicine(TCM).Among the associated mental disorders,XYS is also used for disorders such as perimenopausal syndrome(PMS).The decline in estrogen levels in the body of middle-aged women is the root cause of PMS.Based on clinical practice and experiments,it seems that XYS can alleviate the symptoms of PMS,with a possible mechanism of providing estrogenic compounds to counter the fluctuating estrogen levels.Here an NMR technique,waterLOGSY,was used to identify elements in the decoction of XYS that could be bind to estrogen receptors(ER)to attenuate ER’s activity and compensate for the fluctuating and declining level of estrogen.Results showed that the active ingredients binding to ER were solely from Paeoniae radix alba,one of the herbs used to make XYS.Further NMR spectroscopic analysis with model compounds identified paeoniflorin and gallic acid as the active compounds.Luciferase reporter assay indicated that paeoniflorin was agonistic while gallic acid was antagonistic to estrogen receptor α.The binding energy between ERa and paeoniflorin was-8.7 kcal/mol and that for gallic acid was-5.8 kcal/mol.This is the first demonstration of ER as the molecular target for gallic acid and paeoniflorin.These findings clearly established that XYS contained active ingredients to modulate the activity of estrogen receptors and showed that the prescription of XYS can not only smooth the mental state of women suffering from PMS but can also directly compensate for the lost function of estrogen receptors.The identification of these novel ingredients that could modulate the activity of estrogen receptors not only opens the door for better prescription of TCM for PMS,but also provides new lead compounds for developing therapeutics to treat ER-associated diseases such as breast and ovarian cancers.Epstein-Barr virus(EBV),a ubiquitous human gamma herpesvirus that can cause life-long and largely asymptomatic latent infections in humans,is associated with various malignancies in B lymphocytes and epithelial cells.During lytic infection,EBV expresses BHRF1 and BALF1,the viral analogue to human Bcl-2.BHRF1 has been demonstrated to be an enhancer to the survival of EBV-infected cells,mainly involved in regulating apoptosis of host cells and inducing malignant transformation of infected B lymphocyte,which contributes to EBV establishment of life-long persistent latent infection in the host.There are currently no specific inhibitors targeting BHRF1.Unlike BHRF1,the transmembrane location and structure of BALF1 are poorly understood.In addition,the role of BALF1 in apoptosis has been controversial.There is also currently no specific antibody for BALF1,due to the lack of viable strategies to achieve stable and efficient heterologous expression of BALF1 as a high-quality and soluble protein.Consequently,it is difficult to accurately identify and evaluate the expression of BALF1 at different stages of its infection,as well as its function.A chemical compound,BH231,binding to BHRF 1 was obtained from a compound library(containing 2268 compounds)by using thermal shift assay(TSA)and NMR HSQC.Isothermal titration calorimetry(ITC)showed that BH231 bind to BHRF1 with a Kd value of 861 μM.The binding site of BH231 in BHRF1 was identified by modeling and mutations in NMR HSQC experiments.The BH231 binding site is not at the canonical BH3-binding hydrophilic groove of anti-apoptotic Bcl-2 proteins but at the pocket that comprises four α-helices(α1(Ile14～His20),α2(Val28-Leu29),α6(Leul15～Cys116),α7(Tyr123～Val126))connected respectively by two loops(αlα2 loop(Gly21～Pro27)and α6-α7 loop(Cys117～Pro122)).The NMR HSQC data indicated that BH231 binding could significantly induce change the overall conformation of BHRF1.Taken together,compound BH231 may be a promising lead for the development of novel inhibitors targeting BHRF1 for treating EBV-related diseases,such as EBV-associated cancers.For BALF1,we constructed over 100 recombinant plasmids for various length of BALF1 for expression in E.coli.Through thousands of expression trials,we were able to stably express the soluble recombinant protein,as was supported by LC/MS.The putative transmembrane domain(TM)of BALF1 that have to be truncated for expression seemed not at the carboxyl-terminus,suggesting that BALF1 might be distinct from other members of the Bcl-2 family.Dynamic light scattering(DLS)and negative-staining electron microscopy showed that the fusion MBPmut-BALF1 protein is a homogeneous yet large oligomer.Attempts to crystallize MBPmut-BALF1 were not fruitful.The current effort results in experssing valuable proteins for the production of BALF1 specific antibodies,and lays the foundation for elucidating the BALF1 structure by cryo-electron microscopy(cryo-EM).