A Novel Selective Estrogen Receptor Modulator Isolated From Anoectochilus Endophytic Fungi

Over the past 30 years,more than 60%of small molecule drugs approved for marketing have been directly or indirectly derived from natural products.Anoectochilus has been known as "Lunaophaga" and "Shenzheng" in folk,and it has anti-tumor,anti-aging and other efficacies,but no report has been found on the activity related to estrogen receptor(ER)and osteoporosis.The extract of the endophytic fungi of Anoectochilus roxburghii is a natural "new-type compound library",which can not only be used for the secondary metabolism of natural products of Anoectochilus,but also provide effective separation and extraction of active compounds from the endophytic fungiAfter discovering that the crude extract of an endophytic fungus of Anoectochilus can specifically activate the reporter gene of estrogen receptor alpha(ERα),we further used ERa reporter gene assay to monitor the activity of the ingredients from gradual isolation and purification and finally identified Norlichexanthone(NOR)as one of the pure compounds that activated ERα.The structure of NOR was determined by mass spectrum and nuclear magnetic resonance approaches.We further confirmed that NOR selectively activated ERa transactivation using yeast-one hybrid assay and ERE reporter gene assay.All of our fluorescens titration assay,SPR assay and ITC assay showed that NOR directly bound to ERα.The KD value measured by fluorescens titration assay was at the nM level.It was suggested from mimic docking and derivative examination that the conjugated system of NOR structure and the three substituted hydroxyl groups on the benzene ring played key role for NOR-ERa binding.As to biological activities,we found that NOR did not significantly stimulate the proliferation of MCF-7 cells by MTT assay.Nor did it stimulate the pro-proliferation ERa targeted genes in MCF-7 cells.Interestingly,we found that NOR dramatically inhibited osteoclast differentiation and reduced osteoclast numbers.The number and activity of osteoclasts are highly related to the occurrence of osteoporosis.Thus,our result suggested that NOR may be of therapeutic effect on osteoporosis but without increasing the risk of breast cancer as estrogen.Therefore,our study unraveled a natural compound NOR isolated and purified from an endophytic fungus of Anoectochilus roxburghii acted as ERa cognate ligand.NOR was able to specifically target the ERa and was of a nM binding constant.NOR strongly reduced osteoclast number.Moreover,NOR had certain tissue selectivity in terms of its ERa activation.Thus,NOR and its derivatives have the potential to be the next generation of selective estrogen receptor modulators for clinical use.