Notum,a negative feedback regulator of the Wnt signaling,has emerged as a promising target for treating glucocorticoid-induced osteoporosis(GIOP).This study showcases an efficient strategy for discovering the anti-Not...Notum,a negative feedback regulator of the Wnt signaling,has emerged as a promising target for treating glucocorticoid-induced osteoporosis(GIOP).This study showcases an efficient strategy for discovering the anti-Notum constituents from herbal medicines(HMs)as novel anti-GIOP agents.Firstly,a rapid-responding near-infrared fluorogenic substrate for Notum was rationally engineered for high-throughput identifying the anti-Notum HMs.The results showed that Bu-Gu-Zhi(BGZ),a known anti-osteoporosis herb,potently inhibited Notum in a competitive-inhibition manner.To uncover the key anti-Notum constituents in BGZ,an efficient strategy was adapted via integrating biochemical,phytochemical,computational,and pharmacological assays.Among all identified BGZ constituents,three furanocoumarins were validated as strong Notum inhibitors,while 5-methoxypsoralen(5-MP)showed the most potent anti-Notum activity and favorable safety profiles.Mechanistically,5-MP acted as a competitive inhibitor of Notum via creating strong hydrophobic interactions with Trp128 and Phe268 in the catalytic cavity of Notum.Cellular assays showed that 5-MP remarkably promoted osteoblast differentiation and activated Wnt signaling in dexamethasone(DXMS)-challenged MC3T3-E1 osteoblasts.In dexamethasone-induced osteoporotic mice,5-MP strongly elevated bone mineral density(BMD)and improved cancellous and cortical bone thickness.Collectively,this study constructs a high-efficient platform for discovering key anti-Notum constituents from HMs,while 5-MP emerges as a promising antiGIOP agent.展开更多
A titrant for the SARS-CoV-2 main protease(M^(pro))was developed that enables,for the first time,the exact determination of the concentration of the enzymatically active M^(pro) by active-site titration.The covalent b...A titrant for the SARS-CoV-2 main protease(M^(pro))was developed that enables,for the first time,the exact determination of the concentration of the enzymatically active M^(pro) by active-site titration.The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme–titrant complex.Four fluorogenic substrates of M^(pro),including a prototypical,internally quenched Dabcyl-EDANS peptide,were compared in terms of solubility under typical assay conditions.By exploiting the new titrant,key kinetic parameters for the M^(pro)-catalyzed cleavage of these substrates were determined.展开更多
基金supported by the National Natural Science Foundation of China(82273897,U23A20516,82104281,and 32101202)Organizational Key Research and Development Program of Shanghai University of Traditional Chinese Medicine(2023YZZ02,China)+5 种基金Shanghai Municipal Health Commission’s TCM research project(2022CX005,China)Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(ZYYCXTDD-202004,China)Three-year Action Plan for Shanghai TCM Development and Inheritance Program(ZY(2021-2023)-0401,China)the Fundamental Research Funds for the Central Universities(G2024KY05106,China)the State Key Laboratory of Fine Chemicals,Dalian University of Technology(KF2202,China)supported by China Postdoctoral Science Foundation(2024M762108,China).
文摘Notum,a negative feedback regulator of the Wnt signaling,has emerged as a promising target for treating glucocorticoid-induced osteoporosis(GIOP).This study showcases an efficient strategy for discovering the anti-Notum constituents from herbal medicines(HMs)as novel anti-GIOP agents.Firstly,a rapid-responding near-infrared fluorogenic substrate for Notum was rationally engineered for high-throughput identifying the anti-Notum HMs.The results showed that Bu-Gu-Zhi(BGZ),a known anti-osteoporosis herb,potently inhibited Notum in a competitive-inhibition manner.To uncover the key anti-Notum constituents in BGZ,an efficient strategy was adapted via integrating biochemical,phytochemical,computational,and pharmacological assays.Among all identified BGZ constituents,three furanocoumarins were validated as strong Notum inhibitors,while 5-methoxypsoralen(5-MP)showed the most potent anti-Notum activity and favorable safety profiles.Mechanistically,5-MP acted as a competitive inhibitor of Notum via creating strong hydrophobic interactions with Trp128 and Phe268 in the catalytic cavity of Notum.Cellular assays showed that 5-MP remarkably promoted osteoblast differentiation and activated Wnt signaling in dexamethasone(DXMS)-challenged MC3T3-E1 osteoblasts.In dexamethasone-induced osteoporotic mice,5-MP strongly elevated bone mineral density(BMD)and improved cancellous and cortical bone thickness.Collectively,this study constructs a high-efficient platform for discovering key anti-Notum constituents from HMs,while 5-MP emerges as a promising antiGIOP agent.
基金The authors acknowledge support by Dr.Carina Lemke and Marion Schneider.Christa E.Müller and Michael Gütschow were supported by the Volkswagen Foundation(9A894)Rabea Voget,Christian Steinebach,Christa E.Müller and Michael Gütschow by the German Research Foundation(RTG 2873)Norbert Sträter by the Volkswagen Foundation(9A850).We acknowledge DESY(Hamburg,Germany),a member of the Helmholtz Association HGF,and the EMBL for the provision of experimental facilities at synchrotron beamlines P13 and P14 and the MX Laboratory at the Helmholtz Zentrum Berlin(BESSY II)for beam time.We would like to thank Selina Storm for assistance in using the EMBL beamlines.
文摘A titrant for the SARS-CoV-2 main protease(M^(pro))was developed that enables,for the first time,the exact determination of the concentration of the enzymatically active M^(pro) by active-site titration.The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme–titrant complex.Four fluorogenic substrates of M^(pro),including a prototypical,internally quenched Dabcyl-EDANS peptide,were compared in terms of solubility under typical assay conditions.By exploiting the new titrant,key kinetic parameters for the M^(pro)-catalyzed cleavage of these substrates were determined.
