| Heat shock protein 90(Hsp90),known as a highly conserved molecular chaperone,facilitates the maturation of substrates(or clients)involved in various cellular pathways critical for cell growth,invasiveness and survival beyond protein folding.Hsp90 inhibition leads to the proteasome-mediated degradation of oncogenic client proteins,thus making Hsp90 a remarkable therapeutic target for cancer therapy.Therefore,Hsp90 inhibitors may exhibit a broad spectrum of anticancer activity.17-Allylaminogeldanamycin(17-AAG),the first inhibitor,entered clinical trials in 1999.As extensive efforts in rational drug design and discovery of Hsp90 inhibitors from different origins are continually made,currently more than 17 Hsp90 inhibitors are undergoing clinical trials,although there are no approved Hsp90-targeted drugs.Many Hsp 90 inhibitors are compounds derived from herbal plants;obviously,herbal plants continued to be interested for the exploitation of Hsp90 inhibitors by virtue of their extensive research and development space.Tripterygium wilfordii,a radicular xylem of the Celastraceae family,has been used to treat rheumatoid arthritis and psoriasis due to its abundant bioactive components such as alkaloids,diterpenoids,triterpenoids,and sesquiterpenes,but little research has systematically reported bioactive components that can inhibit Hsp 90.Nevertheless,natural products usually contain hundreds or even thousands of components with various concentrations and properties,and only a few of them are potential inhibitors with the pharmacological activity of interest.Thus,the key is to develop high-throughput and high-selectivity methods for discovery of bioactive components in natural products.Conventional screening techniques,based on the use of highthroughput screening for the molecular structure designed on the assumption of libraries of plant-derived compounds,are obviously labor-immense and time-consuming,which are not applicable for complicated herbal extracts.Protein affinity-based ligand fishing screening assay,ascribing to direct interaction with targets(i.e.protein,enzyme and receptor),has emerged as the most efficient and convenient technology to fish out specific ligands from complex matrix.In these strategies,target proteins are usually immobilized on solid surfaces which enable to resist the environmental changes compared with solution phase-based technique,thus various types of solid supporters such as silica nanospheres,magnetic particles and gold nanoparticles were developed for bioactive molecular discovery.In our previous study,we developed a fluorescent ligand fishing utilizing a kind of Hsp90a functionalized fluorescent mesoporous silica-InP/ZnS quantum dots(QDs)nanocomposites as sorbents and fluorescent tracers to screen and identify not only small-molecule ligands of Hsp90 from complex herbal plants,such as Curcuma longa L.crude extracts or Alisma plantago-aquatica Linn extracts,but also client proteins present in a mixture of cellular extracts.Nevertheless,these developed fluorescent ligand fishing approaches are still confronted with challenges such as the susceptible fluorescence to external environments as well as cytotoxicity caused by leakage of cadmium due to the QDs only coating the surface of the mesoporous nanoparticles,thus inducing the formation of microkernel mesoporous fluorescent nanoparticles responsible for protecting QDs in further bioapplications.In order to solve the problems as mentioned above,the fluorescent ligand fishing approach based on InP/ZnS quantum dots on the surface of mesoporous nanoparticles was meliorated.In this study,we tried to construct a "sandwich" multiplelayer mesoporous nanostructure composed of a protective silica coating CdTe QD core and a mesoporous silica shell responsible for protecting QDs,i.e.,microkernel-based mesoporous(SiO2-CdTe-SiO2)@SiO2 fluorescent nanoparticles(MMFNPs),and utilize it to fish and identify Hsp90 small ligands coupling specific enrichment with in situ biomedical imaging from Tripterygium wilfordii crude extraction.1.The first part is to fabricate amino-functionalized(SiO2-CdTe-SiO2)@SiO2 nanoparticles.2.The second part is to establish a fast and effective screening platform for Hsp90 inhibitors based on fluorescence imaging and ligand fishing method.3.The third part is to verify the anti-tumor Hsp90 activity of the fished compounds.Hence,a color-adjustable,highly fluorescent,stable microkernel-based mesoporous nanoparticle of super surface area and porous,i.e.MMFNPs,was successfully fabricated as extraction sorbents and fluorescent tracers.We developed a fluorescent ligand fishing approach coupling with in situ imaging for screening and identification of Hsp 90 inhibitors from complex herbal extracts,Tripterygium wilfordii crude extraction.This screening approach not only can concurrently carry out biomedical imaging and further show in situ screening results,but also could eliminate the interference from a large number of non-active substances and improve the enrichment efficiency of active molecules.A total of twelve specific components selectively screened and identified by LC/MS and GC/MS analysis were performed to dock with the whole binding sites of Hsp90 for determining binding affinity towards Hsp90,three of which,i.e.,celastrol,wilforine and demecolcine,showed a strong affinity,indicating a promis:ing Hsp90 inhibitor.Their respective bioactivities were further verified by the MTT,wound scratch assay and ELISA.These results suggest that this type of fluorescent ligand fishing approaches have great promise for the development of other plant-derived Hsp90 inhibitors and is expected to provide an effective platform for discovery of biologically active molecules in natural products. |
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