| Drug discovery is a long and costly process, which includes: target identification, lead discovery, lead optimization, preclinical safety and toxicity testing and clinical trials. Target identification is the starting stage during the drug discovery process, and it is also one of the most important stages. In addition to the prominent role activity-based probes (ABP) can play in diagnostics and imaging, they provide a powerful mechanism for screening drugs for specificity against multiple targets in vivo.Recently, a chemical strategy referred to as activity-based proteome profiling (ABPP) has been introduced that utilizes active site-directed probes to record variations in the activity of enzymes in whole proteomes. ABPP probes typically possess three general elements: (i) a binding group that promotes interactions with the active sites of specific classes of enzymes, (ii) a reactive group that covalently labels these active sites, and (iii) a reporter group (e.g., fluorophore or biotin) for the visualization and affinity purification of probe-labeled enzymes. To date, ABPs have been developed for many biomedically relevant enzyme classes.A persistent technical challenge facing the utilization of ABPP probes in vivo is the adverse effect of their bulky reporter tags (fluorophore/biotin), which may inhibit cellular uptake and unduly influence probe distribution within the cell. Thus, a general strategy for carrying out ABPP in vivo would require a method for attaching the reporter tag to the probe after proteins have been covalently labeled. Toward this end,“Click chemistry”as a new synthetic method has being developed in recent years. Due to its numerous advantages,its applications have been paid close attentions and developed rapidly including the synthesis of lead compound database,proteome,bioconjugate technology and biomedicine. Recently,click chemistry,and in particular the Cu + -mediated 1,3-dipolar [3 + 2]cycloaddition between azides and alkynes, has also entered the field of ABPP.Geldanamycin (GA) has been demonstrated to be a potent cytotoxic compound, active in tumour cell lines and reducing the size of solid tumours in mouse models. Initially it was thought that its cytotoxic properties were due to its activity as a non-specific protein kinase inhibitor. A protein kinase is an enzyme that modifies other proteins, resulting in changes in enzyme activity, cellular location or interactions with other proteins. Deregulated kinase activity is known to be a frequent cause of disease, particularly cancer, where kinases regulate many aspects that control cell growth, movement and death. However the method of action of some benzoquinone ansamycins, including geldanamycin, has now been re-evaluated following their demonstrated ability to bind to and antagonize the function of the chaperone protein heat shock protein 90 (Hsp90).This study is based on the antitumor structure and bioavailability relationship of Geldanamycin. 17-azide and 17-alkyne GA were designed and synthesized, which were used as activity-based probes of GA. Rhodamine-alkyne (RB1) was also designed and synthesized as reported group (tag). The targeting proteins of GA were labled by Rhodamine reported group through click chemistry.This study includes:1. Production and verification of GA 6 formations of GA with different morphology were discovered, which were GF1, GF2, GF3, MF1, MF2 and MF3 respectively. The GF1 seemed to reach a high production level at day 6.5, but the white culture GF3 began to decrease by this time. On the other hand the black culture was slow growing, older, and had partially exhausted its nutrients, so it may have taken time to get the culture accelerate. Also, it appears that the cultures produce similar levels of GA, comparing both the microforms and the GF cultures. For later productions the GF1 culture was used, harvesting around day 6. 2. Design and synthesis of GA activity-based probes Totally 3 GA activity-based probes were designed and synthesized, and competed with GA-FITC to bind HSP90α. We found G2 present an excellent HSP90αbinding property. Reporter RB1 was designed and synthesized, which is suitable for application of click chemistry. .3. Set up a ABPP-CC based Polarization Assay for the Hsp90 We developed a ABPP-CC based Polarization Assay for the Hsp90: (G2(2nM) Hsp90α(30nM), RB1(5nM), CuI(1nM), and DMSO(v/v) does not exceed 4%)4. Target identification and validation of GA G2 with different concentrations were directly added to the cell culture medium, either alone or in the present of the competing GA. After two hours, the cells were washed by PBS to remove excessive probes, homogenized, incubated with RB1 reported groups under click chemistry condition, separated by SDS-PAGE gel, and analyzed by in-gel fluorescence scanning. The results indicate that G2 could target HSP90, which makes this approach... |
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