(13−15) Covalent chemical probes and drugs have been developed against diverse proteins, including hydrolases, (16,17) kinases, (18−20) nuclear export proteins, (21) and oncogenic GTPases. (11,12)Ĭovalent ligands are attractive starting points for chemical probe and drug development for several reasons, including the potential to achieve improved potency for shallow binding pockets and increased residency time leading to more durable pharmacological action. (7−10) A subset of these approaches focuses on covalent ligand discovery, where electrophilic compounds are screened for reactivity against thousands of nucleophilic residues (e.g., cysteines, lysines) in the proteome. In recent years, chemical proteomic strategies have emerged that enable the discovery of ligands for proteins on a global scale directly in native biological systems. (1−3) While high-throughput screening methods that expose purified proteins to large compound libraries offer a well-established approach to chemical probe discovery, (4−6) many proteins are challenging to recombinantly express and purify and may further lack sufficient characterization for functional assay development. Selective chemical probes are available for only a modest fraction of the human proteome. Our findings provide a road map to optimize covalent fragments into more advanced chemical probes without requiring protein purification or structural analysis.Ī strategy is described to convert electrophilic fragment-cysteine interactions discovered by chemical proteomics into high-throughput ELISA screens that do not require purified proteins. We use these assays to expediently optimize a weak potency fragment hit into a sub-μM inhibitor that selectively engages an active-site cysteine in the retinaldehyde reductase AKR1B10. Here, we show that broadly reactive electrophilic fragments, or “scouts”, can be converted into site-specific target engagement probes for screening small molecules against a wide array of proteins in convenient gel- and ELISA-based assay formats. Further optimization of these initial ligandability events without resorting to the time-consuming process of individualized protein purification and functional assay development, however, presents a persistent technical challenge. Chemical proteomic methods have been introduced that leverage electrophilic fragments to globally profile the covalent ligandability of nucleophilic residues, such as cysteine and lysine, in native biological systems.
The protocol used in the present study.Covalent ligands are a versatile class of chemical probes and drugs that can target noncanonical sites on proteins and display differentiated pharmacodynamic properties. When they are treated together with a very intensive induction chemotherapy such as In achieving complete remission and survival in patients with advanced neuroblastoma, Gross complete resection of a primary tumor and lymph nodes has a significant value The incidence of local recurrence was lower in patients who underwent grossĬomplete resection than in those who had partial resection. Preserved at surgery had an outcome superior to that of those with associated nephrectomy Patients in whom the ipsilateral kidney was
Years for 31 patients who similarly underwent complete resection but when evidence Resection after 3 cycles of A 1 when resolution of all metastases was obtained, whereas the survival was 52% at 2 Overall survival rate was 62% at 2 years for 27 patients who underwent complete Had a better prognosis than those 11 patients with partial resection ( P <. The 81 patients with gross complete resection achieved (90%): 30 cases during the first 3 cycles of A 1 chemotherapy and 62 cases after that, with gross complete resection accomplished Surgical intervention at the primary site was performed in 92 of the 102 patients Were 79%, 70%, and 70% at 2 years, 3 years, and 4 years, respectively.
Gross complete resection of primary tumor and regional lymph nodes wasįeasible in 17 of the 19 patients (89%), and the survival rate for the 17 patients Intervention at the primary site was performed in 18 of the 19 patients, 9 duringĪnd 9 after the first three cycles of A 1 regimen, consisting of high-dose cyclophosphamide, vincristine, THP-adriamycin, andĬis-platinum. Of Japan between January 1985 and March 1990. The role of surgery was evaluated in 19 stage III and 102 stage IV neuroblastoma patients,Īll of whom were treated with intensive induction chemotherapy by the Study Group
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