title: The natural product avrainvillamide binds to the oncoprotein nucleophosmin.
abstract: Here we present evidence that (+)-avrainvillamide, a naturally occurring alkaloid with antiproliferative effects, binds to the nuclear chaperone nucleophosmin, a proposed oncogenic protein that is overexpressed in many different human tumors. Among other effects, nucleophosmin is known to regulate the tumor suppressor protein p53. A synthetic biotin-avrainvillamide conjugate, nearly equipotent to the natural product in inhibiting the growth of cultured T-47D cells, was used for affinity-isolation of a protein identified as nucleophosmin by MS sequencing and Western-blotting. Affinity-isolation of nucleophosmin was inhibited in the presence of iodoacetamide (10 mM), free (+)-avrainvillamide (100 microM), and a series of closely related structural analogues of (+)-avrainvillamide, the latter with inhibitory effects that appear to correlate with measured growth-inhibitory potencies. Using fluorescence microscopy, a synthetic dansyl-avrainvillamide conjugate was observed to localize within the nucleoli and the cytosol of treated cancer cells. Site-directed mutagenesis of each of the three cysteine residues of a truncated nucleophosmin coexpressed with native nucleophosmin in COS-7 cells revealed that the mutation cys275 --> ala275 effectively and uniquely reduced affinity-isolation of the truncated protein, suggesting that avrainvillamide targets cys275 of nucleophosmin. Finally, we show that treatment of adhered LNCaP or T-47D cells with (+)-avrainvillamide leads to an increase in cellular p53 concentrations, and that siRNA-promoted depletion of nucleophosmin in a population of HeLa S3 cells leads to increased sensitivity of that population toward apoptotic death upon treatment with (+)-avrainvillamide. Although potentially desirable as lead compounds for the development of novel anticancer therapies, nonpeptidic, synthetic small molecules that bind to nucleophosmin have not been described, prior to this report.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: Jeremy E
authors.initials: JE
authors.lastname: Wulff
authors.forename: Romain
authors.initials: R
authors.lastname: Siegrist
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Myers
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D001688:Biological Products / Q000494:pharmacology*; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D000090243:Nucleophosmin; D015513:Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D001688:Biological Products; D007211:Indoles; C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D015513:Oncogene Proteins; C480558:avrainvillamide; D000090243:Nucleophosmin; D001710:Biotin; D003545:Cysteine; D000409:Alanine