The natural product avrainvillamide binds to the oncoprotein nucleophosmin.
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.
Wulff
Jeremy E
JE
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Siegrist
Romain
R
Myers
Andrew G
AG
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
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