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.

Structure of Nucleophosmin DNA-binding Domain and Analysis of Its Complex with a G-quadruplex Sequence from the c-MYC Promoter

“…Cys 275 is targeted and alkylated by the natural antitumoral compound (ϩ)-avrainvillamide (47). Treatment of LNCaP or T-47D cells with (ϩ)-avrainvillamide leads to an increase in cellular p53 concentrations and promotes apoptosis.…”

Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications

“…30 Avrainvillamide, a naturally occurring alkaloid with anti-proliferative activity, binds to the NPM1wt C-terminal domain through Cysteine-275. 31 Interestingly, Cysteine-275 is located on the same face as Lysine residues 263 and 267. 22 Orientated freely on the C-terminus domain surface, these Lysines appear to play an important functional (rather than structural) role in NPM1wt nucleolar localization, 22 suggesting this protein domain area is involved in ligand binding.…”

Deciphering the folding transition state structure and denatured state properties of Nucleophosmin C-terminal domain

“…So far, in fact, the region comprising helix H2 has been mainly envisaged for ligand binding, as it includes important functional residues (e.g. K263 and K267) (46) and the Cys 275, which is responsible for the binding of the natural antitumoral compound Avrainvillamide (47).…”