Semisynthetic aurones inhibit tubulin polymerization at the colchicine-binding site and repress PC-3 tumor xenografts in nude mice and myc-induced T-ALL in zebrafish

Ting Yu ,M. S. Frasinyuk ,Haining Zhu ,Jessica S. Blackburn ,Agripina G. Deaciuc ,Xifu Liu ,Linda P. Dwoskin ,Jing Chen ,David S. Watt ,Elizabeth S. Hausman ,Chang‐Guo Zhan ,Przemysław Wyrębek ,Yanqi Xie ,Zachary M. Martin ,K. M. Kondratyuk ,Chunming Liu ,S. P. Bondarenko ,Vitaliy M. Sviripa ,Liliia M. Kril ,Wen Zhang

doi:10.1038/s41598-019-42917-0

Abstract

Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model.

Highlights

The aurones comprise a family of plant-derived flavonoids that arise out of a mixed polyketide-shikimate pathway, contribute to the yellow coloration of certain flowers[1] and possess a range of biological properties[2,3,4] affecting organisms ranging from protazoans to mammals
Level: drug efflux modulators[2,9,10,11,12,13,14,15] of P-glycoprotein (P-gp) or ATP-binding cassette sub-family G member 2 (ABCG2), modifiers of adenosine-receptor interactions[16,17], DNA sission-promoters[18], teleomerase inhibitors[19], sphingosine-kinase inhibitors[20], phosphatidylinositol-3-kinases (PI3−α) inhibitors[21], cyclin-dependent kinase inhibitors[22], inducers of cytoprotective NAD(P)H:quinone oxidoreductase-123 (NQO1), and scavengers of reactive-oxygen-species[24] (ROS). These findings suggested that aurones would disrupt biological systems non-our studies of the aurone pharmacophore identified heterocyclic variants of the (Z)2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed the low nanomolar in vitro potency, encouraging in vivo potency in mouse xenograft and zebrafish models, selectivity for the colchicine-binding site in tubulin[25,26,27,28,29,30,31], and the absence of appreciable toxicity
Our studies focused on developing agents for the treatment of prostate cancers, the prior report that aurones with 2-(coumarin-4-yl)methylene groups[32] or 2-(furan-2-yl)methylene groups[33] displayed in vitro activity against human leukemia K562 cells prompted a study of myc-induced T-cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model where these aurones exhibited minimal toxicity

Summary

Introduction

The aurones comprise a family of plant-derived flavonoids that arise out of a mixed polyketide-shikimate pathway, contribute to the yellow coloration of certain flowers[1] and possess a range of biological properties[2,3,4] affecting organisms ranging from protazoans to mammals. Using a competition assay with mass spectrometry as an analytical tool, we established that these aurones functioned at a molecular level as tubulin polymerization inhibitors by binding to the colchicine-binding site. A reiterative process of synthesis and screening using in vitro prostate cancer PC-3 cell proliferation assays identified an intersection of modifications at the C-2 and C-6 positions in semisynthetic aurones that were the most promising for further study (Table 1).

Results

Conclusion