Unambiguous Identification of β‐Tubulin as the Direct Cellular Target Responsible for the Cytotoxicity of Chalcone by Photoaffinity Labeling

Abstract

Chalcone is a simple and potentially privileged structure in medicinal chemistry with a diverse repertoire of biological activities, among which cytotoxicity is of particular interest. The sharp structure-activity relationship (SAR) for chalcone's cytotoxicity suggests structure-specific target interactions. Despite the numerous putative targets proposed, evidence for direct target interactions in cells is unavailable. In this study, guided by the sharp cytotoxic SAR, we developed a cytotoxic chalcone-based photoaffinity labeling (PAL) probe, (E)-3-(3-azidophenyl)-1-[3,5-dimethoxy-4-(prop-2-yn-1-yloxy)phenyl]-2-methylprop-2-en-1-one (C95; IC50 : 0.38±0.01 μm), along with two structurally similar non-cytotoxic probes. These probes were used to search for the direct cellular target responsible for chalcone's cytotoxicity through intact cell-based PAL experiments, in which β-tubulin was identified to specifically interact with the cytotoxic probe (i.e., C95) but not the non-cytotoxic probes. A set of phenotypical and biochemical assays further reinforced β-tubulin as the cytotoxic target of chalcones. Peptide mass quantitation by mass spectrometric analysis revealed one peptide potentially labeled by C95, providing information on chalcone's binding site on β-tubulin.

An Indole–Chalcone Inhibits Multidrug-Resistant Cancer Cell Growth by Targeting Microtubules

“…Additional data, including the melting temperature (biochemical binding), COMPARE (correlative analysis), and molecular modeling (Supplementary Figs. S1–S3) data, as well as our previous whole-cell-based photoaffinity labeling data 12 , collectively support this mechanism of action. A whole-cell microtubule dynamics assay was used, instead of a tubulin polymerization/depolymerization biochemical assay, to confirm the direct interaction of FC77 with tubulin, mainly because the concentrations needed to induce polymerization or depolymerization could be more than 1,000-fold higher than cytotoxic concentrations and because there might be a limited correlation, if any, between the biochemical potency and in vitro cytotoxicity, as has been reported for tubulysin by Fecik et al 40 .…”

Natural Benzo/Acetophenones as Leads for New Synthetic Acetophenone Hybrids Containing a 1,2,3-Triazole Ring as Potential Antifouling Agents

“…The newly synthesized compounds 3a-9b were characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS) (Supplementary material, Figure S1-S28). Compounds 2a and 2b were already described in the literature and NMR data are in accordance with the data already published [23,24].…”

Kava as a Clinical Nutrient: Promises and Challenges

“…Flavokavain A was shown to induce G2/M arrest in a panel of cancer cell lines, including breast cancer cell lines (MDA-MB231 at 65 μM) [ 22 ], high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197 at 40 μM) [ 122 ] and prostate cancer cells (PC3 at 50 μM) [ 123 ]. Indeed, chalcone-based compounds have been well documented to induce cell cycle arrest at G2/M phase through inhibition of microtubule polymerization [ 126 , 127 ]. Flavokavain A also demonstrated anticancer activities in several lab animal models.…”