Abstract 5227: Synthesis of a biotin probe for identification of the ajoene protein targets in cancer cells ajoene protein targets in cancer cells

Abstract

Abstract Garlic (Allium sativum) is a medicinal plant belonging to the lily family which has been used since ancient times for its beneficial health effects which include protection against infections and cancer. There are a cluster of bioactive compounds found in crushed garlic which contain sulfide or polysulfide functional groups. One of these compounds, ajoene, is able to interfere with biological processes by undergoing thiolysis exchange reactions with biological thiols, for example glutathione and cysteine residues on proteins. Importantly, ajoene is cytotoxic to cancer cells in the low micromolar range. We have previously found that ajoene targets multiple proteins in cancer cells and the aim of this project is to identify the specific protein targets of ajoene. To achieve this aim, we have synthesized a “tagged” ajoene analogue containing a biotin functional group. As biotin may alter the biological properties of ajoene, our strategy was to initially synthesize an azide-containing ajoene analogue (called azide-ajoene) using our 4-step synthetic route to ajoene analogues previously published. Azide-ajoene was found to retain cancer cell cytotoxicity similar to that of the parent ajoene. We then treated cancer cells (MDA-MB-231 breast and WHCO1 oesophageal cancer) with azide-ajoene to allow transfer of an azide label to the protein targets during thiolysis exchange with ajoene. We then collected lysate from the treated cells and performed an in vitro click reaction with a separately synthesized biotin-alkyne partner which was synthesized via a series of substitution and amide coupling reactions. Click chemistry covalently links biotin to the ajoene-tagged protein via a triazole linkage; and the feasibility of this method was validated by western blot. The biotinylated protein targets were purified using immobilised streptavidin beads and identified by MALDI-TOF mass spectrometry. It is anticipated that knowledge of the specific ajoene protein targets will provide valuable insights into the cytotoxic mechanism of action of ajoene in cancer cells. Citation Format: Catherine H. Kaschula, Daniel A. Kusza, Georgia Schafer, Arieh A. Katz, Shivendra V. Singh, Roger Hunter. Synthesis of a biotin probe for identification of the ajoene protein targets in cancer cells ajoene protein targets in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5227. doi:10.1158/1538-7445.AM2017-5227