Cellular drug target discovery is an important step in any drugs journey from bench to bedside. This is true for our labs molecule of interest, the Chalcone. The Chalcone molecule and its derivatives have been identified as small, plant‐derived secondary metabolites that, when interacting with human cancer cell lines, trigger apoptotic pathways leading to varying levels of cell death. One derivative in particular, 4‐Trifluoromethoxy Chalcone (4TFM), was identified through screenings as inducing the highest death rate in A549 cancer cells, in conjunction with having the lowest IC50, making it a good candidate to use in searching for the currently unknown cellular target of the Chalcone. Using Drug Affinity Response Target Stability (DARTS) Method, we have begun that process, leveraging the fact that a protein's ligand is able to shield its target from proteolysis at a specific concentration. Incubation with and without drug can produce conserved bands observable via gel electrophoresis, providing potential targeted and protected proteins that can be identified through Mass Spectrometry. For 4TMF, we are currently narrowing in on a library of potential protein targets in the hope to ultimately establish the cellular component the small molecule is interacting with, creating its signature anti‐cancer effects.
Chalcones are polyphenols composed of two aromatic rings linked by an aliphatic three carbon chain. Chalcones and their derivatives have been shown to have numerous types of biological activity including anti‐inflammatory, antioxidant, anti‐infective, antiviral, anticancer and antitumor. Previous work in our lab discovered that anti‐cancer activity benefitted from electron donating substitutions to the B ring. Here, we are exploring the flexibility of the bridge structure using tetralone and indanone to generate a more rigid link between the aromatic rings. Using tetralone and indanone combined with benzaldehydes bearing carious electron donating groups a diverse library of chalcones was generated. These different compounds were first tested in noncancerous HEK 293 cells and have continued being tested in breast cancer cells (MCF7). An initial screening assay is performed at 50 μM. Compounds showing fifty percent or greater inhibition are taken on for more detailed analysis to determine an IC50. In both cases, cells are counted and seeded at specific densities, followed by drug treatment forty‐eight hours later, then cell viability is measured using an MTS assay on a visible spectrophotometer forty‐eight hours after drug treatment. Our results indicate the flexibility is an important feature for biological activity.Support or Funding InformationBellarmine University Chemistry and Physics DepartmentThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chalcones are a diphenyl compound that serves as a natural precursor to flavanones in plants.Chalcones have been shown to have anticancer and antimicrobial activities. Chemoprevention activity of chalcones are of high interest in medicinal chemistry because of the simple laboratory synthesis and modification via aldol condensation. Previously this lab created and screened a library of synthetic chalcones against A549 lung adenocarcinoma cell line for antiproliferation properties. We identified a strong drug candidate (4′‐Trifluoromethoxy substituted chalcone) for A549 growth inhibition. However, the cause of inhibition by the substituted chalcone remains to be identified. We began to explore the mechanism of action of this drug by looking at physical characteristics of cell death with microscopy. Then, we conducted viability and cytotoxic assays such as MTS and Trypan blue for cell death quantification. Cytotoxicity was determined with a Lactate Dehydrogenase Assay. Western Blots were ran to identify dose dependency on the specific apoptotic proteins PARP and Caspase‐3. Using flow cytometry, we investigated a more accurate quantification of cells undergoing apoptosis versus necrosis. Here we present the results of our investigation into the apoptotic and necrotic cell death of lung cancer A549 cells by a chalcone derivative.Support or Funding InformationProject Funded by Bellarmine University Department of Chemistry and Physics and Bellarmine University Student Government Association.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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