| The study reports the discovery and rational design of natural product (NP) scaffolds for the selective control of breast malignancies. Different NP sources, including lichen, plant and marine extracts were screened against multiple clonal lines of breast cancer cells in various anticancer screening platforms. The bioassay-guided approach was implemented to identify the bioactive hits. Sensitivity of a particular breast cancer cell line(s), phenotypic and morphological changes, virtual screening and Western blot analysis were all used to uncover the potential macromolecular targets mediating, at lest in part, the anticancer activity of the isolated hits. Two major macromolecular targets were identified: The mechanistic target of rapamycin (mTOR) and proto-oncogene receptor tyrosine kinase c-Met.;Rapamycin and analogs (rapalogs) are allosteric mTOR inhibitors that are approved by the FDA for multiple malignancies. However, feedback activation of Akt/PKB limited the use of rapalogs as monotherapy and triggered the need to develop additional ATP-competitive inhibitors (second-generation mTOR inhibitors) that target the kinase domain of both complexes. Numerous small-molecule inhibitors, including INK128, AZD8055 and AZD2014, have been developed and have progressed to clinical trials.;c-Met is a membrane-bound receptor tyrosine kinase (RTK) that is normally expressed in cells of epithelial origin of many organs. However, c-Met and its high-affinity ligand hepatocyte growth factor (HGF) have been found to be dysregulated and associated with aggressive tumor growth, induction of angiogenesis, and metastasis of many cancers, including the triple-negative breast cancer (TNBC) subtype. The U.S. FDA approval of two c-Met inhibitory cancer drugs (crizotinib and cabozantinib) affirmed the validity of c-Met as a novel target for cancer therapy.;(+)-Usnic acid is a lichen-derived metabolite with a dibenzofuran scaffold. Screening of usnic acid against a panel of breast cancer cell lines revealed low microM antiproliferative activity levels. Moreover, usnic acid treatment potently induced autophagy in multiple breast cancer cells, suggesting mTOR as a potential macromolecular target. Western blot analysis revealed the upregulation of principle autophagic markers and the significant inhibition of mTOR downstream effectors. A molecular extension strategy was proposed, based on the binding mode of usnic acid at the mTOR kinase domain, to probe stacking interaction with a Tyr2225 in a deep hydrophobic pocket at the core of the kinase cleft. Claisen-Schmidt coupling with aromatic aldehydes afforded benzylidene analogs that were able to interact with the phenolic side-chain of the Tyr2225 residue at the targeted mTOR pocket. The most active analog 52 potently (at nM levels) inhibited the proliferation, migration and invasion of multiple breast cancer cells. Moreover, analog 52 also exhibited potent mTOR inhibition and autophagy induction according to results obtained by Western blot analysis. Additionally, analog 52 showed robust in vivo antitumor activity against in two different breast cancer xenografts in nude mice.;Norstictic acid is a depsidone-derived metabolite commonly found in lichens that was identified to significantly suppress the triple negative breast cancer (TNBC) MDA-MB-231 cell proliferation, migration and invasion. Molecular modeling, Z'-LYTE kinase assay and Western blot analysis identified c-Met as a potential molecular target. Furthermore, administration of norstictic acid to nude mice grafted with MDA-MB-231/GFP cells significantly attenuated the in vivo tumor growth without any signs of toxicity. Therefore, norstictic acid was identified as a novel depsidone-based c-Met inhibitory lead.;Members of an in-house marine natural product (MNP)-based mini-library were submitted for biological screening to the Lilly's Open Innovation for Phenotypic Drug Discovery (PD2-OIDD). The results for the phenotypic screening assays in the area of angiogenesis and biochemical kinase profiling supported by molecular docking studies for the active hits are reported. Furthermore, virtual analogs were devised to enhance hit-target interactions based on favorable binding modes of their parent MNPs.;(1S,2E,4S,6 R,7E,11E)-2,7,11-cembratriene-4,6-diol and its 4-epi-analog are the cembranoid precursors to several key flavor ingredients in most Nicotiana (tobacco) species. Nearly 40-60% of these cembranoids are purposely degraded during the commercial tobacco fermentation. However, they display promising bioactivities, including anticancer. In particular, (1S,2E,4 S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol displayed antiproliferative, anti-migratory and anti-invasive effects against the c-Met overexpressing TNBC MDA-MB-231 cells at moderate microM concentrations. The Z'-LYTE kinase platform and Western blot analysis identified c-Met as a potential mechanistic target. Rationally designed analogs were proposed to probe additional c-Met interactions and improve the cellular potency. The chemistry efforts guided by cell-based and kinase assays led to the creation of analog 20 that showed a potent in vitro anticancer activity against the MDA-MB-231 breast cancer cells at low microM concentrations. The hit 20 significantly inhibited in vivo tumor growth of the grafted MDA-MB-231/GFP cells, when administered to hosting nude mice and was therefore promoted to the lead rank. (Abstract shortened by ProQuest.). |
