Elva Morretta (EM) obtained a Master’s degree in Pharmaceutical Chemistry and Technology (110/110 cum laude) in July 2016 at Salerno University (bio-organic chemistry thesis, tutor Prof. Monti). In September, EM obtained a fellowship for the Drug Discovery and Development Ph.D. program (tutor Prof. Casapullo). In 2018, EM spent six months at Prof. Heck Biomolecular Mass Spectrometry and Proteomics Lab. On March 2020, EM defended her Ph.D., discussing a thesis entitled “Interactome Analysis of Bioactive Molecules: Optimization of a Functional Proteomics Platform”. From January 16th 2020, EM works as a Post-Doctoral Researcher at the Pharmacy Department of Salerno University.
Label-free functional proteomics links the anti-angiogenic properties of the pyrazolyl-urea GeGe-3 to Calreticulin binding
n the last twenty years, 5-pyrazolyl-ureas have been largely investigated for their poly-pharmacological potential. In this scenario, ethyl 1-(2-hydroxypentyl)-5-(3-(3-(trifluoromethyl) phenyl)ureido)-1H-pyrazole-4-carboxylate (GeGe-3) emerged as a promising anti-angiogenic compound, inhibiting Human Umbilical Vein Endothelial Cells (HUVEC cells) proliferation and endothelial tube formation, impairing inter-segmental angiogenesis during zebrafish embryos development and blocking tumour growth in transplanted subcutaneous Lewis Lung Carcinomas. Regrettably, although different primary targets implicated in cell division and/or calcium homeostasis have been hypothesized for this compound, all the binding tests gave negative results. Thus, to link GeGe-3 anti-angiogenic potential to a suitable protein partner, the molecule interactome has been deeply investigated in HUVEC cells through label-free functional proteomics approaches, namely Drug Affinity Responsive Target Stability (DARTS) and targeted Limited Proteolysis coupled to Multiple Reaction Monitoring Mass Spectrometry (t-LiPMRM). These approaches share the principle that, interacting with a molecule, a protein undergoes conformational changes that result in its lower sensitivity to limited proteolysis, when performed in native conditions. Thus, in a first step, the coupling of DARTS with high resolution mass spectrometry allowed the identification of GeGe-3 most reliable interacting protein, Calreticulin, as later on validated by Western Blotting. Subsequently t-LiP-MRM, which allows to discover the target protein structural alterations due to complex formation with GeGe-3, served the purpose of pinpointing Calreticulin regions directly or distally involved in the interaction with the compound. T-LiP-MRM obtained results were corroborated by molecular docking analyses. Calreticulin is a major Ca2+ binding protein involved in intracellular Ca2+ homeostasis, cells adhesion, migration, proliferation, differentiation and apoptosis, as well as in cell-cell interactions. To shed light on the biological consequences of GeGe-3 interaction with such an interesting protein partner, in cell assays were performed. The obtained results disclosed GeGe-3 potential mechanism of action as anti-angiogenic factor: due to its binding to calreticulin, the molecule is able to alter Ca2+ intracellular shift in HUVEC cells, consequently modifying their cytoskeletal proteins organization.
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