Vascular Endothelial Growth Factor Receptor, fms-Like Tyrosine Kinase-1 (Flt-1), as a Novel Binding Partner for SARS-CoV-2 Spike Receptor-Binding Domain

Abstract

Angiotensin-converting enzyme 2 (ACE2) and neuropilin 1, a vascular endothelial growth factor (VEGF) receptor, were identified to bind to the SARS-CoV-2 spike receptor-binding domain (spike RBD). In silico analysis based on 3D structure, multiple sequence alignment, and molecular docking of second domain of soluble Flt-1 (sFlt-1) and spike RBD revealed structural similarities, sequence homology, and protein-protein interaction. Interaction and binding of recombinant spike RBD (rspike RBD) and recombinant sFlt-1 (rsFlt-1) in vitro induced a conformational change, as revealed by spectrofluorimetric data, with increased fluorescence intensity in emission spectra as compared to either of the proteins alone. Results on ELISA confirmed the binding and cross-reactivity of rspike-RBD and rsFlt-1 as determined by using either specific antibodies towards each protein or immunized human serum. We found that polyclonal or monoclonal anti-spike RBD antibodies can recognize either rsFlt-1 or rspike RBD, showing cross-reactivity for the two proteins in a dose-dependent binding response. Recognition of bound rspike RBD or rsFlt-1 by anti-Flt-1 or anti-spike RBD antibodies, respectively, as observed by immunoblotting, further confirmed interaction between the two proteins. Immunoprecipitation and immunoblot analysis demonstrated the identification of rspike RBD binding to the Flt-1 receptor on A549 cells. Further, the binding of rspike RBD to Flt-1 receptor was shown using immunofluorescence on 2D-culture or 3D-spheroid of MDA-MB-231 cells, which over-express Flt-1 receptor. Together, our study concludes that the Flt-1 receptor is a novel binding partner for SARS-CoV-2 spike RBD.

Determination of 5-Fluorouracil and Doxorubicin Concentrations Required for Effective Induction of Cytotoxicity and Morphological Responses in 3D Tumor Spheroids

“…Tumor spheroids (MDA-MB 231) were generated in ULA-96 well round-bottom plate (2000cells/well) as described earlier [13]. Generated tumor spheroids (3 days old) were treated either with 5-FU (100µM and 300µM) or doxorubicin (0.1µM and 0.3µM).…”