Inhibition of SARS-CoV-2 infection in cellular systems using engineered trimeric receptor-binding domain of spike protein.

Here, we provide a protocol for the design, expression, purification, and functional studies of an engineered trimeric version of the receptor-binding domain (tRBD) of SARS-CoV-2 spike protein. We describe the use of tRBD to block SARS-CoV-2 spike pseudovirus and true virus binding to cellular angiotensin converting enzyme-2 (ACE2), thereby blocking viral infection. This protocol is applicable to generate a trimeric version of any protein of interest. For complete details on the use and execution of this protocol, please refer to Basavarajappa et al. (2022).1.

Trimeric receptor-binding domain of SARS-CoV-2 acts as a potent inhibitor of ACE2 receptor-mediated viral entry.

The COVID-19 pandemic has caused over four million deaths and effective methods to control CoV-2 infection, in addition to vaccines, are needed. The CoV-2 binds to the ACE2 on human cells through the receptor-binding domain (RBD) of the trimeric spike protein. Our modeling studies show that a modified trimeric RBD (tRBD) can interact with three ACE2 receptors, unlike the native spike protein, which binds to only one ACE2. We found that tRBD binds to the ACE2 with 58-fold higher affinity than monomeric RBD (mRBD) and blocks spike-dependent pseudoviral infection over 4-fold more effectively compared to the mRBD. Although mRBD failed to block CoV-2 USA-WA1/2020 infection, tRBD efficiently blocked the true virus infection in plaque assays. We show that tRBD is a potent inhibitor of CoV-2 through both competitive binding to the ACE2 and steric hindrance, and has the potential to emerge as a first-line therapeutic method to control COVID-19.

HBD-2 binds SARS-CoV-2 RBD and blocks viral entry: Strategy to combat COVID-19.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19-related deaths and medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell-derived host defense peptide that has anti-viral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical measurements confirm that hBD-2 indeed binds to the CoV-2-receptor-binding domain (RBD) (KD ∼ 2µM by surface plasmon resonance), preventing it from binding to ACE2-expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSVG-mediated infection, of ACE2-expressing human cells with an IC50 of 2.8 ± 0.4 µM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as agents to prevent SARS-CoV-2 infection.