Binding of ACE-2 to the viral spike protein receptor binding domain

The WAVEsystem, a Creoptix technology, is a cutting-edge optical biosensor built upon the GCI technology. The WAVEsystem measures binding affinity and kinetics label-free using a sensor chip, the WAVEchip. Because of the unique design of the WAVEchip, the WAVEsystem can measure affinity and kinetics from weak to tight binders (k_a = 10² – 3x10⁹ M-1 sec^–1; K_d = 10^–6 – 10 sec^–1 and binding affinities (KD) from low pM to low mM). The WAVEchip has all the microfluidics integrated (not within the device core) making it virtually clog-free: compatible sample types include 100% serum or plasma, cell lysates, crude membranes preparations, large drug targets, virus-like particles (VLPs), liposomes, and aggregates (fibrils).

When measuring an interaction between two components, one component (ligand) is immobilized onto the sensor chip surface. The other component (analyte) is flown past by microfluidics. Typically the WAVEchip is available with two different basis matrices: quasi-planar polycarboxylate hydrogel (PCP) and thick polycarboxylate hydrogel (PCH). In addition, different surface chemistries are available to suit different ligand immobilization strategies. Here we introduce the regenerable streptavidin surface (RST), a new surface chemistry for the WAVEchip, with data from a proof-of-concept study on ACE-2 binding spike protein receptor binding domain.

The regenerable streptavidin WAVEchips are based on the PCH or PCP matrices. These RST WAVEchips enable capture of biotinylated ligands on a streptavidin-covered surface with undetectable dissociation (k_d < 10^-7 s^-1), while permitting complete surface regeneration. Regenerable streptavidin WAVEchips provide an alternative to permanent ligand capture on streptavidin chips (PCP-STA and PCH-STA) for applications where reversible ligand capture is required.

Binding of ACE-2 with different RBD variants on PCP-STA and PCP-RST WAVEchips

Angiotensin-converting enzyme 2 (ACE2) as bound in the membrane of cells plays a role in regulating blood pressure and has thus been a drug target for cardiovascular diseases. However, the ACE2 protein also serves as an entry point into the cell for some coronaviruses, among which the SARS-CoV-2 causing the global pandemic. Proteins that interact with ACE2 might compete with the SARS-CoV-2, thus preventing cell entry and infection of the cell by the virus. Thus, research into the interaction between ACE-2 with the viral spike receptor binding domain (RBD) protein and potential inhibitors of that interaction spiked after the COVID pandemic broke in 2019. 

As published in August 20221, the effect of amino acid changes in the SARS-CoV-2 binding domain on the binding of ACE-2 was measured on the WAVEsystem using an PCP-STA WAVEchip (pre-immobilized streptavidin on a quasi-planar polycarboxylate hydrogel coated WAVEchip). 

This experiment was repeated on the new regenerable streptavidin PCP-RST WAVEchip. Each analyte was measured nine times. The results are highly reproducible and in excellent agreement with the results obtained on the PCP-STA chip.

The plot shows the variation in obtained association and dissociation rate (resulting in the K_D) is also shown (same colors are nine replicates of same interaction).

Materials and methods

ACE-2 (Avi-tagged) is 87.7 kDa, the wt RBD and variants are ~27 kDa. For the experiment with the PCP-STA chip, ACE-2 was diluted to 500 ng/ml in HBS-EP buffer and captured to a density of ≈75 pg/mm². Analyses were performed in 2 repeats over the same surface. Further details see https://doi.org/10.3389/fimmu.2022.966236. The experiments were repeated on the PCP-RST chip. For capturing, RST Capture reagent was diluted 1:70 in HBS-EP buffer. ACE-2 was diluted to 500 ng/ml in HBS-EP and captured to 75 pg/mm² using the target-level function. A total of 51 RST/ACE-2 capture cycles were performed. Each analyte was analyzed in nine repeats following the scheme: RST capture – ACE-2 capture – analyte (waveRAPID kinetics) - regeneration. Pulsed waveRAPID injections of compounds were done from a 500 nM sample solution for 60 s, followed by buffer injection for 100 s with a flow rate of 30 μl/min per flow cell. Surface regeneration was performed by 2 consecutive injections of 50 mM NaOH, 1 M NaCl followed by injection of running buffer for surface equilibration. Data were double-referenced by subtracting the signal of the reference channel and blank injections. The apparent concentration of the analytes in the sensing area over the six injection pulses was calibrated with a pulsed injection of running buffer supplemented with additional 0.5% DMSO, using the same injection parameters as for the analytes. Kinetic analysis was done using a 1:1 binding model. 

Please also refer to the protocol page in the regenerable streptavidin kit (product code PCP-RST 9060019; PCH-RST 9060020).

Acknowledgements

With special thanks to Benjamin Zimmer, Anna Lena Schmidt and Caroline Gabriel from 2Bind GmbH for running and analyzing the experiments with the PCP-RST chip and David Peterhoff, Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, for kindly providing the constructs. 

References

1) Magnus, C. L., Hiergeist, A., Schuster, P., Rohrhofer, A., Medenbach, J., Gessner, A., Peterhoff, D., Schmidt, B. “Targeted escape of SARS-CoV-2 in vitro from monoclonal antibody S309, the precursor of sotrovimab.”. Frontiers in Immunology, 2022. 

Currently available WAVEchip surfaces

PC- = polycarboxylate, DX- = Carboxymethyldextran
DX coated sensors available upon request