In a recent and timely paper, currently available on the bioRxiv* preprint server, researchers from Israel and France demonstrated how in vitro evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) follows contagious mutation spread – but can also generate an efficient infection inhibitor.
Both structural and functional research endeavors have revealed that a single RBD of the SARS-CoV-2 homotrimer spike glycoprotein is sufficient for interaction with angiotensin-converting enzyme 2 (ACE-2) viral receptor.
The binding process and subsequent cleavage by the host protease transmembrane serine protease 2 (TMPRSS2) lead to the fusion between the cell and viral membranes and results in cell entry. Consequently, this can be circumvented by blocking the ACE2 receptors with specific antibodies.
There is also the role of the increased affinity towards the receptor, which is (at least partially) responsible for SARS-CoV-2 infectivity. Recently evolved viral mutations within the spike glycoprotein's RBD gave additional credence to this hypothesis.
And we are now faced with specific examples. The "British" mutation (N501Y or variant B.1.1.7) was suggested to enhance binding to ACE2 from deep sequencing mutation analysis. Likewise, the "South African" variant (501.V2) that includes K417N, E484K, and N501Y is spreading swiftly and turning into a dominant lineage in the Eastern Cape and Western Cape Provinces.
Hence, potential treatment targets that aim to halt the viral entry in cells should entail molecules blocking the spike glycoprotein, the ACE2 receptor or the TMPRSS2 protease. Accordingly, multiple high-affinity neutralizing antibodies have recently been suggested and developed.
A yeast display selection strategy
Remarkably, the RBD domain itself may be utilized as an effective competitive inhibitor of the ACE2 receptor binding site. Nonetheless, its affinity has to be substantially optimized in order to work – basically, it has to reach picomolar affinity.
Recently, an enhanced strategy for yeast display was developed based on C- and N- terminal fusions of exceptionally bright fluorescence colors that can observe expression at minute levels, enabling the selection to proceed down to picomolar bait concentrations.
In this study, a research group (led by Dr. Jiří Zahradník from the Weizmann Institute of Science in Rehovot, Israel) decided to use two different detection strategies based on surface exposure tailored reporters eUnaG2 and DnbALFA, and eliminate tedious DNA purification step.
For optimal surface expression, various sizes of the RBD were tested prior to library construction. High-resolution cryo-electron microscopy was employed for structure determination of RBD, which was then used to appraise electrostatic complementarity.
Global comparison between RBD-WT and RBD-62. A) The RBD-62 preserves its typical twisted five-stranded antiparallel β sheet (β1, β3-β5, and β10) with an extended insertion containing the short β5-β9 strands, α4, and η3 helices and loops. The biggest differences are pronounced between M470 and F490 (black circle). B) The upper part comprised of three segments: R357-S371 (β2, α2), G381-V395 (α3), and F515-H534 (β11) is not resolved in the electron density map (blue ribbon, added from PDB ID: 6M0J).
Tighter binding to ACE2
This study has shown that naturally selected mutations S477N, E484K and N501Y of the spike glycoprotein RBD that are known to confer higher infectivity were actually selected by yeast surface display affinity maturation in the first round, giving rise to the E484K, N501Y, South-African and British variants with 3.5-fold tighter binding to ACE2 in comparison to wild type RBD.
Moreover, after three additional rounds of yeast display selection, there was 600-fold tighter binding compared to wild-type RBD. This selection process capitalized on combinatorial selection without compromising protein stability.
"Plotting the binding affinity to ACE2 of selected RBD mutations against their incidence in the population shows a strong correlation between the two", say the authors of this study. "Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity", they add.
In addition, the researchers also appraised the role of high-affinity binder RBD-62 as a potential therapeutic agent and showed it could competently block ACE2 – without influencing its prominent and necessary enzymatic activity.
Intriguing questions and implications
Notwithstanding the fact that natural virus selection is not as efficient a process when compared to in vitro selection, the obtained information on the more critical mutations can be implemented as a tool for quick identification of emerging mutations.
"An intriguing question is whether the spreading of the tighter binding SARS-CoV-2 variants in humans is accidental", say study authors in this bioRxiv paper. "From the similarity to yeast display selection, where stringent conditions are used, one may hypothesize that stringent selection is also driving the rapid spread of these mutations," they add.
The most abundant low-quality face masks would actually provide such selection conditions since they decrease exhaled viral titers, providing tighter binding variants an advantage over wild types for rapid spread in the population.
And this is something that should be urgently looked into, as higher quality face-masks may become mandatory to reduce viral titers below infection levels (which is actually the case with medical personal) and halt the spread of these tighter binding virus mutations.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.