De novo protein decoys that block SARS-CoV-2
With the current COVID-19 pandemic having already claimed hundreds of thousands of lives worldwide, scientists are working to bring out effective antiviral strategies that can be quickly deployed, not only for the current scenario but to counter future threats.
Now, a new study published on the preprint server bioRxiv* in August 2020 shows that computational design technology can be efficiently harnessed towards this end, producing new molecules to inhibit viral entry and replication. The researchers developed protein mimics that block the virus attachment to the cell receptor by acting as decoys and prevent viral escape via new mutations.
The Principle
The current study is based on the principle of designing a protein surface that is identical to that cell surface targeted by the SARS-CoV-2 virus but with a higher binding affinity for the viral RBD than the host cell. This will also need to inhibit viral attachment to the cell. The end result will be that these “de novo designer proteins can outcompete the viral interaction and act as neutralizing agents.” Moreover, the very design of the decoy ensures that mutational escape is impossible since any mutation that weakens viral binding to the decoy will automatically also weaken its ability to bind to the receptor surface of which the decoy is a copy.
The Process
The scientists first identified the characteristic repeating structures that make up the protein surface
actually bound to by the viral RBD. This is based on three structures made publicly available, reflecting
the possible structure of the RBD-ACE2 complex for SARS-CoV-1 and SARS-CoV-2. They picked out
the four structural components that make up the binding interface of the ACE2 protein and built the de
novo decoy with three of them – two long alpha-helices and one short beta-hairpin. They avoided any
part of the biologically active part of the molecule, such as the enzymatic catalytic site or the site of interaction with the cell membrane.
The target elements were then mounted on a new standalone support structure that allows proper folding
of the binding site into the globular shape required and stabilizes the interface. The Rosetta software was
used to develop about 35,000 fully connected protein topologies with all these elements, without altering
even the conformation of the amino acids of the target binding interface. The amino acid sequences
were then generated such that they could fold into the target structures. The designs were then evaluated
using an automatic filter, and the top 196 were tested for binding to the virus. They found that one
molecule, which they named CTC-445.2d (ConquerTheCorona445.2duo), had a nanomolar and very
specific affinity of binding to the SARS-CoV-2 RBD, but failed to inhibit its binding to ACE2.
The researchers then carried out targeted mutagenesis to incorporate a planned combination of the most
useful mutations found to stabilize the protein and further enhance its affinity of binding. This resulted in
CTC-445.2, with five substituted amino acids, none at the binding interface. This was found to be very
stable and a potent inhibitor of infection in vitro. Finally, they performed a domain duplication to make
it bivalent. This step resulted in CTC-445.2d, with tenfold binding affinity and 100 times the neutralizing capacity of the earlier version of the protein.
The Protein
CTC-445.2d is a 160 amino acid protein which binds the SARS-CoV-2 RBD at nanomolar
concentrations, is very stable, and is also reactive for SARS-CoV-1 RBD. Though it is such a
strong competitor of viral infection, cell viability remains unaffected as does the enzymatic activity
of ACE2. Complete neutralization of viral infection was shown in three in vitro systems, including in
a lung epithelial cell-derived system Calu-3.
Further, its efficacy was greatest when present throughout the course of infection, that is, when it was
incubated along with the virus and in cell media, indicating that it inhibited the infection by extracellular
neutralization. The researchers also instilled a high dose of the protein intranasally to an experimental
mouse model, and found that the molecule could be found in a fully functional form for over 24 hours,
and also detectable in blood. This points to the possibility of systemic exposure as well at some level.
The Advantages
The use of soluble decoy proteins is quite different from that of vaccines, small molecule inhibitors or
neutralizing antibodies, and overcomes the problem of mutational escape. These molecules also avoid
the stability and unwanted biological effects, as well as potential autoimmunity, implicit in the use of
natural proteins. Being quite different from natural proteins in their amino acid sequence and structure,
they are unlikely to elicit autoimmune responses.
These small molecules are easy to produce at large scale in available bacterial systems, are stable over
a wide range of conditions, and can be further refined to increase their potential for affinity and
neutralization potency. The decoys are functionally resilient to viral escape mutations as well. The
underlying principle is validated by the cross-reactivity of the decoy proteins with SARS-CoV-1,
because the differences in the RBD of the two viruses involve the binding interface at least at 17 sites
but fail to prevent neutralization of both by the same protein.
The researchers say, “Using our novel platform, in less than ten weeks, we engineered, validated, and*Important Notice
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.