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The Indian Institute of Science (IISc) scientists have designed a new class of artificial peptides or miniproteins that provides an alternative mechanism to render viruses like SARS-CoV-2 inactive. The miniprotein was also found to be stable for months at room temperature without deteriorating.
To test if SIH-5 would be useful for preventing Covid-19 infection, the team first tested the miniprotein for toxicity in mammalian cells in the lab and found it to be safe. They noted that the protein-protein interaction is often like that of a lock and a key. It can be hampered by a lab-made miniprotein that mimics, competes with, and prevents the 'key' from binding to the 'lock', or vice versa, they said.
The team used this approach to design miniproteins that can bind to, and block the spike protein on the surface of the SARS-CoV-2 virus, which helps it to enter and infect the human cells. In a study published in Nature Chemical Biology, the researchers indicated that miniproteins that can not only block virus entry into cells but also reduce the ability to infect.
The researchers said that the binding was further characterised extensively by cryo-electron microscopy (cryo-EM) and other biophysical methods.
These miniproteins are helical, hairpin-shaped peptides, each capable of pairing up with another of its kind, forming what is known as a dimer. Each dimeric ‘bundle’ presents two faces to interact with two target molecules.
The researchers assumed that the two faces would bind to two separate target proteins locking all four in a complex and blocking the targets’ action. "But we needed proof of principle," said Jayanta Chatterjee, Associate Professor, Molecular Biophysics Unit (MBU), IISc, and the lead author of the study.
The team decided to test their hypothesis by using one of the miniproteins called SIH-5 to target the interaction between the spike protein of SARS-CoV-2 and ACE2 protein in human cells.
The spike protein is a complex of three identical polypeptides, each of which contains a Receptor Binding Domain (RBD) that binds to the ACE2 receptor on the host cell surface, facilitating viral entry into the cell.
The SIH-5 miniprotein was designed to block the binding of the RBD to human ACE2. When a SIH-5 dimer encountered an S protein, one of its faces bound tightly to one of the three RBDs on the S protein trimer, and the other face bound to an RBD from a different S protein. This ‘cross-linking’ allowed the miniprotein to block both S proteins at the same time. "Several monomers can block their targets," said Chatterjee.
Under cryo-EM, the S proteins targeted by SIH-5 appeared to be attached head-to-head. “We expected to see a complex of one spike trimer with SIH-5 peptides. But spotted a structure that was much more elongated,” said Somnath Dutta, Assistant Professor, MBU and a corresponding authors of the study.
The spike proteins were being forced to form dimers and clumped into complexes with the miniprotein. This type of clumping can simultaneously inactivate multiple spike proteins of the same virus and even multiple virus particles, stated Dutta.
The other experiments carried out at lab of Raghavan Varadarajan, Professor, MBU, hamsters were dosed with the miniprotein, followed by exposure to SARS-CoV-2. These animals showed no weight loss and indicated to have lower viral load, much less cell damage in the lungs, compared to hamsters exposed only to the virus.
The team of researchers concluded that with a bit of modifications and peptide engineering, the lab-which developed the miniprotein could inhibit other protein-protein interactions.
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