According to an official statement released on Monday, June 6, researchers at the Indian Institute of Science (IISc) have identified an alternative mechanism to render viruses like SARS-CoV-2 inactive. The researchers revealed the creation of a novel class of manmade peptides or miniproteins that can not only impede virus entry into our cells but also cluster virions (virus particles) together, decreasing their potential to infect, in a paper published in Nature Chemical Biology, as stated in an IANS report.
A protein-protein interaction is frequently compared to a lock and key mechanism. A lab-made miniprotein that mimics, competes with and inhibits the 'key' from binding to the 'lock', or vice versa, can thwart this interaction. The scientists used this strategy in the latest work to create small proteins that bind to and block the spike protein on the surface of the SARS-CoV-2 virus. Cryo-electron microscopy (cryo-EM) and other biophysical approaches were used to further characterise this interaction.
These miniproteins are helical, hairpin-shaped peptides that can form dimers when they couple up with another of their kind. Each dimeric 'bundle' has two 'faces' on which two target molecules can engage. The researchers hypothesised that the two faces would attach to two different target proteins, forming a complex that would lock all four together and prevent the targets from acting. "However, we needed proof of principle," says Jayanta Chatterjee, Associate Professor at IISc's Molecular Biophysics Unit (MBU) and the study's primary author. The researchers decided to put their theory to the test by targeting the interaction between the SARS-CoV-2 spike (S) protein and the ACE2 protein in human cells using one of the miniproteins, dubbed SIH-5.
The S protein is a trimer — a complex of three identical polypeptides. Each polypeptide contains a Receptor Binding Domain (RBD) that binds to the ACE2 receptor on the host cell surface. This interaction facilitates 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 encounters an S protein, one of its faces is bound tightly to one of the three RBDs on the S protein trimer, and the other face is 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," says Chatterjee. "(But) cross-linking of S proteins blocks their action many times more effectively. This is called the avidity effect."
Under cryo-EM, the S proteins targetted 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 I saw a structure that was much more elongated," says Somnath Dutta, Assistant Professor at MBU and one of the corresponding authors. Dutta and the others realised that 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. "I have worked with antibodies raised against the spike protein before and observed them under a cryo-EM. But they never created dimers of the spikes," says Dutta. The miniprotein was also found to be thermostable — it can be stored for months at room temperature without deteriorating.
The next step was to ask if SIH-5 would be useful for preventing COVID-19 infection. To answer this, the team first tested the miniprotein for toxicity in mammalian cells in the lab and found it to be safe. Next, in experiments carried out in the lab of Raghavan Varadarajan, Professor at MBU, hamsters were dosed with the miniprotein, followed by exposure to SARS-CoV-2. These animals showed no weight loss and had greatly decreased viral load as well as much less cell damage in the lungs, compared to hamsters exposed only to the virus. The researchers believe that with minor modifications and peptide engineering, this lab-made miniprotein could inhibit other protein-protein interactions as well.
