Dottorato in BIOLOGIA UMANA E GENETICA MEDICA

Titolo della tesi: Temporal reconstruction of the interaction interface between SARS-CoV-2 Spike glycoprotein and human ACE2 and assessment of the functionality of key-player mutant amino acids

Since its first outbreak in Wuhan, China, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread across the world as a tremendous pandemic, claiming more than 5 million lives and probably infecting more than 400 million people. Today, thanks to the efforts of the worldwide scientific community, we have several therapeutic approaches and a formidable toolkit of vaccines available. However, vaccine hesitancy and the current spread of viral variants of concern (VoCs) demonstrate that the threat is far from over. Most current treatments focus on the SARS-CoV-2 Spike glycoprotein, which is responsible for the viral entry into host cells using the ACE2 receptor as a first binding site. The use of recombinant ACE2 decoys represents another promising approach, not only for their natural potency in the virus recognition but also because the virus may not escape the ACE2-mediated neutralization without a simultaneous decrease in the affinity for the native ACE2. However, their design is complex, with hundreds of thousands of residue-wise modifications possible. Here, we developed an advanced computational workflow with the double aim of explaining, at the atomic level, the molecular mechanisms of SARS-CoV-2 infection and, thus, guide a rational design of a new potent ACE2 decoy able to act as a competitive kidnapper of SARS-CoV-2. Our approach grounds on the unusual research workflow of first generating hypotheses in-silico and, then, providing the best performing ACE2 mutants to further validate experimentally. Hence, we have simulated and explored the whole recognition pathway of SARS-CoV-2 and ACE2 using enhanced Molecular Dynamics (MD) techniques. We have traced the occurrence of multiple intermediate states that chronologically anticipate the experimental high-resolution bound complex and, thus, evaluated the role of each transient interaction in the formation of the final binding interface. We could then spot the most critical ACE2 residues, design and simulate the most effective set of mutants in terms of enhanced binding affinity, and finally compare them with a competitor decoy and some widespread viral mutants.