Evaluating the effect of a new antiviral compound on the interaction between ACE2 and the SARS-CoV-2 spike protein
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- The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a single-stranded RNA virus that emerged in December 2019 from Wuhan city (China). Since then, the epidemic caused by its spread has impacted the whole world and an impressive number of variants emerged of which seven have been considered as Variant of concern (VOC) by the World Health Organization. The illness caused by SARS-CoV-2 is the coronavirus disease 2019 (COVID-19) and has officially caused the death of almost 7 million people. In a race against time, vaccine development has accelerated with the first large-scale production for human of RNA vaccines. However, other antiviral compounds have also been studied as concerns raised about vaccine protection against VOCs given the impressive number of mutations they carry. A better understanding of how SARS-CoV-2 enters the host cell to establish its infection and proliferation is key to developing our knowledge of this family of viruses that has already triggered epidemics twice since the beginning of the 21th century. In order to spread, viruses need to enter a host cell and hijack their cellular machinery to replicate themselves. The SARS-CoV-2 virus enters host cells through interactions between its spike glycoprotein (S), divided into two sub-units (S1-S2), and host cell surface molecules. The main host cell surface molecule interacting with S is the angiotensin-converting enzyme 2 (ACE2) receptor although other functional receptors have been described for SARS-CoV-2. Therefore, several parties are involved in mediating the entry of SARS-CoV-2 into the host cell which means that many targets exist for the development of antiviral compounds. In this context, recent work has identified two lactam molecules, Lactavir and pyroglutamate, as compounds with antiviral effects on SARS-CoV-2 infection. Moreover, these molecules are similar to the pyroglutamyl function present on many extracellular proteins following the cyclisation of an N-terminal glutamine, including the S protein and the ACE2 receptor. The work carried out during this thesis focused on identifying the mechanism of action of Lactavir and its effect both on in vitro interactions between S1 and ACE2, and on the in cellular infectivity of Spike pseudotyped Vesicular Stomatitis Viruses (S*VSVs) in a human cell line (A549). Also, we aimed at gaining some insights into the importance of the N-terminal pyroglutamate (pGlu) function present on the S protein for the S1-ACE2 interaction. Concerning the in vitro experiments, we verified the sequence of two commercial proteins by mass spectrometry and confirmed the sequence of S1(Val) but established that the sequence given for the S1(pGlu) protein was truncated. By using BioLayer Interferometry (BLI), we found that the addition of Lactavir has no impact on the interaction between S1 and ACE2 regardless the S1 used. Also, we measured that the presence or absence of the N-ter pGlu residue on S1 has no impact on the affinity constant of the S1-ACE2 interaction. We highlight also that Lactavir does not act as an inhibitor on ACE2 enzymatic activity. For the in cellular experiments, we improved the VSV production and we determined that there are no significant effects of Lactavir on the infectivity of VSVs pseudotyped with the S from the Wuhan strain. Preliminary results with a VOC (Delta) were obtained but no conclusions can be drawn for these. Overall, this thesis has improved our knowledge concerning the impact of Lactavir on the Spike-ACE2 interaction. These results can be used for further research to identify and characterize antiviral compounds.