Synthesis of SARS-CoV-2 Mpro inhibitors bearing a cinnamic ester warhead with in vitro activity against human coronaviruses.

COVID-19 now ranks among the most devastating global pandemics in history. The causative virus, SARS-CoV-2, is a new human coronavirus (hCoV) that spreads among humans and animals. Great efforts have been made to develop therapeutic agents to treat COVID-19, and among the available viral molecular targets, the cysteine protease SARS-CoV-2 Mpro is considered the most appealing one due to its essential role in viral replication. However, the inhibition of Mpro activity is an interesting challenge and several small molecules and peptidomimetics have been synthesized for this purpose. In this work, the Michael acceptor cinnamic ester was employed as an electrophilic warhead for the covalent inhibition of Mpro by endowing some peptidomimetic derivatives with such a functionality. Among the synthesized compounds, the indole-based inhibitors 17 and 18 efficiently impaired the in vitro replication of beta hCoV-OC-43 in the low micromolar range (EC50 = 9.14 µM and 10.1 µM, respectively). Moreover, the carbamate derivative 12 showed an antiviral activity of note (EC50 = 5.27 µM) against another hCoV, namely hCoV-229E, thus suggesting the potential applicability of such cinnamic pseudopeptides also against human alpha CoVs. Taken together, these results support the feasibility of considering the cinnamic framework for the development of new Mpro inhibitors endowed with antiviral activity against human coronaviruses.

Discovery of a Novel Trifluoromethyl Diazirine Inhibitor of SARS-CoV-2 Mpro.

SARS-CoV-2 Mpro is a chymotrypsin-like cysteine protease playing a relevant role during the replication and infectivity of SARS-CoV-2, the coronavirus responsible for COVID-19. The binding site of Mpro is characterized by the presence of a catalytic Cys145 which carries out the hydrolytic activity of the enzyme. As a consequence, several Mpro inhibitors have been proposed to date in order to fight the COVID-19 pandemic. In our work, we designed, synthesized and biologically evaluated MPD112, a novel inhibitor of SARS-CoV-2 Mpro bearing a trifluoromethyl diazirine moiety. MPD112 displayed in vitro inhibition activity against SARS-CoV-2 Mpro at a low micromolar level (IC50 = 4.1 µM) in a FRET-based assay. Moreover, an inhibition assay against PLpro revealed lack of inhibition, assuring the selectivity of the compound for the Mpro. Furthermore, the target compound MPD112 was docked within the binding site of the enzyme to predict the established intermolecular interactions in silico. MPD112 was subsequently tested on the HCT-8 cell line to evaluate its effect on human cells' viability, displaying good tolerability, demonstrating the promising biological compatibility and activity of a trifluoromethyl diazirine moiety in the design and development of SARS-CoV-2 Mpro binders.

Considerations around the SARS-CoV-2 Spike Protein with Particular Attention to COVID-19 Brain Infection and Neurological Symptoms.

Spike protein (S protein) is the virus "key" to infect cells and is able to strongly bind to the human angiotensin-converting enzyme2 (ACE2), as has been reported. In fact, Spike structure and function is known to be highly important for cell infection as well as for entering the brain. Growing evidence indicates that different types of coronaviruses not only affect the respiratory system, but they might also invade the central nervous system (CNS). However, very little evidence has been so far reported on the presence of COVID-19 in the brain, and the potential exploitation, by this virus, of the lung to brain axis to reach neurons has not been completely understood. In this Article, we assessed the SARS-CoV and SARS-CoV-2 Spike protein sequence, structure, and electrostatic potential using computational approaches. Our results showed that the S proteins of SARS-CoV-2 and SARS-CoV are highly similar, sharing a sequence identity of 77%. In addition, we found that the SARS-CoV-2 S protein is slightly more positively charged than that of SARS-CoV since it contains four more positively charged residues and five less negatively charged residues which may lead to an increased affinity to bind to negatively charged regions of other molecules through nonspecific and specific interactions. Analysis the S protein binding to the host ACE2 receptor showed a 30% higher binding energy for SARS-CoV-2 than for the SARS-CoV S protein. These results might be useful for understanding the mechanism of cell entry, blood-brain barrier crossing, and clinical features related to the CNS infection by SARS-CoV-2.