Capillary electrokinetic chromatography for chiral separation of potential SARS-CoV-2 3CL protease inhibitors.

In this work, a capillary electrophoresis method was developed as a quality control tool to determine the enantiomeric purity of a series of five chiral compounds evaluated as potential severe acute respiratory syndrome coronavirus 2 3CL protease inhibitors. The first cyclodextrin tested, that is, highly sulfated-ß-cyclodextrin, at 6% (m/v) in a 25 mM phosphate buffer, using a capillary dynamically coated with polyethylene oxide, at an applied voltage of 15 kV and a temperature of 25°C, was found to successfully separate the five derivatives. The limits of detection and quantification were calculated together with the greenness score of the method in order to evaluate the method in terms of analytical and environmental performance. In addition, it is noteworthy that simultaneously high-performance liquid chromatography separation of the enantiomers of the same compounds with two different columns, the amylose tris(3,5-dimethylphenylcarbamate)-coated and the cellulose tris(3,5-dichlorophenylcarbamate)-immobilized on silica stationary phases, was studied. Neither the former stationary phase nor the latter was able to separate all derivatives in a mobile phase consisting of n-heptane/propan-2-ol 80/20 (v/v).

NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment.

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.

N-acylbenzimidazoles as selective Acylators of the catalytic cystein of the coronavirus 3CL protease.

The 3CL protease (3CLpro, Mpro) plays a key role in the replication of the SARS-CoV-2 and was validated as therapeutic target by the development and approval of specific antiviral drugs (nirmatrelvir, ensitrelvir), inhibitors of this protease. Moreover, its high conservation within the coronavirus family renders it an attractive therapeutic target for the development of anti-coronavirus compounds with broad spectrum activity to control COVID-19 and future coronavirus diseases. Here we report on the design, synthesis and structure-activity relationships of a new series of small covalent reversible inhibitors of the SARS-CoV-2 3CLpro. As elucidated thanks to the X-Ray structure of some inhibitors with the 3CLpro, the mode of inhibition involves acylation of the thiol of the catalytic cysteine. The synthesis of 60 analogs led to the identification of compound 56 that inhibits the SARS-CoV-2 3CLpro with high potency (IC50 = 70 nM) and displays antiviral activity in cells (EC50 = 3.1 µM). Notably, compound 56 inhibits the 3CLpro of three other human coronaviruses and exhibit a good selectivity against two human cysteine proteases. These results demonstrate the potential of this electrophilic N-acylbenzimidazole series as a basis for further optimization.

Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses.

Since end of 2019, the global and unprecedented outbreak caused by the coronavirus SARS-CoV-2 led to dramatic numbers of infections and deaths worldwide. SARS-CoV-2 produces two large viral polyproteins which are cleaved by two cysteine proteases encoded by the virus, the 3CL protease (3CLpro) and the papain-like protease, to generate non-structural proteins essential for the virus life cycle. Both proteases are recognized as promising drug targets for the development of anti-coronavirus chemotherapy. Aiming at identifying broad spectrum agents for the treatment of COVID-19 but also to fight emergent coronaviruses, we focused on 3CLpro that is well conserved within this viral family. Here we present a high-throughput screening of more than 89,000 small molecules that led to the identification of a new chemotype, potent inhibitor of the SARS-CoV-2 3CLpro. The mechanism of inhibition, the interaction with the protease using NMR and X-Ray, the specificity against host cysteine proteases and promising antiviral properties in cells are reported.