Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives

Abstract

The SARS-CoV-2 pandemic has triggered global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro), critical for viral replication, is a key target for therapeutic development. An organoselenium drug called ebselen has been demonstrated to have potent Mpro inhibition and antiviral activity. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of derivatives. A free selenium atom bound with cysteine of catalytic dyad has been revealed in crystallographic structures of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct and formation of a phenolic by-product, confirmed by mass spectrometry. The target engagement with selenation mechanism of inhibition suggests wider therapeutic applications of these compounds against SARS-CoV-2 and other zoonotic beta-corona viruses.

Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays

“… N.A. Not a M pro inhibitor Hits from drug repurposing Calpain inhibitor II 0.97 ± 0.27 2 8.98 ± 2.0 26 6.65 2 >60 0.60 ± 0.11 2.07 ± 0.76 2 27 ± 1.4 26 6XA4 3 Validated M pro Inhibitor Cell-type dependent Calpain inhibitor XII 0.45 ± 0.06 2 6.48 ± 3.4 26 7.86 2 38.71 ± 5.66 0.79 ± 0.10 0.49 ± 0.18 2 1.3 ± 0.57 26 6XBH 3 Validated M pro Inhibitor Cell-type dependent Ebselen >60 41 0.67 ± 0.09 16 , 42 >100 26 0.14 41 >60 >60 4.67 ± 0.80 16 , 42 >100 26 7BAK 42 Not a M pro inhibitor Disulfiram >60 41 9.35 ± 0.18 16 0.21 41 >60 >240 not active 16 N.A. Not a M pro inhibitor Carmofur 28.2 ± 9.5 41 1.82 ± 0.06 16 >1...…”

“…Despite the accumulating evidence to support the promiscuous mechanism of action of ebselen, several studies continue to explore ebselen and its analogs as SARS-CoV-2 M pro and PL pro inhibitors 42 , 61 , 62 . A number of ebselen analogs were designed and found to have comparable enzymatic inhibition and antiviral activity as ebselen.…”

“…X-ray crystallization of SARS-CoV-2 M pro with MR6-31-2 (PDB: 7BAL) and ebselen (PDB: 7BAK) revealed nearly identical complex structures. It was found that selenium coordinates directly to Cys145 and forms a S–Se bond 42 . Accordingly, a mechanism involving hydrolysis of the organoselenium compounds was proposed.…”

Discovery and Mechanism of SARS-CoV-2 Main Protease Inhibitors

“…Binding is further stabilized by a hydrogen bond between the carbonyl oxygen atom of ebselen and Glu 166. These results are consistent with a recently reported crystal structure of SARS-CoV-2 M pro in complex with ebselen, which indicates that it primarily binds to the catalytic site of the enzyme and forms a selenyl sulfide bond with Cys 145; this was further confirmed by LC/MRM-MS. 24 …”

Ebselen and Analogues: Pharmacological Properties and Synthetic Strategies for Their Preparation

“…At the time the present review article was almost complete, a very important paper was published by Yang and coworkers who, in an attempt to obtain co-crystal structures between ebselen, its analogues and M pro , found that after protein/small molecule incubation only the selenium atom is present in the enzyme active site, suggesting the mechanism summarized in Scheme 5 . In particular, once the selenenyl sulfide intermediate is formed, it reacts through a SNAr-like mechanism with water, leading to the formation of 2-hydroxy- N -phenylbenzamide and the selenenylated Cys145 [ 54 ].…”