Structural and Biochemical Analysis of the Dual Inhibition of MG-132 against SARS-CoV-2 Main Protease (Mpro/3CLpro) and Human Cathepsin-L

Abstract

After almost two years from its first evidence, the COVID-19 pandemic continues to afflict people worldwide, highlighting the need for multiple antiviral strategies. SARS-CoV-2 main protease (Mpro/3CLpro) is a recognized promising target for the development of effective drugs. Because single target inhibition might not be sufficient to block SARS-CoV-2 infection and replication, multi enzymatic-based therapies may provide a better strategy. Here we present a structural and biochemical characterization of the binding mode of MG-132 to both the main protease of SARS-CoV-2, and to the human Cathepsin-L, suggesting thus an interesting scaffold for the development of double-inhibitors. X-ray diffraction data show that MG-132 well fits into the Mpro active site, forming a covalent bond with Cys145 independently from reducing agents and crystallization conditions. Docking of MG-132 into Cathepsin-L well-matches with a covalent binding to the catalytic cysteine. Accordingly, MG-132 inhibits Cathepsin-L with nanomolar potency and reversibly inhibits Mpro with micromolar potency, but with a prolonged residency time. We compared the apo and MG-132-inhibited structures of Mpro solved in different space groups and we identified a new apo structure that features several similarities with the inhibited ones, offering interesting perspectives for future drug design and in silico efforts.

Gain-of-Signal Assays for Probing Inhibition of SARS-CoV-2 M pro /3CL pro in Living Cells

“…Biochemical activity of M pro was analyzed using a quenched fluorescent peptide substrate DABCYL-KTSAVLQ↓SGFRKM-EDANS (Bachem; catalog no. 4045664), which has been used in multiple recent studies ( 33 , 34 , 60 ). M pro cleavage between Gln and Ser liberates fluorescence, which was quantified by excitation and emission at 350 and 490 nm, respectively.…”

In Silico Drug Repositioning to Target the SARS-CoV-2 Main Protease as Covalent Inhibitors Employing a Combined Structure-Based Virtual Screening Strategy of Pharmacophore Models and Covalent Docking

“…Therefore, these types of enzymes that work in a proteolytic network are proposed as therapeutic targets to reduce the rate of viral infection. Even in this sense, inhibitors with Cathepsin/Mpro dual activity have been proposed, such as Calpain Inhibitor I and II [ 71 ], which were identified in our virtual screening data ( Figure 5 L, entry L in Table 2 ); recently, the dual activity of the inhibitor M-132 (Cbz-Leu-Leu-Leu-al) was also reported [ 72 ].…”

Potential drug discovery for COVID-19 treatment targeting Cathepsin L using a deep learning-based strategy

“…Based on the predicted score ranking and availability, we selected 50 molecules for experimental validation and eventually identified 12 compounds with more than 50% inhibition against CTSL activity at 100 μM in a cell-free system, five of which, Mg-132, Z-FA-FMK, Leupeptin Hemisulfate, Mg-101 and Calpeptin, were able to exert inhibition at nanomolar concentrations. It is reported that proteasome inhibitor, Mg-132, can also cross-inhibit CTSL [51] , [52] . Cysteine protease inhibitors, Z-FA-FMK, Leupeptin Hemisulfate, MG-101 and calpain inhibitor, calpeptin were also reported to inhibit CTSL [53] , [54] , [55] , [56] , [57] , [58] , [59] , [60] , [61] .…”