Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies

Abstract

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

Mass Spectrometric Assays Reveal Discrepancies in Inhibition Profiles for the SARS‐CoV‐2 Papain‐Like Protease

“…The results reveal the nsp2/3 peptide ( 2 ) is a substantially more efficient PL pro substrate compared to the nsp1/2 ( 1 ) and nsp3/4 ( 3 ) peptides, for which only low levels of hydrolyzed product peptides were observed (Supporting Figure S2). The reduced activity of PL pro with 1 and 3 is consistent with prior reports that the isolated catalytic domain of SARS‐CoV‐2 PL pro itself does not efficiently hydrolyze nsp1/2, but requires the presence of specific non‐catalytic domains present in nsp3, [4a,8a,14] and with similar observations for SARS‐CoV PL pro [15] . The endpoint assays were used to identify preferred reaction conditions suitable for developing a direct SPE‐MS‐based PL pro assay (0.2 μM PL pro and 2.0 μM nsp2/3 peptide 2 in 50 mM Tris, pH 8.0).…”

“…The SPE‐MS inhibition assay was then applied to investigate effects of selected reported PL pro inhibitors on catalysis (Table 2). The results reveal that GRL0617, which was originally developed as a SARS‐CoV PL pro inhibitor in 2008 [7a] and which was subsequently shown to efficiently inhibit SARS‐CoV‐2 PL pro in vitro and in cells by non‐covalent binding at the P3 and P4 pockets as manifested by crystallographic studies, [5a,17b] inhibits PL pro in the SPE‐MS assay with similar potency to that reported using fluorescence‐based assays [4a,5d,20] (IC 50 ∼3.8 μM, Table 2, entry 1). Analysis of the Hill coefficients of the inhibition curves reveals values in the range of −1, as predicted for single molecules competing with the substrate for binding (Figure 3a).…”

“…[ 8a , 10 ] A matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS‐based deubiquitinase assay [11] has been applied to investigate small‐molecules for PL pro inhibition, however, this assay monitors only the deubiquitinase activity of PL pro and requires additional sample manipulation for matrix formation. [4a] Recently, we have reported a solid phase extraction (SPE) coupled to MS assay for SARS‐CoV‐2 M pro which is suitable for screening small‐molecule inhibitors. [12] We now report a high‐throughput SPE‐MS‐based SARS‐CoV‐2 PL pro assay which uses oligopeptides as substrates and which is orthogonal to the typically employed fluorescence‐based PL pro assays.…”

“…They monitor PL pro activity, e. g ., by analyzing the cleavage of C‐ and N‐terminal protein‐tagged peptides using protein mass spectrometry (MS) and/or SDS‐PAGE [8a,10] . A matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS‐based deubiquitinase assay [11] has been applied to investigate small‐molecules for PL pro inhibition, however, this assay monitors only the deubiquitinase activity of PL pro and requires additional sample manipulation for matrix formation [4a] . Recently, we have reported a solid phase extraction (SPE) coupled to MS assay for SARS‐CoV‐2 M pro which is suitable for screening small‐molecule inhibitors [12] .…”

Precursors of Viral Proteases as Distinct Drug Targets

“…Allosteric modulators and other compounds with alternative mechanisms of action could be more suitable to avoid off-target binding. Yeast-surface display nanobodies (a single-domain antibody) were developed as potential inhibitors of PLpro [ 251 ]. This strategy represents an interesting alternative to classical inhibitors.…”