Therapeutic potential of metal ions for COVID-19: insights from the papain-like protease of SARS-CoV-2

Abstract

Coronaviruses have been responsible for multiple challenging global pandemics, including coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Papain-like protease (PLpro), one of two cysteine proteases responsible for the maturation and infectivity of SARS-CoV-2, processes and liberates functional proteins from the viral polyproteins and cleaves ubiquitin and ISG15 modifications to inhibit innate immune sensing. Consequently, PLpro is an attractive target for developing COVID-19 therapies. PLpro contains a zinc-finger domain important for substrate binding and structural stability. However, the impact of metal ions on the activity and biophysical properties of SARS-CoV-2 PLpro has not been comprehensively studied. Here, we assessed the impacts of metal ions on the catalytic activity of PLpro. Zinc had the largest inhibitory effect on PLpro, followed by manganese. Calcium, magnesium, and iron had smaller or no effects on PLpro activity. EDTA at a concentration of 0.5 mM was essential for PLpro activity, likely by chelating trace metals that inhibit PLpro. IC50 values for ZnCl2, ZnSO4, and MnCl2 of 0.42 ± 0.02 mM, 0.35 ± 0.01 mM, and 2.6 ± 0.3 mM were obtained in the presence of 0.5 mM EDTA; in the absence of EDTA, the estimated IC50 of ZnCl2 was 14 µM. Tryptophan intrinsic fluorescence analysis confirmed the binding of zinc and manganese to PLpro, and differential scanning calorimetry revealed that zinc but not manganese reduced ΔHcal of PLpro. The results of this study provide a reference for further work targeting PLpro to prevent and treat COVID-19.

Identification of Novel Allosteric Sites of SARS-CoV-2 Papain-Like Protease (PLpro) for the Development of COVID-19 Antivirals

“…The protein concentration and purity were determined by the Bradford assay 51 and SDS-PAGE, respectively. PLpro enzyme titration and initial velocity analyses were performed using a Cytation 5 multimode microplate reader (BIOTEK Instruments, Winooski, VT) as described previously 15 . The peptide substrate contained the PLpro cleavage site Leu-Arg-Gly-Gly flanked by a fluorescent AMC (7-amino-4-methyl coumarin) tag and corresponding quencher CBZ (carbobenzoxy) (i.e., CBZ-LRGG-AMC).…”

“…The peptide substrate contained the PLpro cleavage site Leu-Arg-Gly-Gly flanked by a fluorescent AMC (7-amino-4-methyl coumarin) tag and corresponding quencher CBZ (carbobenzoxy) (i.e., CBZ-LRGG-AMC). The proteolytic reaction was initiated by adding PLpro to the peptide substrate in buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, and 1 mM EDTA) containing 2% DMSO (v/v) to enhance the solubility and solubility of the peptide 15 . The PLpro reaction was monitored in a 96-well microplate at 25 °C for 10 min at excitation and emission wavelengths of 340 nm and 487 nm, respectively.…”

“…Studying the catalytic function and structural stability of PLpro may provide a foundation for developing novel drugs that target viral infection and the immune evasion machinery of multiple coronaviruses, such as SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV. 14,15 Structural and mutagenesis studies have provided insights into the domains and residues responsible for the catalytic function and stability of PLpro [16][17][18] . The tertiary structure of the SARS-CoV-2 PLpro monomer adopts a conformation resembling an extended right-handed architecture and comprises two main domains: the ubiquitin-like (Ubl) domain and the catalytic domain [16][17][18][19] .…”

“…Several repurposed protease inhibitors with promising effects on PLpro in vivo or in vitro, such as simeprevir, tanshinones, famotidine, and ebselen, are either clinically ineffective or still in clinical trials [32][33][34][35][36] . Another class of inhibitors of interest against PLpro is FDA-approved zinc-ejecting drugs 15,17,37,38 . These drugs target the zinc-finger motif common to metalloenzymes such as PLpro and displace zinc from the zinc-cysteine tetrahedral complex.…”

“…Studying the catalytic function and structural stability of PLpro may provide a foundation for developing novel drugs that target viral infection and the immune evasion machinery of multiple coronaviruses, such as SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV. 14, 15…”