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.

Hexokinase (HK) catalyzes the first step in glucose metabolism, making it an exciting target for the inhibition of tumor initiation and progression due to their elevated glucose metabolism. The upregulation of hexokinase-2 (HK2) in many cancers and its limited expression in normal tissues makes it a particularly attractive target for the selective inhibition of cancer growth and the eradication of tumors with limited side effects. The design of such safe and effective anticancer therapeutics requires the development of HK2-specific inhibitors that will not interfere with other HK isozymes. As HK2 is unique among HKs in having a catalytically active N-terminal domain (NTD), we have focused our attention on this region. We previously found that NTD activity is affected by the size of the linker helix-α13 that connects the N- and C-terminal domains of HK2. Three non-active site residues (D447, S449, and K451) at the beginning of the linker helix-α13 have been found to regulate the NTD activity of HK2. Mutation of these residues led to increased dynamics, as shown via hydrogen-deuterium exchange analysis and molecular dynamic simulations. D447A contributed the most to the enhanced dynamics of the NTD, with reduced calorimetric enthalpy of HK2. Similar residues exist in the C-terminal domain (CTD) but are unnecessary for HK1 and HK2 activity. Thus, we postulate these residues serve as a regulatory site for HK2, and may provide new directions for the design of anticancer therapeutics that reduce the rate of glycolysis in cancer through specific inhibition of HK2.