Abstract
MS is a powerful methodology for chemical screening to directly quantify substrates and products of enzymes, but its low throughput has been an issue. Recently, an acoustic liquid-handling apparatus (Echo®) used for rapid nano-dispensing has been coupled to a high-sensitivity mass spectrometer to create the Echo® MS system, and we applied this system to screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease inhibitors. Primary screening of 32033 chemical samples was completed in 12 h. Among the hits showing selective, dose-dependent 3CL-inhibitory activity, 8 compounds showed antiviral activity in cell-based assay.
Introduction
Worldwide efforts are underway to find therapeutic agents for coronavirus disease 2019 (COVID-19). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease, which contributes to viral replication, is considered a target molecule for antiviral drugs.1,2) In order to find inhibitors, virtual screening3–5) and fluorogenic enzymatic assays6,7) have been carried out, resulting in the discovery of several candidate drugs, most of which are still under investigation for in vivo efficacy against SARS-CoV-2. Cell-based phenotypic assays have also resulted in the identification of some natural compounds as candidate antiviral drugs.8) Oridonin, which had been reported as a covalent NLRP3 inhibitor with strong anti-inflammasome activity,9) was proposed to be a potential antiviral drug, although its target molecule was not established. As of this writing, there is no specific oral antiviral drug globally approved for clinical use.
Chemical screening based on MS has the advantage that the target analyte can be detected without labeling, but throughput is limited by the time and effort required for the clean-up process prior to MS, using LC or other methods. The acoustic droplet ejection technique itself is familiar in the screening field as a method for dispensing nanoliter amounts of chemical compounds onto assay plates, using acoustic waves to precisely transfer samples in a non-contact manner. This technology is also used as a means of transferring samples in assay plates to a mass spectrometer. Acoustic ejection mass spectrometry (AEMS) is a combination of acoustic droplet ejection, a transfer method involving solvent flow in a coaxial tube, and electrospray ionization (ESI)-MS/MS analysis, and is commercially available as the Echo® MS system. In this system, the reaction mixture is introduced from each well of the microplate directly into the MS, allowing quantification of the target component without pretreatment on a timescale of less than one second per well.10) It is as simple as mix-and-read assays based on fluorescence or luminescence, and has the advantage of being label-free.
We applied this system to screening of 32033 diverse samples to find new inhibitors of the SARS-CoV-2 3CL protease.
Results and Discussion
The 3CL protease assays were performed in 1536-well AEMS dedicated plates at room temperature. Dimethyl sulfoxide solutions of test compounds (2 mM; 10 nL/well) were dispensed in advance, and the reaction was initiated by adding 2 µL of peptide substrate (final 20 µM), followed by 2 µL of protease (final 0.1 µM). The plates were incubated for 16 h in a humidity box containing a wet towel, and the protease reaction was stopped by addition of 1 µL of formic acid (final 0.1%).
Parameters for MS measurements are summarized in Table 1. MS conditions were generated semi-automatically by means of the “MRM Method” on the SCIEX OS for the protease-substrate peptides and the product peptide. Acoustic ejection (AE) conditions, such as mobile phase flow rate and composition, sample injection interval, and injection volume, were optimized to obtain good peak shapes. The peak shapes and intensity were quite sensitive to AE conditions and assay buffer components. The assay buffer components were optimized in terms of detection sensitivity and MS signal stability, and were set to 25 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) pH 8.0, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.005% Tween20, and 25 µM reduced glutathione. The effect of assay buffer components, especially Tris salt, on ion suppression in the ESI process is significant, so HEPES salt, which has marginally less impact, was used (Supplementary Fig. S1). The amount of Tris salt carried from the protease solution was only about 10 µM and had little effect on the detection, so the protease was used without changing the buffer. The stability of the analytes was checked overnight in the reaction stop solution (Supplementary Fig. S2). The detection limit of the product was about 80 nM. The assay conditions, such as the concentrations of 3CL protease and substrate, and the duration of the reaction, were determined so that the protease reaction progressed by about 30% and remained in the linear range (Supplementary Fig. S3). After 1-min centrifugation at 240 × g, the sample plates were loaded on the TEMPO™-controlled ACCESS™ Laboratory Workstation linked with the AEMS, which executed a series of operations such as peeling off the plate seal, and loading and unloading the plates.
Table 1. Echo
® MS Parameters
| Analyte | MRM transition | Dwell | DP | EP | CE | CXP |
|---|
| Q1 (m/z) | Q3 (m/z) | Time (ms) | (V) | (V) | (V) | (V) |
|---|
| Substrate | 523.9 | 721.3 | 33 | 70 | 10 | 32 | 44 |
| Product | 472.2 | 326.2 | 33 | 40 | 10 | 21 | 21 |
| Promenton | 226 | 142 | 33 | 51 | 10 | 29 | 16 |
| MS conditions | GS1 90 psi, GS2 50 psi, Curtain gas 20 psi, CAD gas 9 psi, Temp. 300 °C |
| Mobile phase | 0.1% Formic acid in acetonitrile : methanol = 1 : 1, Flow rate: 420 µL/min |
| AE conditions | Normal mode, Additional time = 0 ms, Injection volume = 2.5 nL |
MRM: Multiple reaction monitoring, DP: declustering potential, EP: entrance potential, CE: collision energy, CXP: collision cell exit potential, GS1: nebulizer gas, GS2: heater gas, CAD gas: collisionally activated dissociation gas.
The screening results are summarized in Fig. 1. Screening of 32033 compounds was completed in about 12 h. A scatterplot of the primary screening results is shown in Supplementary Figure S4. From the primary screening, 242 compounds were selected with an inhibition rate of 49% or higher. Confirmation and dose-dependency assay, also using AEMS, yielded 24 compounds exhibiting dose-dependent inhibition. A small number of false positive compounds were generated. They might be due to problems in mixing the compounds or dispensing the enzyme solution.
Of these, 23 compounds remained after enzyme specificity testing by means of fluorescence assay using another cysteine protease, recombinant human cathepsin L (rh-cathepsin L), and its fluorescent substrate, N-carbobenzyloxy-Leu-Arg-7-amino-4-methylcoumarin (Z-LR-AMC; R&D Systems, Minneapolis, MN, U.S.A.). The assay was performed in 10 µL volume in 384-well plates at room temperature. Final concentrations of 40 µM fluorescent substrate and 0.01 ng/µL rh-cathepsin L were added to the plate containing test compounds, and the fluorescence intensity (Ex. 350 nm/Em. 450 nm) was measured after 90 min. Except for one compound, none of the candidates showed rh-cathepsin L inhibition of more than 15% at 5 µM.
Next, the compounds were incubated with VeroE6/TMPRSS2 cells infected with the SARS-CoV-2 WK-521 strain for virus inhibition assay. The assay indicated that 8 compounds, listed in Fig. 2, were effective. Their dose–response curves are shown in Supplementary Figure S5. Cytotoxicity was tested in the same way using virus-uninfected cells. The 50% cell death-inhibitory concentration (EC50) of virus-infected cells and the 50% cell death concentration (CC50) of virus-uninfected cells were calculated. The results are summarized in Table 2. Oridonin was included among these compounds, so the target molecule for its reported antiviral effect might be 3CL protease. Fluorescent compounds, such as DDI-6 and ZM39923, could be also identified as inhibitors, because the assay is not based on conventional fluorescence methods.
Table 2. Potency of SARS-CoV-2 3CL Protease Inhibitors
| Compound name | Enzyme inhibitory activity | Virus inhibitory activity | Cytotoxicity |
|---|
| IC50 (µM) | EC50 (µM) | CC50 (µM) |
|---|
| Oridonin | 3.2 | 0.6 | 6.9 |
| DDI-2 | 3.4 | 1.2 | >10.0 |
| Ebselen | <0.25 | 2.1 | 9.1 |
| DDI-4 | 3.2 | 2.2 | 6.4 |
| DDI-5 | 2.7 | 3.1 | 7.8 |
| DDI-6 | 2.9 | 4.1 | 10.0 |
| ZM39923 | 1.2 | 4.5 | 7.3 |
| DDI-8 | 2.9 | 4.9 | 5.2 |
Thus, the AEMS system allowed for high-throughput chemical screening of the 3CL protease inhibitors in a 1536-well format based on mass spectrometry. Our results suggest that this system would also be applicable for screening inhibitors of other enzymes.
Acknowledgments
We thank National Institute of Infectious Diseases, Japan for providing SARS-CoV-2 WK-521. This research was supported by the Platform Project for Supporting Drug Discovery and Life Science Research from AMED under Grant Number JP21am0101086 and JST CREST Grant Number JPMJCR20H8.
Conflict of Interest
The author A.S. is an employee of Shionogi & Co., Ltd. The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Supplementary Materials
This article contains supplementary materials.
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