TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS

Abstract

SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here, we report the development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay for monitoring the TMPRSS2-SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance. To enable large-scale compound screening, we further miniaturized the assay into 1536-well ultrahigh-throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein-protein interaction.

Keywords: COVID-19; SARS-CoV-2; SPIKE; TMPRSS2; TR-FRET; high-throughput screening; protein–protein interaction.

Figure 1

TMPRSS2–SPIKE TR-FRET interaction assay and workflow. (A) TR-FRET model. SPIKE and TMPRSS2 are fused to tags that interact with antibodies conjugated to fluorophores. When the proteins are in close proximity (<10 nm), excitation of a donor fluorophore can cause excitation of the acceptor fluorophore. (B) Workflow of TR-FRET assay development and screening. Expression of various TMPRSS2 and SPIKE proteins and different tags were tested with different donor and acceptor combinations in TR-FRET. For the pair chosen, assay evaluation for HTS and miniaturization were done followed by a small-scale pilot screen of bioactive compounds.

Figure 2

Expression of TMPRSS2 and SPIKE constructs and confirmation of binding in HEK-293T cells. (A) Expression of SPIKE constructs determined by western blotting. C-terminal tagged SPIKE expressed well and was therefore selected for this study. No expression of N-GST was detected (data not shown). NC, no construct; N, N-terminal tag; C, C-terminal tag. (B) Expression of TMPRSS2 constructs determined by western blotting. N-terminal tagged TMPRSS2 expressed well and was therefore selected for this study. (C) GST pulldown (PD) results of SPIKE-VF and GST-TMPRSS2 overexpressed in HEK-293T cells. PD and whole-cell lysate (WCL) samples were subjected to western blotting as indicated.

Figure 3

TMPRSS2–SPIKE TR-FRET assay development in 384-well format. (A) Various tags and donor/acceptor fluorophore-conjugated antibodies were used to find the optimal combination for TR-FRET. Combinations are as follows: (1) His-TMPRSS2–SPIKE-VF with FLAG-Tb/His-D2; (2) His-TMPRSS2–SPIKE-FLAG with FLAG-Tb/His-D2; (3) His-TMPRSS2–SPIKE-VF with His-Tb/FLAG-D2; (4) His-TMPRSS2–SPIKE-FLAG with His-Tb/FLAG-D2; (5) GST-TMPRSS2–SPIKE-VF with FLAG-Tb/GST-D2; (6) GST-TMPRSS2–SPIKE-FLAG with FLAG-Tb/GST-D2; (7) GST-TMPRSS2–SPIKE-VF with GST-Tb/FLAG-D2; (8) GST-TMPRSS2–SPIKE-FLAG with GST-Tb/FLAG-D2; (9) VF-TMPRSS2–SPIKE-His with FLAG-Tb/His-D2; (10) VF-TMPRSS2–SPIKE-His with His-Tb/FLAG-D2; (11) FLAG-TMPRSS2–SPIKE-His with His-Tb/FLAG-D2; (12) GST-TMPRSS2–SPIKE-His with His-Tb/GST-D2; and (13) GST-TMPRSS2–SPIKE-His with GST-Tb/His-D2. Combination 2 was chosen for further evaluation. (B) TR-FRET signal at 2-fold dilution of lysate from HEK-293T cells overexpressing His-TMPRSS2 and SPIKE-FLAG. Cells were lysed in 1% NP-40 lysis buffer, and cell lysate was serially diluted in FRET buffer. Error bars represent standard deviation of n = 3 values. (C) TR-FRET S/B ratio and Z-factor (Z′) at 2-fold dilution of lysate from cells overexpressing His-TMPRSS2 and SPIKE-FLAG. Circles represent S/B ratios, and triangles represent Z′ (n = 3). Error bars represent standard deviation of n = 3 values.

Figure 4

TMPRSS2 and SPIKE TR-FRET assay optimization in 384-well format. (A) Stability of TR-FRET signal in different concentrations of DMSO. Error bars represent standard deviation of n = 3 values. (B) Temporal stability of TR-FRET signal over 48 h. Error bars represent standard deviation of n = 3 values. TR-FRET signals were generated from cell lysate expressing His-TMPRSS2–SPIKE-FLAG with a total protein concentration at 10 μg/ml for both A and B.

Figure 5

TR-FRET assay miniaturization for 1536-well format. (A) Scaling down from 384-well plates used for assay development, which uses 30 μl of TR-FRET reaction buffer per well, to 1536-well plates for uHTS, which utilizes 5 μl of TR-FRET reaction buffer. (B) Comparison of TR-FRET signal in 384- and 1536-well plates. TR-FRET signals were generated with same TR-FRET reaction mixture containing 10 μg/ml total protein and antibodies at working concentration (FLAG-Tb at 1:1000 and His-D2 at 1:500). Error bars represent standard deviation of n = 5 values. (C) S/B ratios and Z′ of TR-FRET assay in 384- and 1536-well-plate formats. Error bars represent standard deviation of n = 5 values.

Figure 6

Pilot screening in 1536-well uHTS format. (A) S/B for each 1536-well plate used for pilot screening. (B) Z′ for each 1536-well plate used for pilot screening. (C) TR-FRET signal of compounds from EEBL and LOPAC libraries, expressed as percent of the control signal. Compounds with TR-FRET signal <50% of control were selected as primary hits. Screening details are found in Table 1.

Figure 7

DR confirmation and orthogonal assay validation of reordered primary hits. (A–C) DR curves from TR-FRET assay showing the disruption of TMPRSS2–SPIKE interaction by CAPE (A), gossypol acetate (B), and verteporfin (C). Cell lysate expressing His-TMPRSS2 and SPIKE-FLAG was treated with compounds at a dose range from 0.01 to 100 μM. Compound structures were generated using the PyMol software. Error bars represent standard deviation of n = 4 values. (D–F) Dose-dependent curves from the NanoBiT assay showing the disruption of TMPRSS2–SPIKE interaction by CAPE (D), gossypol acetate (E), and verteporfin (F). HEK-293T cells (3500 cells/well) expressing SmBiT-TMPRSS2 and SPIKE-LgBiT were replaced into a 1536-well plate for compound treatment at a dose range from 0.01 to 100 μM. Error bars represent standard deviation of n = 4 values. (G–I) Thermal shift analysis of SPIKE-S2 domain with CAPE (G), gossypol acetate (H), and verteporfin (I). Purified His-tagged SPIKE-S2 was applied to determine the protein thermal stability by measuring fluorogenic readout from SYPRO Orange dye. Error bars represent standard deviation of n = 2 values.

Figure 8

Evaluation of eltrombopag. (A) Predicted three-dimensional structure of eltrombopag generated in Maestro of the Schrodinger Small Molecule Drug Discovery suite of software. Light blue: carbon; dark blue: nitrogen; red: oxygen. (B) Eltrombopag in TR-FRET assay with an IC₅₀ of ∼20 μM using a nonlinear fit of inhibitor vs. response with a variable slope. Error bars represent the standard deviation of n = 4 values. (C) Eltrombopag with His-SPIKE-S2 in the SYPRO Orange thermal shift assay with a ΔT_m of 1°C compared to DMSO control at 25 μM when fit with the Boltzmann sigmoidal equation. (D) SiteMap identified three main binding sites of eltrombopag on SPIKE (red) when in complex with ACE2 (blue) and TMPRSS2 (purple). Site 2 is located at the interface of TMPRSS2 and SPIKE. (E) Eltrombopag can fit all three binding sites. (F) The molecular surface of the predicted eltrombopag binding site. Blue and red colors indicate positively and negatively charged atoms, respectively. (G) The predicted interactions of eltrombopag with SPIKE at the TMPRSS2–SPIKE PPI interface binding site.