Fig 1: 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.
Fig 2: 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.
Fig 3: 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.
Fig 4: 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.
Fig 5: 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.
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