L4100

The cloning vectors used to create LgBiT and SmBiT protein fusions were provided in the Nano-Glo^® Live Cell Assay System (Promega, Madison, WI, USA). The vpu and BST-2 genes were subcloned into pBiT1.1-C [TK/LgBiT], pBiT2.1-C [TK/SmBiT], pBiT1.1-N [TK/LgBiT], and pBiT2.1-N [TK/SmBiT] vectors at the XhoI and NheI sites, respectively, to generate the fusion protein expression vector. A library of 1117 bioactive compounds was obtained from Selleck Chemicals (L4100 and L2000; Houston, TX, USA). Y-39983 HCl (S7935), brazilin (S9428), tangeretin (S2363), and AS1517499 (S8685) were also obtained from Selleck Chemicals. The structure of 4 compounds is shown in Figure S1.

HEK293T cells were plated in 96-well plates at a concentration of 1 × 10⁵ cells/mL and co-transfected with the plasmids encoding the Vpu and BST-2 fusion proteins at a ratio of 1:1. The empty vector (HaloTag with SmBiT) was used as a negative control. At 12 h post-transfection, the cells were treated with bioactive compounds (L2000 and L4100; Selleck Chemicals) at a final concentration of 10 μmol/L and incubated for 24 h. Then, Nano-Glo Live Cell Substrate (N2012; Promega) was added and luminescence was measured using an EnSpire Multilabel Plate Reader 2300 (PerkinElmer, Waltham, MA, USA). The inhibition rate on Vpu-BST-2 binding was calculated as, inhibition rate (%) = (1 − experimental OD value/control OD value) × 100%. A heat map of the inhibition rate of all compounds was generated using GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA).