Figure 8.

Inhibitory impact of hit warheads on the HCMV core NEC interaction. (A) 293T cells were transiently transfected with the constructs LgBiT::UL53-Flag, LgBiT::UL53(1–87)-Flag or SmBiT::UL50-HA for assaying in the NanoBiT system. At 1 d p.t., biological triplicates of cells were transferred into a 96-well plate. After addition of NanoGlo Live Cell Reagent, measurements were immediately performed to quantify luminescence for 2 h. Each of the constructs, expressed separately together with empty vectors, served as controls of background signals, and RFP served as a transfection control. In (B), the construct LgBiT::UL53-Flag served as the primary binding component, while in (C) the truncated construct LgBiT::UL53(1–87)-Flag was alternatively applied. Assay conditions were identical to those for panel A. Compound treatment was started directly before NanoGlo Live Cell Reagent addition, and compound concentrations referring to 5×, 1× or 0.2× anti-HCMV EC₅₀ levels were applied. (D) As a control, which was not supposed to interact with the drug ibrutinib, viral pUL97 interaction with cyclin H was used in the respective pair of test constructs (LgBiT::UL97(231–280)-Flag + SmBiT::CycH-HA). Note the lack of concentration-dependent inhibitory impact of ibrutinib on this pUL97–cycH interaction signal. Mean values ± SDs are given.