Fig. 1: High throughput screening utilizing human monocytic THP-1 reporter cells identified PVP-057, a thiazole benzamide that activated IRF- and NF-κB.
A Schematic diagram depicting high-throughput screening (HTS) of small molecules with adjuvant effect utilizing human THP-1-Dual cells. Down selecting from ~200,000 molecules using HTS, ~1300 potential hits were identified using a no-wash 384-well quantitative Luminescent (NF-kB) and Fluorescence (Adherence) immunoassay. Among those, 167 were established hits as confirmed with ELISA-based titration assays. Created in BioRender. Dowling, D. (2025) https://BioRender.com/vcd4k52. B THP-1-Dual NF-κB/IRF reporter cells were stimulated for 18 h with THP-1 screening hits at 33 µM and NF-κB and IRF activities were plotted as median fold over DMSO vehicle. C Chemical structure of the thiazole benzamide PVP-057, which was identified as one of the top 5 ranked small molecules in inducing adherence in THP-1 reporter cell lines at a concentration of 33 µM. These THP-1 cells were harvested and stained for expression of the co-stimulatory molecules CD80 and CD86 (D), as well as the apoptotic marker Annexin V (E). Median fluorescence intensity (MFI) of CD80 and CD86 and the percentage of cells positive for Annexin V are shown. PMA was used as a positive control while DMSO was used as a negative control. Their activities are shown as dashed lines if not otherwise indicated. Kruskal–Wallis test with post-hoc Dunn’s test were used to compare the activity of PVP-057 and/or PMA with that of DMSO. B–E N = 3 technical replicates. F PVP-057 NF-κB-driven SEAP expression and ISG-driven luciferase expression in THP-1 cells for three concentrations. Results are shown as fold activity over DMSO in a 4-fold serial dilution with a top concentration of 25 µM. Data are presented as mean values ± SEM. N = 14 technical replicates per concentration. NS p > 0.05, *p ≤ 0.05. Source data are provided as a Source Data file.
