Journal: bioRxiv

Article Title: Discovery of Small Molecules and a Druggable Groove That Regulate DNA Binding and Release of the AP1 Transcription Factor ΔFOSB

doi: 10.1101/2025.10.21.683675

Figure Lengend Snippet: A) Cell viability of JPC0661 comm and HTS10307 assessed in the range of 0-20 μM after 24 h (top) and 72 h (bottom) in neuronal progenitor cells (NPC) using the CellTiter-Glo viability assay. Cell viability measures are normalized to the negative control containing no compound but 0.6% DMSO. Dotted lines indicate 100% cell viability. The mean of n replicates is shown for 24 h (n=3) and 72 h (n=2) with error bars indicating the SEM. B) Stability of JPC0661 comm and HTS10307 in mouse liver microsomes. C) and D) Effects of JPC0661 comm (0–100 μM; C ) and JPC0661 (0–100 μM; D ) on AP1-driven luciferase activity in AP1-luc HEK293 cells. The dose-dependent activation of the AP1-driven luciferase reporter was measured based on changes in the luciferase signal and expressed as relative fluorescence units (RFU). Each compound was tested twice (n = 4 wells per experiment; total n = 6–8 wells for JPC0661 comm and n = 7–8 wells for JPC0661 per concentration), with results normalized to the luciferase signals of blank wells from corresponding experiments (n = 8 wells). Nonlinear regression using a three-parameter model was applied to fit the luciferase signal and calculate IC 50 values, which are reported with a 95% confidence interval (CI), and data points are presented as the mean ± SEM. E) Effect of serially diluted JPC0661 (0.003-100 μM) on the viability of AP1-luc HEK293 cells using the Celltiter-Go viability assay (2 h). Cell viability was normalized to the DMSO control, which contained 0.5% DMSO but no compound (n = 7 wells). Data points are presented as the mean ± SEM, with a total n = 10–12 wells per concentration. F) Effect of JPC0661 (at doses 12.5, 25, and 50 μM) on the viability of Neuro 2A cells (72 h) using the Celltiter-Go viability assay to assess potential toxicity in mouse neuronal cells. Cell viability was normalized to the DMSO control, which contained 0.5% DMSO but no compound (n = 8 wells). Data points are presented as the mean ± SEM, with a total n = 12 wells per concentration.

Article Snippet: The AP1-luciferase reporter human embryonic kidney (HEK293) cell line was obtained from BPS Bioscience (USA) and maintained in growth medium 1B (BPS Bioscience) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Hyclone) under standard incubation conditions of 37 °C with 5% CO 2 .

Techniques: Viability Assay, Negative Control, Luciferase, Activity Assay, Activation Assay, Fluorescence, Concentration Assay, Control

Journal: bioRxiv

Article Title: Discovery of Small Molecules and a Druggable Groove That Regulate DNA Binding and Release of the AP1 Transcription Factor ΔFOSB

doi: 10.1101/2025.10.21.683675

Figure Lengend Snippet: A) A panel of JPC0661 analogs designed to probe the role of the sulfonic acid group and the amino-pyrazolone group. B) Table summarizing the JPC0661 analogs tested in AP1-reporter assays using AP1-luc HEK293 cells, yielding cell-based IC50 values and FP-DRC assays. Lower IC 50 values are marked with more plus signs (+) and indicate higher activity. The plots from the FP-DRC and cell-based DRC assays for these compounds are shown in Supplementary Fig. S1 . C) ΔFOSB/JUND bZIP incubated with compounds (0.5 mM) with 100 μM diamide (‘ox’, oxidized) or without diamide (‘red’, reduced) and assessed by SDS-PAGE (with or without reducing agent in the loading buffer). D) ΔFOSB/JUND bZIP protein incubated with compounds (0.5 mM) with no diamide (‘red) or protein alone (no compound) incubated with diamide (‘ox’) as a control and then assessed by SDS-PAGE (with or without reducing agent in the loading buffer). In C) and D) ‘cntrl’ denotes the ΔFOSB/JUND bZIP protein in absence of compound.

Article Snippet: The AP1-luciferase reporter human embryonic kidney (HEK293) cell line was obtained from BPS Bioscience (USA) and maintained in growth medium 1B (BPS Bioscience) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Hyclone) under standard incubation conditions of 37 °C with 5% CO 2 .

Techniques: Activity Assay, Incubation, SDS Page, Control