Extended Data Fig. 6: VVD-118313 functional activity and proteomic selectivity in primary immune cells.
From: Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine
a-e, Western blots corresponding to Fig. 4a-e, showing effects of VVD-118313 (5a), stereoisomeric mixture 5, and tofacitinib (Tofa) on JAK-STAT signaling pathways in human PBMCs and PHA-P/IL-2 generated T-blasts. Cells were treated with compounds at the indicated concentrations for 2 h prior to stimulation with IFNα (a; 100 ng/mL, 30 min), IL-6 (b; 25 ng/mL, 30 min), IL-2 (c; 20 U/mL, 15 min), GM-CSF (d; 0.5 mg/mL, 15 min), or IL-12 (e; 12.5 ng/mL, 15 min). Blots are representative of n = 3 (IL-6, IL-2, IL-12) or n = 4 (IFNα, GM-CSF) independent experiments. f, Left, Global cysteine reactivity profile for VVD-118313 (5a; 10 µM, 3 h, in situ) in primary human PBMCs. Reactivity values for JAK1_C817 (red) and TYK2_C838 (blue) are highlighted, and dashed horizontal line marks boundary for > 75% engagement by VVD-118313 at 10 µM. Right, heat map showing the reactivity profiles for cysteines in PBMCs treated with the indicated concentrations of VVD-118313. Only cysteines that were engaged >75% by VVD-118313 at 10 µM are shown. Data in both panels represent mean ratio values (DMSO/VVD-118313) for IA-DTB-labeled, cysteine-containing peptides quantified from n = 2 replicate cell treatments analyzed in a single MS-ABPP experiment. g, Left, Global cysteine reactivity profile for VVD-118313 (1 µM, 1 h, in vitro) in mouse splenocyte lysates. Jak1_C816 shown in red. Right, Reactivity of JAK1_C816 in mouse splenocyte lysates treated with the indicated concentrations of VVD-118313 (5a; 1 h). Data in both panels represent mean ratio values (DMSO/VVD-118313) for IA-DTB-labeled, cysteine-containing peptides quantified from n = 2 replicate cell treatments analyzed in a single MS-ABPP experiment.
