Fig. 4: Expanding the engagement profile on a known drug target.
From: Proteome-wide ligandability maps of drugs with diverse cysteine-reactive chemotypes
a Schematic showing the canonical JAK1/Stat6 and JAK1/Stat1 pathways, triggered by cytokines (IL4) and (IFNα), respectively. The elements are obtained from Biorender. b Dot plot showing the RH/L values for all quantified cysteines of JAK1, highlighting C988 as the sole engaged site. Each data point represents a unique DPCC-cysteine interaction quantified via QTRP from 3 biological replicates and 2 technical replicates. c Representative XIC showing the change in probe-labeled JAK1C988 from cells treated with or without Natamycin, with light and heavy-labeled peptide profiles shown in red and blue, respectively. The average RH/L values from four replicate experiments are indicated below the XIC. d Representative western blot analysis showing the competition of Natamycin against biotin-iodoacetamide (100 μM, 1 h) labeling of JAK1. HEK293T cells overexpressing JAK1 and the C988S mutant were treated with varying concentrations of Natamycin for 4 h. The immunoblots are representative of three independent experiments. e Synthesis of Natamycin-Alkyne. f Representative western blots showing the labeling of JAK1 or JAK1-C988S with Natamycin-alkyne in cellulo, with a decrease observed in the presences of the parent compound. The immunoblots are representative of three independent experiments. g CorrSite2.0-based prediction showing that JAK1C988 is localized within a potential allosteric pocket. h In vitro activity of Natamycin, Tofacitinib (Tofa), and their combination against recombinant JAK1 (residues 438–1154). Data are presented as mean ± s.d. from three independent experiments. i Representative western blots showing the effects of Natamycin on JAK1 phosphorylation (p-JAK1 at Y1034/Y1035), as well as IL-4-stimulated Stat6 phosphorylation (p-Stat6 at Y641 phosphorylation). j Representative western blots showing the effects of Natamycin on JAK1 phosphorylation (p-JAK1 at Y1034/Y1035), IFNα-stimulated Stat1 phosphorylation (p-Stat1 at Y701). k Representative western blots showing that the C988S mutation abrogates the inhibitory effect of Natamycin on the IL4/JAK1/Stat6 pathway. l Representative western blots showing that the C988S mutation abolished the inhibitory effect of Natamycin on the IFNα/JAK1/Stat1 pathway. Quantification results from three independent experiments for (i–l) are provided in Supplementary Fig. 10. Source data are provided as a Source Data file.
