Extended Data Fig. 2: Chemical optimization, characterization and proteome-wide reactivity of VVD-118313 (5a).
From: Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine
a, Engagement and inhibitory activity of covalent ligands targeting JAK1_C817. Engagement (TE50, μM, 1 h, in vitro) for JAK1_C817 or TYK2_C838 determined by targeted TMT-ABPP in human cell lysates. Data are mean values ± S.D. from n = 2-3 independent experiments with the exception of values marked with †, which were from a single experiment. JAK1 inhibition (IC50) determined using HTRF assays measuring IFNα (100 ng/mL, 30 min)-stimulated STAT1 phosphorylation or IL-6 (25 ng/mL, 30 min)-stimulated STAT3 phosphorylation in human PBMCs pretreated with compounds for 2 h. Compounds were tested as single stereoisomers except where noted §. Absolute configuration not assigned for (-)-1a, (-)-2a and (-)-3a. For 5a, JAK1 inhibition following IFNα stimulation was also measured in PBMCs cultured in media supplemented with 10% v/v fetal bovine serum (FBS). Data are mean values ± S.D. from n = 2 independent experiments except where noted (‡n = 3, †n = 1). ND – not determined. NA – not applicable for a non-covalent orthosteric inhibitor. b, IC50 values for JAK1_C817 engagement by VVD-118313 (5a) and enantiomer 5b in Jurkat T-cell lysate at 10, 30 and 60 minutes. JAK1_C817 engagement was measured by targeted MS-ABPP, where iodoacetamide desthiobiotin (200 μM) was added to cell lysates at the indicated timepoints after incubation with 5a or 5b. Data are mean values ± S.D. of n = 2 independent experiments. TE50 values were estimated by fitting data to a 4PL model and are reported as 95% confidence interval. c, Left, Global cysteine reactivity profile for VVD-118313 (1 µM, 1 h, in vitro) in primary human PBMC lysates (2 mg/mL proteome). Data 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. Ratio values for JAK1_C817 (red) and TYK2_C838 (blue) are highlighted. Quantified cysteines with ratios ≥ 4 (≥ 75% engagement) are marked. Right, Concentration-dependent reactivity profile for VVD-118313 reactivity with TOR4A_C21 in human PBMCs (0.01-10 μM, 3 h, in situ) or PBMC lysates (0.01-10 µM, 1 h, in vitro). Bars show mean values from VVD-118313-treated samples shown as a percentage of DMSO-treated samples from n = 2 replicate cell treatments analyzed in a single MS-ABPP experiment.
