Chemical proteomics reveals target selectivity of clinical Jak inhibitors in human primary cells

Kinobeads are a set of promiscuous kinase inhibitors immobilized on sepharose beads for the comprehensive enrichment of endogenously expressed protein kinases from cell lines and tissues. These beads enable chemoproteomics profiling of kinase inhibitors of interest in dose-dependent competition studies in combination with quantitative mass spectrometry. We present improved bead matrices that capture more than 350 protein kinases and 15 lipid kinases from human cell lysates, respectively. A multiplexing strategy is suggested that enables determination of apparent dissociation constants in a single mass spectrometry experiment. Miniaturization of the procedure enabled determining the target selectivity of the clinical BCR-ABL inhibitor dasatinib in peripheral blood mononuclear cell (PBMC) lysates from individual donors. Profiling of a set of Jak kinase inhibitors revealed kinase off-targets from nearly all kinase families underpinning the need to profile kinase inhibitors against the kinome. Potently bound off-targets of clinical inhibitors suggest polypharmacology, e.g. through MRCK alpha and beta, which bind to decernotinib with nanomolar affinity.


Supplementary table legends and descriptions:
Supplementary (IPI , G , Targetclass, Subclass) using the old matrix from one representative experiment, including proteinscore, total peptide to spectra matches (totalpsm), quantified spectra (q ) q (q ) 2 " " contain the relative abundance for each kinase in the first binding step (relative to of the two binding events), the two columns rebinding contain the relative abundance (to the first binding event) of each kinase in the rebinding step. The column "I 5 2 " " " cribed above, but for the new capturing matrix.
Supplementary table 4: Kinome coverage using lipidkinobeads. This table summarizes the kinome coverage from duplicate depletion experiments using the lipidkinobeads. For each kinase (Gene name, Targetclass, IPI identifier) the table lists for both replicates quantified peptide matches (qpm), quantified spectra matches (qusm) and proteinscore. In addition, relative abundance for binding (in n2), rebinding (in n2) as well as competition using 50 µm and 5 µM free lipidkinobeads mix and the depletion factor F (IC502KD) are reported.

General Chemistry Procedures
Unless otherwise stated, all solvents and were used as purchased without further purification. Preparative mass directed high performance liquid chromatography (prep HPLC) was preformed on a XBridge BEH C18 OBD 5µm Prep Column (19 x 150 mm) at a flow rate of 30 mL/min, eluting with acetonitrile in water (0.2% formic acid as modifier). Purifications were conducted on a waters autopurification system [detectors: 3100 Mass Detector and a 2996 Photodiode Array Detector]. The purity of all final compounds was 95% or higher as determined by either, Method A: analytical high performance liquid chromatography (HPLC) using a Waters XBridge BEH C18 5µm Column (4.6 x 150 mm) at a flow rate of 1.75 mL/min with a linear gradient over 9 min (1 to 99% acetonitrile in water, 0.2% formic acid as modifier). The instrument used for analysis was a waters autopurification system [detectors: 3100 Mass Detector and a 2996 Photodiode Array Detector] or Method B: analytical ultra performance liquid chromatography (UPLC), using a Waters Acquity BEH C18 1.7 µm Column (2.1 x 50 mm) at a flow rate of 1 mL/min with a linear gradient over 2 min (3 to 99% acetonitrile in water, 0.1% formic acid as modifier). The instrument used for analysis was a waters Acquity system [detectors: Acquity SQD, Acquity ELSD, Acquity PDA]. Proton (1H) NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz) using DMSO-d6 or CDCl3 as solvents. Chemical shifts are ( ) (δ 1H).
The combined organics were washed with water (400 ml), brine (400 ml), dried over Na2SO4, filtered and concentrated. The residue was triturated with methanol and the solid collected by filtration. This was then further purified by column chromatography (methanol in ethyl acetate containing 1% triethyl amine). This afforded 3-(1-ethyl-1Himidazo[4,5-c]pyridin-2-yl)pyrazin-2-amine as a mixture which was taken on to the next step without further purification.
Two drops of water was added to a solution of 5-bromo- 63 mmol), K2CO3 (1.04 g, 7.52 mmol) and PdCl2(dppf)-CH2Cl2 (0.205 g, 0.251 mmol) in N,N-dimethylformamide (15 mL) and the reaction mixture was heated to 150ºC for 13 min in a microwave (solution was pre-stirred 8 min). The solution was then partitioned between ethyl acetate (200 ml) and water (200 ml), and the organic phase was washed with further water (2 x 200ml) and then brine (200 ml). It was then dried over Na2SO4, filtered and concentrated in vacuo. The resulting solid was washed with ethyl acetate to remove the unreacted boronic ester to afford tert-butyl (3- carbamate as a yellow solid (0.50 g, 40 % yield).