In-cell NMR spectroscopy K562 cells were maintained in IMDM media supplemented with 10% FBS. For NMR measurements cells were harvested by centrifugation, washed and resuspended in OptiMEM serum-free media to obtain a suspension with 65% v/v cell content. 10% D2O and 500 µM prodrug in DMSO-d6 were added (0.5% 8inal DMSO concentration) and incubated on a spinning wheel for 5 min. A 200 µl suspension was transferred into a 3mm Shigemi tube (without insert) matched to the magnetic susceptibility of water. 1D F spectra with inverse-gated decoupling of protons were collected at 37 °C with 2048 scans (experiment time 37 min) on a Bruker AVANCE III spectrometer with an 11.7 T magnet using a QCI-F cryoprobe at 470 MHz Lamour frequency. After 4 h the sample was lysed by freeze-thawing and a further spectrum of the lysate was collected. To con8irm the identity of the observed species, the lysate was sequentially spiked with 500 µM MN551 and prodrug and measured again. For comparison, 1D MN551 was incubated with the pre-cleared lysate for 1.5 hours at room temperature. Pull-down experiments were performed using biotinylated peptides, GHR pY595 (Biotin-aminohexanoic acid-PVPDpYTSIHIV-amide) or GHR Y595(Biotin-aminohexanoic acid-PVPDYTSIHIV-amide). The peptides were immobilized using 30 µl/IP high-capacity streptavidin agarose beads (Pierce High-Capacity Streptavidin Agarose, 20357) by incubating for 30 minutes a 4°C. Separate tubes of peptide incubation was performed for GHR pY595 and GHR Y595. Unbound peptide was washed off through 3x cycles of buffer wash and spin. Beads were resuspended to a total volume of 100 µl per IP sample. 100 µl beads with immobilized peptides was added to the MN551 lysate tubes. Samples were incubated for 40 minutes at room temperature. Samples were spun and unbound supernatant was removed.