Ligand-observed T1ρ and STD NMR experiments were used to verify compound-protein interactions. All NMR spectra were acquired at 25°C on a Bruker Avance III 600 MHz (proton frequency) spectrometer equipped with a cryogenically cooled probe (Bruker BioSpin, Germany). For ligand-observed T1ρ measurements, 200 μM compound with 0, 1, 5, and 20 μM Hsp90 were prepared as samples. For the STD experiments, 100 μM compound with or without 5 μM Hsp90 was prepared as samples. Compounds and proteins were dissolved in phosphate buffer [20 mM sodium phosphate (pH 7.4), 150 mM NaCl, and 5% DMSO] for NMR data acquisition.

Two-dimensional HSQC experiments were performed on a Bruker 800-MHz NMR spectrometer, and samples involving 50 μM 15N-labeled Hsp90 protein with or without the indicated concentration of compound (250 μM and 500 μM) were investigated in assay buffer containing 20 mM sodium phosphate (pH 7.4), 150 mM NaCl, and 5% DMSO. NMR data processing and analysis used the SPARKY (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco) program. Images of the structures were generated by PyMOL. Chemical shift changes were calculated using the combined shift changes of amide nitrogens and protons, Δω = (ΔωN2 + ΔωH2)1/2, where ΔωN and ΔωH are the differences in 15N and 1H frequencies, respectively, between WT protein and compound-bound protein in hertz.

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