Ligand-Based Competition Binding by Real-Time 19F NMR in Human Cells

The development of more effective drugs requires knowledge of their bioavailability and binding efficacy directly in the native cellular environment. In-cell nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for investigating ligand–target interactions directly in living cells. However, the target molecule may be NMR-invisible due to interactions with cellular components, while observing the ligand by 1H NMR is impractical due to the cellular background. Such limitations can be overcome by observing fluorinated ligands by 19F in-cell NMR as they bind to the intracellular target. Here we report a novel approach based on real-time in-cell 19F NMR that allows measuring ligand binding affinities in human cells by competition binding, using a fluorinated compound as a reference. The binding of a set of compounds toward Hsp90α was investigated. In principle, this approach could be applied to other pharmacologically relevant targets, thus aiding the design of more effective compounds in the early stages of drug development.

Thin layer chromatography was conducted with 5 × 10 cm plates coated with Merck Type 60 F254 silica gel to a thickness of 0.25mm.All reagents obtained from commercial sources were used without further purification.Anhydrous solvents were obtained from the Sigma-Aldrich Chemical Company Ltd., and used without further drying.

PREPARATIVE HPLC.
Preparative HPLC purifications were performed on a Waters FractionLynx MS Autopurification system with a Gemini® 5 μM C18(2), 100 mm × 20 mm i.d.column from Phenomenex, running at a flow rate of 20 mL min-1 with UV diode array detection (210 -400 nm) and mass-directed collection.Representative Gradients used are shown in Table S3.

Figure S1 .
Figure S1.Intracellular localization of Hsp90 N .SDS-PAGE analysis of the nuclear, cytosolic and mitochondrial fractions of HEK293T cells overexpressing Hsp90 N .The nuclear and cytosolic fractions are diluted 1:10 and 1:40 with respect to the mitochondrial fraction, respectively.

Figure S2 .
Figure S2.Binding of compounds 1-4 to intracellular Hsp90 N .Background-subtracted 1 H-15 N SOFAST-HMQC spectra of human cells (A) and corresponding lysates (B) expressing [U-15 N]-Hsp90 N in the absence (black) and in the presence (blue) of a fluorinated compound ( 1-4 from top to bottom).Ligand binding is revealed by the shifted crosspeaks in the 1 H-15 N NMR spectra of the cell lysates.

Figure S3 .
Figure S3.Binding of compounds 5-7 to intracellular Hsp90 N .Background-subtracted 1 H-15 N SOFAST-HMQC spectra of human cells (A) and corresponding lysates (B) expressing [U-15 N]-Hsp90 N in the absence (black) and in the presence (magenta) of a test compound ( 5-7 from top to bottom).Ligand binding is revealed by the shifted crosspeaks in the 1 H-15 N NMR spectra of the cell lysates.

Figure S4 .
Figure S4.Competition binding of compound 5. Waterfall plot of time-resolved 1D 19 F NMR spectra recorded on cells expressing Hsp90 N , perfused in the bioreactor with a constant concentration of compound 2 at increasing concentrations of compound 5 over the course of ~60 hours.Spectral intensity (a.u.) is color-coded from blue (lowest) to yellow (highest).Hsp90 N :2 + 5: signal arising from the Hsp90 N :2 complex as it is displaced by compound 5; free 2: signal arising from free compound 2 in the extracellular medium.

Figure S5 .
Figure S5.Competition binding of compound 6.Waterfall plot of time-resolved 1D 19 F NMR spectra recorded on cells expressing Hsp90 N , perfused in the bioreactor with a constant concentration of compound 2 at increasing concentrations of compound 6 over the course of ~60 hours.Spectral intensity (a.u.) is color-coded from blue (lowest) to yellow (highest).Hsp90 N :2 + 6: signal arising from the Hsp90 N :2 complex as it is displaced by compound 6; free 2: signal arising from free compound 2 in the extracellular medium.

Figure S6 .
Figure S6.Competition binding of compound 7. Waterfall plot of time-resolved 1D 19 F NMR spectra recorded on cells expressing Hsp90 N , perfused in the bioreactor with a constant concentration of compound 2 at increasing concentrations of compound 7 over the course of ~56 hours.Spectral intensity (a.u.) is color-coded from blue (lowest) to yellow (highest).Hsp90 N :2 + 7: signal arising from the Hsp90 N :2 complex as it is displaced by compound 7; free 2: signal arising from free compound 2 in the extracellular medium.

Figure S7 .
Figure S7.In-cell, leakage control and lysate NMR spectra.Projections along the 1 H axis of 1 H-15 N SOFAST-HMQC spectra of cells (black), supernatants (grey) and cell lysates (re d) from samples of cells expressing Hsp90 N treated with compounds 1-4.

Figure S8 .
Figure S8.Saturation of the 19 F signals.(A) Signal arising from the intracellular Hsp90 N :2 complex recorded with an interscan delay (D1) of 1 s (left) and 10 s (right); (B) T 1 relaxation analysis of Hsp90 N :2 by inversion recovery; (C) Signal arising from free compound 2 in DMEM medium recorded with an interscan delay (D1) of 1 s (left) and 10 s (right); (D) T 1 relaxation analysis of compound 2 by inversion recovery.

Table S1 . Durations and ligand concentrations of each step of the bioreactor runs reported in Figure 4A-C. For
each channel, ligand concentrations in the reservoir and flow rates are also reported.

(Figure 4C
1 (400 MHz) and 13 C (100.6 MHz) Nuclear magnetic resonance (NMR) analyses were performed using a Bruker DPX-400 MHz NMR spectrometer.1HNMR Spectra were also recorded at 250 MHz on a Bruker AC250 and at 500 MHz on a Bruker 500 MHz Ultrashield spectrometer. Th spectral reference was the known chemical shift of the sample solvent.1H NMR data is reported indicating the chemical shift (d) as parts per million (ppm), the multiplicity, (s, singlet; d, doublet; t, triplet; q, quartet; sept, septet; m, multiplet; br, broad; dd, doublet of doublets etc.) the integration (e.g.1H), the coupling constant (J) in Hertz (Hz) (app implies apparent coupling on broadened signals).13C NMR data is reported indicating the chemical shift (d) as parts per million (ppm), and i n some cases annotated with the carbon multiplicity: (CH3) for primary carbon, (CH2) for secondary carbon, (CH) for tertiary carbon and (C) for quaternary carbon.Deuterated solvents were obtained from the Sigma-Aldrich Chemical Company or Fluorochem.LCMS analyses were performed on an HP1100 instrument (method A), with a Luna 3 DM, C18(2), 30 mm × 4.6 mm i.d.column from Phenomenex at a temperature of 22 °C, with a flow rate of 2 mL min-1 using the following solvent systems (solvents purchased from Romil UK, HPLC was performed on a Perkin Elmer series 200 quaternary pump and 235C DAD instrument, with a Gemini 5 DM, C18 110A 50 mm × 4.6 mm i.d. clumn from Phenomenex (part number 00B-4435-E0) at a temperature of 22 °C, at a flow rate of 2 mL min-1 using the following solvent systems (Solvents purchased from Romil UK, Waterbeach, UK) Solvent A: HPLC grade Water + 10 mM ammonium acetate adjusted to pH 7.5 with ammonium hydroxide.

Table S2 :
Solvent gradient used for analytical HPLC method B.

Table S3 .
Preparative HPLC gradients, showing 4 representative gradients (W-Z) used for purifying certain compounds described below.