Targeting secondary protein complexes in drug discovery: studying the druggability and chemical biology of the HSP70/BAG1 complex† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7cc01376k

A non-nucleotide FP-probe was designed to study the mechanism of action and druggability of the secondary HSP70/BAG1 complex.

S3 mM TRIS base pH 7.4, 100 mM NaCl, 1 mM DTT, 1% (v/v) glycerol) or BAG1 replacement buffer (5 μL, 20 mM TRIS base pH 7.4, 100 mM NaCl, 1 mM DTT, 1% (v/v) glycerol) was added to the corresponding wells of the assay plate. Fluorescence polarisation values for probe and HSP72 ± BAG1 were plotted using GraphPad prism 6. K D determination with BAG1: To each well, 5 μL containing probe molecule (20 nM in assay buffer) and BAG1 protein (1.4 μM in assay buffer) and increasing concentrations of HSP70 protein (5 μL, two-fold dilution series) were added. Fluorescence polarisation values for tracer control wells (10 nM probe in assay buffer only) were subtracted from each data point prior to data analysis. K D determination was performed using non-linear regression analysis (GraphPad Prism 6, one site-specific binding model).
Compound screen with and without BAG-1: Compounds (0.2 μL at 50 x screening concentration in DMSO) were dispensed using an ECHO 550 Liquid Handler (Labcyte Inc.). To the corresponding wells was added, 5 μL of probe molecule (20 nM in assay buffer), 2.5 μL of HSP72 (four times final concentration in assay buffer) and either 2.5 µL of BAG-1 (2.8 µM in 20 mM TRIS base pH 7.4, 100 mM NaCl, 1 mM DTT, 1% (v/v) glycerol) or 2.5 µL of BAG1 replacement buffer (20 mM TRIS base pH 7.4, 100 mM NaCl, 1 mM DTT, 1% (v/v) glycerol). The assay was performed in duplicate. Tracer controls (10 nM probe molecule only) and bound tracer controls (10 nM probe in the presence of appropriate protein concentration) were included on each assay plate.
IC 50 determination was performed using non-linear least squares curve fitting (GraphPad Prism 6, log(inhibitor) vs. responsevariable slope (four parameters)). bisaryl-ATTO-488 (blue) binding to HSP72; b) Representative binding isotherms for ATP-ATTO-488 with 20 nM to 10 μM HSP72 at 10 minutes incubation (red) and 6 hours incubation (blue). K D determination was performed using non-linear regression analysis (one site-specific binding model), all data points were tested in triplicate and are represented as the arithmetic mean ± SEM. pK D =-log K D (M). pK D values are quoted as the geometric mean±SEM of n independent repeats. . Bisaryl-ATTO-488 binding to HSP72 is not detergent dependent. Representative binding isotherms for bisaryl-ATTO-488 binding to HSP72 in the presence of 0.1% (w/v) CHAPS (red) or 0.01% Triton X-100 (v/v) (blue) detergent. K D determination was performed using non-linear regression analysis (one site-specific binding model), all data points were tested in triplicate and were represented as the arithmetic mean ± SEM. pK D =-log K D (M). pK D values are quoted as the geometric mean±SEM of n independent repeats. IC 50 = 137 nM (pIC 50 = 6.87 ± 0.05, n = 3) Fig. S4. Competitive displacement curve for bisarryl-ATTO-448 and HSP72 with compound 2. IC 50 determination was performed using non-linear least squares curve fitting (GraphPad Prism 6, log(inhibitor) vs. response-variable slope (four parameters)), all data points were tested in triplicate and are represented as the arithmetic mean ± SEM. pK D =-log K D (M). pK D values are quoted as the geometric mean±SEM of n independent repeats. Representative binding isotherms for bisaryl-ATTO-488 (blue) and ATP-ATTO-488 (red) binding to TR-HSP72 (aa 3-382) (•) and TR-HSC70 (aa 4-381) (x). K D determination was performed using non-linear regression analysis (one site-specific binding model), all data points were tested in triplicate and are represented as the arithmetic mean ± SEM. pK D =-log K D (M). pK D values are quoted as the geometric mean±SEM of n independent repeats. with 700 nM BAG1 (blue) or without BAG1 (red). K D determination was performed using non-linear regression analysis (one sitespecific binding model), all data points were tested in triplicate and are represented as the arithmetic mean ± SEM. Competition experiments in the presence of BAG1 (red) were performed using 10 nM biaryl-ATTO-488, 700 nM BAG1 and 140 nM HSP70 to give a 50% bound fraction. Competition experiments in the absence of BAG1 (blue) were performed using 10 nM biaryl-ATTO-488 and 180 nM HSP70 to give a 50% bound fraction. IC 50 determination was performed using non-linear regression analysis (log(inhibitor) vs. response -Variable slope (four parameters)), all data points were tested in duplicate and are represented as the arithmetic mean ± SEM.  Purification by semi-preparative HPLC was carried out using one of the following sets of conditions: Method A) Chromatographic separation was carried out at room temperature using a Gilson GX-281 Method C) Chromatographic separation was carried out at room temperature using a 1200 Series Preparative HPLC (Agilent, Santa Clara, USA) with a Phenomenex Gemini column (5 µm, 250 x 10 mm, C18, Phenomenex, Torrance, USA) using a 15 minute gradient elution (Grad15min.m) from 10:90 to 100:0 methanol:water (both modified with 0.1% formic acid) at a flow rate of 5.0 mL/min. UV-Vis spectra were acquired at 254nm and 280nm on a 1200 Series Prep Scale diode array detector (Agilent, Santa Clara, USA). Collection was triggered by UV and collected on a 1200 Series Fraction Collector (Agilent, Santa Clara, USA).
Chemical shifts are quoted to 0.1 ppm, unless greater accuracy is required. * Denotes peaks observed only in HSQC spectra.

1 M TBAF, THF
Compound 8 (90 mg, 0.12 mmol) was dissolved in THF (2.5 mL) and TBAF (1.0 M in THF, 3.1 eq) was added and the reaction stirred until complete by TLC analysis. The reaction mixture was diluted with MeOH, the solvent removed under reduced pressure to give the crude product. 63% of the isolated crude material was dissolved in EtOH (1.5 mL). Triethylamine (1.5 eq.) and quinolone-6-methyl amine (1.5 eq.) in EtOH (1.5 mL) were added and the reaction stirred at 40 °C for 16 hours under N 2 .