Supporting Information Labeled Ligand Displacement: Extending NMR-based Screening of Protein Targets Steven L. Swann, Danying Song, Chaohong Sun, Philip J. Hajduk, and Andrew M. Petros Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064 1
Experimental Section NMR sample preparation and spectroscopy. The ATPase domain from Hsp90 was prepared as previously described 12 with the exception that the growth media used was unlabeled ( 12 C, 14 N). Bovine erythrocyte carbonic anhydrase was purchased from Sigma- Aldrich Co. (catalog number C3934). NMR samples for Hsp90 were prepared in 50 mm sodium phosphate, ph 7.5, 30 mm sodium chloride, 10 mm magnesium chloride, 100% D 2 O, and carbonic anhydrase samples were in 50 mm sodium phosphate, ph 7.5, 30 mm sodium chloride, 100% D 2 O. Bcl-x L which lacks the putative transmembrane helix was prepared as previously described 17. Bcl-x L samples were in 40 mm sodium phosphate buffer, ph 7.0, 100% D 2 O. Uniformly 13 C-labeled ATP was purchased from Cambridge Isotope Labs (catalog number CNLM-4265). The 13 C-leucine labeled Bak peptide was purchased from Genescript. Ligands were added from stock solutions prepared in DMSO. All compounds except 2 and 4 (see below) were from our corporate compound repository. One-dimensional 13 C-selected spectra were recorded on a Bruker DRX600 spectrometer with a cryoprobe accessory at 298 K. One-dimensional 13 C-selected spectra were run as the first slice of a 13 C-HSQC spectrum. Total acquisition time for each spectrum was typically from 15 to 30 minutes. STD experiments were carried out on a Bruker DRX600 spectrometer with a cryoprobe accessory at 298 K. For these experiments, Hsp90 was at 10 um and the probe (compound 1) was at 100 um. Saturation was carried out with a shaped pulse train for for both on-resonance (0.00 ppm) and off-resonace (-8.45 ppm) spectra. The saturation time in each case was 2.5 s. A 36 ms spin-lock was used to suppress signals from the protein. 2
Chemistry All final products were purified by preparative HPLC on a Phenomenex Luna C8(2) 5 um 100Å AXIA column (30mm 75mm). A gradient of acetonitrile (A) and 0.1% trifluoroacetic acid in water (B) was used, at a flow rate of 70mL/min (0-0.5 min 10% A, 0.5-12.0 min linear gradient 10-95% A, 12.0-15.0 min 95% A, 15.0-17.0 min linear gradient 95-10% A). Samples were injected in 2.5mL dimethyl sullfoxide:methanol (1:1). A custom purification system was used, consisting of the following modules: Waters LC4000 preparative pump; Waters 996 diode-array detector; Waters 717+ autosampler; Waters SAT/IN module, Alltech Varex III evaporative light-scattering detector; Gilson 506C interface box; and two Gilson FC204 fraction collectors. The system was controlled using Waters Millennium32 software, automated using an Abbott developed Visual Basic application for fraction collector control and fraction tracking. Fractions were collected based upon UV signal threshold and selected fractions subsequently analyzed by flow injection analysis mass spectrometry using positive APCI ionization on a Finnigan LCQ using 70:30 methanol:10 mm NH 4 OH (aqueous) at a flow rate of 0.8 ml/min. Loop-injection mass spectra were acquired using a Finnigan LCQ running LCQ Navigator 1.2 software and a Gilson 215 liquid handler for fraction injection controlled by an Abbott developed Visual Basic application. 4-methyl-6-( 13 C 6 phenylpyrimidin-2-amine (2) A mixture of 4-chloro-6- methylpyrimidin-2-amine (0.1 g, 0.697 mmol), Aqueoeus (2N) Cs2CO3 (1. ml, 2.79 mmol), 13 C 6 Phenyl Boronic Acid (Cambridge Isotope Labs) and Pd(dppf) chloroform adduct (4.5mg, 0.035mmol) in dioxane (3.48mL) was stirred at 95 C for 4 hours. The cooled reaction was diluted with water and washed with CH 2 Cl 2 (3 X 10mLs). The 3
organic layer was dried and purified using HPLC and the desired product was isolated as a white solid (91mg, 71% yield) 1 H NMR (500 MHz, METHANOL-D4) δ 8.16 (d, J = 5.6, 1H), 7.85 (s, 1H), 7.62 (s, 1H), 7.30 (s, 1H), 7.03 (d, J = 1.7, 1H), 2.38 (s, 3H); ESI+MS m/z M+H = 192.2. 4-fluoro- 13 C 6 benzenesulfonamide (4) A mixture of 4-fluoro- 13 C 6 benzenesulfonylchloride (0.1 g, 0.514 mmol), and 7N ammonia in methanol (0.22ml, 1.54mmol) in THF (2.57mL) was stirred at 25 C for 12 hours. The reaction was concentrated to a clear oil which was purified using HPLC and the desired product was isolated as a white solid (45mg, 50% yield) 1 H NMR (500 MHz, METHANOL-D4) δ 1 H NMR (300 MHz, CDCL3) δ 8.26 (d, J = 14.5, 1H), 7.64 (dd, J = 4.8, 14.7, 1H), 7.55 7.40 (m, 1H), 6.91 (dt, J = 5.9, 13.6, 1H), 4.80 (s, 2H); ESI-MS m/z M-H = 180.1.. 4
A B C D Figure 1S. Effect of increasing protein (Hsp90, ATPase domain) concentration on the 13 C-selected, proton NMR spectrum of 13 C-labeled ATP (30 um). (A) No protein. (B) 60 um protein. (C) 120 um protein. (D) 180 um protein. The dissociation constant for the binding of ATP to the Hsp90, ATPase domain is about 132 um. 18 5
A B C D Figure 2S. Effect of increasing protein (Bcl-x L ) concentration on the 13 C-selected, proton NMR spectrum of a mutant Bak peptide (GQVGAQLAIIGDDINR) which is 13 C-labeled at the leucine residue. Peptide is at 20 um. (A) No protein. (B) 30 um protein. (C) 50 um protein. (D) 70 um protein. This peptide binds to Bcl-x L with a dissociation constant of ~ 0.5 um. 17 6
Table 1: Hsp90, ATPase Domain Competition Assay Ligand K (um) 3 Percent Compound 1 signal recovered 2 i 3 18 11 6 43 5 7 92 0.031 1 Compounds added at 60 um to a solution of 30 um protein plus 20 um probe. 2 Based on intensity of free probe at 20 um 3 TR-FRET assay 12 7
Table 2: Carbonic Anhydrase Competition Assay Ligand K (um) 3 Percent Compound 1 signal recovered 2 i 5 33 48 8 57 3 9 90 1 1 Compounds added at 100 um to a solution of 40 um protein plus 20 um probe. 2 Based on intensity of free probe at 20 um 3 Biacore assay 16 8