Probing SH2-domains using Inhibitor Affinity Purification (IAP)

Background Many human diseases are correlated with the dysregulation of signal transduction processes. One of the most important protein interaction domains in the context of signal transduction is the Src homology 2 (SH2) domain that binds phosphotyrosine residues. Hence, appropriate methods for the investigation of SH2 proteins are indispensable in diagnostics and medicinal chemistry. Therefore, an affinity resin for the enrichment of all SH2 proteins in one experiment would be desirable. However, current methods are unable to address all SH2 proteins simultaneously with a single compound or a small array of compounds. Results In order to overcome these limitations for the investigation of this particular protein family in future experiments, a dipeptide-derived probe has been designed, synthesized and evaluated. This probe successfully enriched 22 SH2 proteins from mixed cell lysates which contained 50 SH2 proteins. Further characterization of the SH2 binding properties of the probe using depletion and competition experiments indicated its ability to enrich complexes consisting of SH2 domain bearing regulatory PI3K subunits and catalytic phosphoinositide 3-kinase (PI3K) subunits that have no SH2 domain. Conclusion The results make this probe a promising starting point for the development of a mixed affinity resin with complete SH2 protein coverage. Moreover, the additional findings render it a valuable tool for the evaluation of PI3K complex interrupting inhibitors.

NMR-spectroscopy: 1 H-NMR and 1 H, 1 H-COSY experiments were recorded using a Bruker DRX 500 instrument ( 1 H-NMR: 500.1 MHz). 13 C-NMR, HSQC and HMBC experiments were recorded using a Bruker Avance 600 instrument ( 1 H-NMR: 600.1 MHz , 13 C-NMR: 150.9 MHz). The recorded data was processed using TopSpin v 2.1, the spectra were evaluated using MestReNova software 6.0.2. Deuterated chloroform was usually used as solvent for NMR measurements with tetramethylsilane (TMS) as internal standard. Other deuterated solvents were referenced to the solvent peak. Structures of unpublished molecules were assigned with the help of 1 H, 1

H-COSY, HSQC and HMBC NMR experiments.
Mass-spectrometry: ESI-MS: ESI/APCI mass spectra were recorded using an Esquire 3000 ion trap mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with a standard ESI/APCI source. Samples were introduced by direct infusion with a syringe pump. Nitrogen served both as the nebulizer gas and the dry gas. Nitrogen was generated by a Bruker nitrogen generator NGM 11. Helium served as cooling gas for the ion trap and collision gas for MS n experiments. The spectra were recorded with the Bruker Daltonik esquireNT 5.2 esquireControl software by the accumulation and averaging of several single spectra. DataAnalysis™ software 3.4 was used for processing the spectra.

MALDI-MS: MALDI experiments were performed using a Fourier Transform Ion Cyclotron
Resonance (FT-ICR) mass spectrometer APEX III (Bruker Daltonik GmbH, Bremen, Germany) equipped with a 7.0 T, 160 mm bore superconducting magnet (Bruker Analytik GmbH -Magnetics, Karlsruhe, Germany), infinity cell, and interfaced to an external MALDI ion source. Nitrogen served both as the nebulizer gas and the dry gas for ESI. Nitrogen was generated by a Bruker nitrogen generator NGM 11. Argon served as cooling gas in the infinity cell and collision gas for MSn experiments. Scan accumulation and fourier transformation were performed with XMASS NT (7.08) on a PC Workstation, for further data processing DataAnalysis™ 3.4 was used. LC-MS: LC-MS analysis was accomplished using a Waters Alliance HT equipped with a Waters Symmetry 3.5 µm column (C 8 , 100 x 2.1 mm, eluent A H 2 O/HCOOH = 100:0.1, eluent B CH 3 CN/HCOOH = 100:0.1, flowrate 0.4 mL⋅min-1 using a gradient from 5-95% B over 10 minutes) coupled with a Waters micromass ZQ2000 ESI-MS.  (30 mL) was added dropwise to a solution of 2-(2-(2-aminoethoxy)ethoxy)ethanamine (14.5 g, 98 mmol) in THF (30 mL) over a period of 5 h. The resulting suspension was stirred overnight at rt prior evaporation of the solvent. The residue was dissolved in water (50 mL) and extracted with DCM (3x20 mL). The organic phases were washed with water (10 mL) dried over MgSO 4 and the solvent was evaporated. The crude product was used without further purification in the next step.

tert-Butyl 2-(2-(2-(pentylamino)ethoxy)ethoxy)ethylcarbamate (3)
Amine 2 (2.5 g, 10 mmol) and DIPEA (1.75 mL, 10 mmol) were prearranged in dry DCM (6 mL). A solution of 1-bromopentane (623 μL, 5 mmol) in DCM (1.5 mL) was added at rt over 4 h. After stirring at rt for 12 h the reaction mixture was dissolved in 1M NaOH solution and extracted with DCM (3x10 mL). The combined organic layers were dried over MgSO 4 and the solvent evaporated to give the crude product that was purified using flash chromatography ( (S)-2-Acetamido-3-(4-iodophenyl)propanoic acid (4) 4-Iodophenylalanine (764 mg, 2.6 mmol) was dissolved in Ac 2 O (30 mL) and treated with TEA (368 µL, 266 mg, 2.6 mmol). The reaction mixture was stirred at rt for 12 h prior to hydrolysis of the excess anhydride with water. The resulting suspension was concentrated to a volume of 5 mL and dissolved in 5% KHSO 4 solution (40 mL). The aqueous phase was extracted with EE (4x10 mL) and the combined organic phases were dried over MgSO 4 . The solvent was evaporated and the crude product was used in the next step without further purification.  (J. Med. Chem. 2007, 50, 856-864) Carboxylic acid 4 (925 mg, 2.8 mmol) was dissolved in MeOH (10 mL) and SOCl 2 (404 µL, 661 mg, 5.6 mmol) was added dropwise over 5 min at 4 °C. After stirring at rt for 2 h H 2 O (10 mL) was added carefully and the reaction mixture neutralized with 1M NaOH. MeOH was evaporated and the residue extracted with EE (5x15 mL). The combined organic layers were washed with 5% KHSO 4 solution, sat. NaHCO 3 solution and brine prior to drying over MgSO 4 and evaporation of the solvent. The title compound 5 was used as obtained in the next step.  (Tetrahedron 1997, 53, 3, 815-822) HCl activated Zn (279 mg, 4.3 mmol) was dissolved in DMAC (1 mL) and treated with diethyl(bromodifluoromethyl)phosphonate (760 µL, 1.1 g, 4.3 mmol). The exothermic reaction was stirred for 2 h without cooling prior to addition of CuBr (614 mg, 4.3 mmol) in one portion. The reaction mixture was stirred at rt for 30 min followed by addition of aryl iodide 5 (634 mg, 1.8 mmol). After stirring at rt for 3 d the reaction mixture was dissolved in EE (20 mL) and washed with 5% KHSO 4 solution (2x5 mL), sat. NaHCO 3 solution (2x5 mL) and brine (1x5 mL). The organic phase was dried over MgSO 4 and the solvent evaporated to give the crude product that was purified using flash chromatography (eluent: DCM/MeOH 99:1) to give the title compound 6. Unreacted starting material was recovered.  (Tetrahedron 1997, 53, 32, 11171-11178) Methyl ester 6 (102 mg, 0.25 mmol) was dissolved in a mixture of MeOH/THF/H 2 O (1:1:1, 2 mL) and treated with LiOH (12.6 mg, 0.3 mmol). The reaction mixture was stirred at rt for 30 min and subsequently quenched with formic acid (1 mL). Volatile components were evaporated and the residue purified using preparative RP-HPLC to give the title compound 7.   Current Medicinal Chemistry, 2000, 7, 1081-1100 The F2PmP probe 1 was synthesized using standard SPPS procedures on 2-chlorotrityl resin. The resin (500 mg) was incubated with a solution of Fmoc-L-Glu-OAll (614 mg, 1.5 mmol) and DIPEA (523 μL, 3 mmol) in DCM (5.0 mL) for 2 h at rt under argon atmosphere. The coupling solution was removed by filtration and the resin washed with DMF (5x10 mL) and DCM (5x10 mL). After incubation with MeOH (10 mL) for 10 min at rt the solvent was removed by filtration and the resin dried under reduced pressure.

Statistical analysis of the pervanadate depletion experiments
Supplementary Figure 4: Volcano plot of the pull-down data from the pervanadate depletion experiments.