SHetA2 interference with mortalin binding to p66shc and p53 identified using drug-conjugated magnetic microspheres

In order to attach compound SHetA2 [1] to a solid support while retaining the majority of the structure intact for protein binding, the known metabolite 2, which has a hydroxyl group available for attachment, was synthesized as described [21] (Structures of 1 and 2 are shown in Fig. 1). The SHetA2 metabolite 2 was then further modified as shown in Fig. 1. Amino-dPEG₄ acid was purchased from Quanta Biodesign Limited, Powell, OH 43065, and used as received. Tetrahydrofuran was dried over potassium hydroxide pellets and distilled from lithium aluminium hydride prior to use. Dichloromethane was used from a freshly opened bottle. All reactions were run under dry nitrogen in oven-dried glassware. The reaction was monitored by thin layer chromatography (TLC) on silica gel GF plates (Analtech No 21521). Purification was performed using preparative thin layer chromatography (PTLC) on a 20-cm × 20-cm silica gel GF plate (Analtech No 02015). Band elution for both methods was monitored using a hand-held UV lamp. The infrared (IR) spectrum was run as a thin film on sodium chloride disks. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were measured in deuteriochloroform at 300 MHz and 75 MHz, respectively, and were referenced to internal tetramethylsilane; coupling constants (J) were reported in Hz. The ultraviolet–visible (UV–vis) spectrum was collected for the sample using a Varian Cary 5000 spectrophotometer. The mass spectrum was collected for the sample using a Shimadzu LC-MS instrument.

A 25-mL, three-necked, round-bottomed flask, equipped with a reflux condenser and a magnetic stirrer was charged with 100 mg (0.24 mmol) of metabolite 2 and 5 mL of tetrahydrofuran. The solution was cooled to −40 °C, 29 mg (0.24 mmol) of 4-(dimethylamino)pyridine was added. The mixture was stirred for 5 min to dissolve the solid. To the resulting yellow solution was added dropwise a solution of 48 mg (0.239 mmol) of 4-nitrophenyl chloroformate in 1 mL of tetrahydrofuran, and the reaction mixture was allowed to stir for 30 min. At this time, TLC eluted with ether:hexane (4:1) confirmed that the reaction was complete (and formed 3).

A solution of exactly 63 mg (0.24 mmol) of amino-dPEG 4-acid in 3 mL of dichloromethane was carefully added to the above reaction mixture containing 3 at −40 °C over a period of 10 min. The reaction mixture was then slowly allowed to warm to room temperature over a period of 1 h. The reaction mixture then was stirred for 20 min, and concentrated under vacuum. Purification was achieved by PTLC eluted with 98:2 chloroform:methanol. After one elution, the product was washed from the silica gel using ethyl acetate followed by chloroform:methanol (95:5) to give 33 mg (20 %) of conjugate 4 as a pale yellow liquid. LCMS: (M+) 732 (Na+ adduct), 418; IR: 3375, 1740, 1640, 1592, 1515, 1475, 1335, 1110 cm⁻¹; ¹H NMR: 8.94 (s, 1 H), 8.76 (s, 1 H), 8.19 (d, J = 9.0 Hz, 2 H), 7.85 (obscured m, 1 H), 7.84 (d, J = 9.0 Hz, 2 H), 7.45 (s, 1 H), 7.13 (d, J = 8.2 Hz, 1 H), 5.46 (brt, J = ca 6.2 Hz, l H), 4.53 and 4.42 (2 AB patterns, J = 11.0 Hz, 1 H), 3.76 (t, J = 5.5 Hz, 2 H), 3.63 (m, 13 H), 3.40 (m, 2 H), 3.22 (m, 2 H), 2.60 (t, J = 5.5 Hz, 2 H), 2.36 (d, J = 14.2 Hz, 1 H), 1.83 (d, J = 14.2 Hz, 1 H), 1.43 (s, 6 H), 1.40 (s, 3 H); UV–vis (H₃COH): ƛmax = 285 nm (ε = 1752) and 335 nm (ε = 1456). The ¹³C NMR experiment could not be carried out due to the instability of the product. The product decomposed rapidly at 20 °C. Thus, the polyether was attached to the microspheres without further purification.

Only a small quantity of conjugate 4 was obtained, and it was transferred in dry ice to SoluLink (San Diego, CA). Compound 4 was converted to compound 5 by a proprietary method of SoluLink who provided a mass spectrum of the compound as MW - Na + 984, the primary m/z occurring at 884.20. Compound 5 was reacted with the magnetic microspheres through an amide linkage. This led to conjugate 6 attached to the NanoLink Amino-Magnetic Microspheres which were utilized to identify SHetA2 binding proteins.

Protein extracts of the A2780 human ovarian cancer cell line grown to 90 % confluency in 10 cm plates were isolated using m-PER-PPI (m-PER solution [Thermo Scientific] containing a protease inhibitor cocktail [Active Motif] and a phosphatase inhibitor cocktail [Active Motif]) and stored at −80 °C until use. The protein concentration of the extracts was measured with the BCA Protein Assay (Thermo Scientific). SHetA2-conjugated NanoLink Amino-Magnetic Microspheres 6 were pelleted with a magnet for 2 min, and the supernatant was removed. The pellet was washed once in 100 μL PBS and re-suspended in 100 μL of m-PER-PPI. Unconjugated NanoLink Amino-Magnetic Microspheres were manipulated under the same conditions in parallel as a negative control. The general experimental procedure is as follows with specific modifications detailed in the subsequent paragraphs and Table 1. Equal volumes of the SHetA2- and unconjugated-microsphere suspensions were pelleted and re-suspended in A2780 protein extract and incubated. The microspheres were pelleted with a magnet and washed one to three times with m-PER-PPI at a volume equal to the incubation volume. Bound proteins were then eluted with excess free SHetA2 in m-PER-PPI, followed by removal of the microspheres with a magnet. Aliquots of the washes and eluents of the SHetA2- and unconjugated microspheres were electrophoresed into 2-D SDS-PAGE gels. Four repeats of the experiment were performed in efforts to optimize differential bands observed in the SDS gel wells corresponding to SHetA2-microspheres in comparison to unconjugated microspheres.

In experiment 1, 100 μL of A2780 protein extract (6.8 mg total) was mixed with a volume of 100 μL of SHetA2 microspheres (containing approximately 5 μg or 12.45 nM SHetA2) from the starting solution and incubated at room temperature in the dark with agitation for 30 min. After 3 washes, bound proteins were eluted by incubating the microspheres in 10 μM SHetA2 for 10 min with agitation in the dark. Differential bands in the lanes corresponding to SHetA2 eluents from the SHetA2- and unconjugated microspheres could not be visualized upon staining the SDS-PAGE gel with Coomassie Blue.

Experiment 2 was designed to reduce the non-specific binding in SDS-gel bands corresponding to unconjugated microspheres by decreasing the amount of protein added and to increase the specific binding in SDS-gel bands corresponding to SHetA2-microspheres by increasing the incubation temperature. Because a limited amount of microspheres was available, both parameters were altered simultaneously. In experiment 2, 50 μL of A2780 protein extract (1.8 mg) was incubated with the SHetA2- or unconjugated magnetic microspheres at 37 °C for 30 min in the dark without agitation, followed by 5 min of slow agitation at room temperature in the dark. After one wash, bound proteins were eluted by incubating the microspheres with 1 mM SHetA2 in 20 μL of m-PER-PPI for 30 min at 37 °C in the dark. Again no differential bands were observed between the lanes corresponding to the SHetA2- and unconjugated microspheres eluents when the gel was stained with Coomassi blue; however, a differential band was observed upon staining of a repeat gel with the Plus One DNA Silver Staining Kit (Amersham Biosciences) (Fig. 2a).

Experiment 4 was designed to increase the yield by increasing the amount of protein added and to reduce the non-specific background binding by increasing the number of washes. Thus, 275 μL of SHetA2-microspheres (13.75 μg or 34.24 nM SHetA2) or unconjugated-microspheres were mixed with 200 μL of A2780 protein extract (approximately 10 μg), agitated for 5 min in the dark at room temperature and then incubated without agitation at 37 °C for 25 min in the dark. Two washes with 300 μL of m-PER-PPI were performed, followed by an elution step with 30 μL of 1 mM SHetA2 in m-PER-PPI for 5 min at 37 °C. A differential band was discerned between the lanes corresponding to the SHetA2- and unconjugated microspheres upon Coomassie blue staining of the SDS-PAGE gel; however, the non-specific binding was very high (Fig. 2b).

Efforts to regenerate and re-use the SHetA2-microspheres were unsuccessful. The specific bands in experiments 2 and 4 were evaluated by QStar Mass spectrometry and frozen aliquots of the eluents were thawed and evaluated by Orbitrap Mass spectrometry.

Identification of proteins that bind to the SHetA2 affinity resin was determined by a standard proteomics analysis as briefly described below. The protein bands were excised and cut into small pieces and destained with 5 mM sodium thiosulfate/15 mM potassium ferricyanide in water. After rinsing with 25 mM ammonium bicarbonate in 50 % acetonitrile, the proteins in the gel were reduced in 55 mM tris[2-carboxylethyl]phosphine in 25 mM ammonium bicarbonate at 60 °C for 10 min, followed by alkylation in 100 mM iodoacetoamide/25 mM ammonium bicarbonate at room temperature for 60 min. After washing the alkylation buffer, the gel pieces were washed in 50 % acetonitrile, followed by 100 % acetonitrile. In-gel tryptic digestion was conducted by adding 100 ng of trypsin (Sequencing Grade Modified Trypsin; Promega, Madison, WI) in 10 μL of 25 mM ammonium bicarbonate. The gel pieces were allowed to swell, and an additional 25 μL of 25 mM ammonium bicarbonate was added to submerge the gel pieces. The digestion reaction was incubated at 30 °C for 16 hrs. Twenty-five microliters of 1 % trifluoroacetic acid was added to stop the digestion reaction, and the tryptic peptides were extracted into 50 % acetonitrile/0.5 % trifluoroacetic acid. The extract was evaporated by a SpeedVac concentrator (Thermo Electron Corporation, Waltham, MA). All water used was an ultra-pure grade. A Dionex UltiMate 3000 HPLC interfaced to an ABI Sciex QStar Elite Hybrid Quadrupole TOF mass spectrometer was used to analyze the tryptic digests by peptide mass fingerprinting. MASCOT (Matrix Science, Boston, MA) analysis of the MS/MS data against NCBInr 20100405 identified three candidates that show Probability Based MOWSE Score higher than 41 (p < 0.05); gi|6470150 (Homo sapiens BiP protein; MOWSE Score, 1202), gi|5729877 (Homo sapiens heat shock cognate 71 kDa protein isoform 1; MOWSE Score, 1030), and gi|292059 (Homo sapiens MTHSP75; MOWSE Score, 423).

Aliquots of eluents from the SHetA2-microspheres and unconjugated microspheres were submitted for mass spectrometry analysis without electrophoresing them into gels. MS-grade solvents were from Burdick and Jackson, or Baker. Sequencing grade trypsin was from Promega. Other solutions were the highest grade available from Sigma-Aldrich. Samples were dissolved in 8 M urea, 100 mM Tris–HCl pH = 8.5 at RT, 5 mM tris(2-carboxyethyl)phosphine, and reduced at room temperature for 20 min. After incubation, 1/20th volume of 200 mM iodoacetamide was added. The alkylation was allowed to proceed for 15 min in the dark at room temperature, after which the samples were diluted with four volumes of 100 mM TrisHCl pH 8.5 and digested with 4 μg/mL trypsin overnight at 37 °C. Digested samples were acidified with 1 % formic acid, and purified by tip-based C18 chromatography (OMIX tips from Agilent). Samples were analyzed on a hybrid LTQ-OrbitrapXL mass spectrometer (Thermo Fisher Scientific) coupled to a New Objectives PV-550 nanoelectrospray ion source and an Eksigent NanoLC-2D chromatography system.

Peptides were analyzed by trapping on a 2.5 cm pre-column, followed by analytical separation on a 15–20 cm 75 μm ID fused silica column, both packed with Magic C18 AQ (Bruker). Columns were terminated with an integral fused silica emitter prepared in house. Peptides were eluted using a 5–40 % ACN/0.1 % formic acid gradient performed over 116 min at a flow rate of 250–300 nL/min. For each full-range Fourier transform mass spectrometry (FT-MS) scan (nominal resolution of 60,000), the six most intense ions were analyzed via data-dependent MS/MS in the linear ion trap using dynamic exclusion for 150 % of the observed chromatographic peak width. MS/MS settings used a trigger threshold of 8,000 counts, monoisotopic precursor selection (MIPS), and rejection of parent ions that had unassigned charge states or were previously identified as contaminants. Centroided ion masses were extracted using the extract_msn.exe utility from Bioworks 3.3.1 and were used for database searching with MASCOT v2.2.04 (Matrix Science) and X Tandem v2007.01.01.1 (www.​thegpm.​org).

Scaffold (Version 3, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95 % probability as specified by the Peptide Prophet algorithm [22]. Protein identifications were accepted if they could be established at greater than 99.0 % probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [23]. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. A t-test was performed to determine significant differences in proteins identified in the eluents from SHetA2-microspheres in comparison to the eluents from unconjugated microspheres. P values of less than 0.05 were considered to be statistically significant.

The A2780 human ovarian cancer cell line (gift of Michael Birrer, Harvard Medical School, Boston, MA) was cultured in RPMI 1640 tissue culture medium supplemented with 10 % fetal bovine serum (FBS), antibiotic/antimycotic, 1 nM sodium pyruvate and 1 mM HEPES buffer. Whole cell protein extracts were prepared from cultures treated with SHetA2 or DMSO for various times using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) or Triton × 100 lysis buffer consisting of 1 % Triton × 100, 10 mM Tris pH 7.4, 5 mM EDTA pH 8.0, and 50 mM NaCl. Cells were incubated and agitated by pipette or vortex every 5 min for 30 min. After incubation, samples were spun down for 3 min at 10,000 g to remove debris. Cell lysates were centrifuged to remove debris for 5 min at 3,000 g using a 1:100 ratio of anti-p66shc antibody (Santa Cruz, Cat # sc-967), anti-p53 antibody (Santa Cruz, Cat # sc-126) or anti-mortalin antibody (Cell signaling Cat # 3593) to lysate was added to each lysate immediately and incubated at 4 °C overnight. The next day, cells were incubated with Protein G-PLUS Agarose microspheres (Santa Cruz) for 1 hr. Microspheres were then washed 3 times with 1 mL of 10 % NP-40/Tris pH 8.0 buffer and centrifuged at 3,000 ×g for 4 min. Twenty microliters of SDS loading buffer was then added to each sample, and the solution was boiled for 5 min before being electrophoresed into a 12–15 % sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked in 5 % milk for 1 hr at room temperature, and then immunoblotted with the desired primary antibody (anti-mortalin antibody (Cell signaling Cat # 3593), anti-p53 antibody (Santa Cruz, Cat # sc-126) or anti-p66shc antibody (Santa Cruz, Cat # sc-967) and then incubated overnight at 4 °C. The membranes were then washed 3 times with 10 mL of PBS-Tween 20 (0.1 %), incubated with the appropriate HRP-conjugated secondary antibody for 30 min at room temperature and twashed 3 times with 10 mL of PBS-Tween 20 (0.1 %). Finally, specifically bound antibody was detected using Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and exposure to X-Ray negative film.