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1 Schenone, M.; Dancik, V.; Wagner, B. K.; Clemons, P. A., Target identification and mechanism of action in chemical biology and drug discovery. Nat. Chem. Biol. 2013, 9 (4), 232-40.
2 Swinney, D. C.; Anthony, J., How were new medicines discovered? Nat.
Rev. Drug Discov. 2011, 10 (7), 507-519.
3 Park, J.; Koh, M.; Koo, J. Y.; Lee, S.; Park, S. B., Investigation of Specific Binding Proteins to Photoaffinity Linkers for Efficient Deconvolution of Target Protein. ACS Chem. Biol. 2016, 11 (1), 44-52.
4 Kleiner, P.; Heydenreuter, W.; Stahl, M.; Korotkov, V. S.; Sieber, S. A., A Whole Proteome Inventory of Background Photocrosslinker Binding.
Angew. Chem. Int. Ed. 2017, 56 (5), 1396-1401.
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5HSURGXFWLRQ RI WKH IROORZLQJ DUWLFOH IRU WKH WKHVLV LV DOORZHG E\ WKH 5R\DO 6RFLHW\ RI &KHPLVWU\ Park, H.; Koo, J. Y.; Srikanth, Y. V. V.; Oh, S.; Le e, J.; Park, J.; Park, S. B., Nonspecific protein labeling of photoaffinity linkers correlates with their molecular shapes in living cells. Chem. Co mmun. 2016, 52 (34), 5828-5831.
1.2. Introduction
Photoaffinity-based approach has received attention over the last few decades for target identification and activity-based protein profiling.1 In the conventional affinity-based protein pull-down approach, bioactive small molecules with excellent potency (subnanomolar range activity) are generally required for successful enrichment of the interacting proteins.2 Even with highly potent bioactive compounds, experimental conditions such as salt concentration, type of detergents, and temperature often interfere with the protein±small molecule interaction. This may lead to unexpected experimental failure in target identification.3 In contrast, the photoaffinity-based target identification approach secures the interaction between small-molecule probes and their binding proteins under any experimental condition via the formation of UV-mediated covalent bonds.
This approach expands the range of target identification from only high- affinity molecules to low-affinity molecules (micromolar range activity).4 However, nonspecific protein labeling by photoaffinity linkers (PLs) has been a major concern in the photoaffinity-based approach.5±9
1.3. Results and discussion
1.3.1. Design and in silico analysis of 5 photoaffinity linkers
Although a list of reports have emphasized the importance of probe design strategy for photoaffinity-based target identification, the molecular shape of PLs has been overlooked in the design of photoaffinity probes. Initially, we hypothesized that the molecular shape of PLs determines their flexibility, resulting in different protein-labeling patterns caused by nonspecific interaction. Following this initial hypothesis, we attempted to find a PL with minimal nonspecific labeling. To investigate molecular shape-dependent protein-labeling, we designed and synthesized 5 different PLs (1, 2, 3, 4, and 5) containing commonly used photoactivatable moieties (benzophenone and diazirine) and an acetylene moiety for bio-orthogonal fluorescence labeling via Click chemistry (Fig. 1.1). Benzophenone-based PLs 1 and 2 have different shapes (1: linear, 2: branched), as do diazirine- based PLs 3 and 4. In addition, we designed and synthesized a branched PL 5 with the same number of total carbon and nitrogen atoms as the linear PL 3, but smaller than another branched PL 4. In silico analysis of surface charges showed clear differences between linear and branched PLs (Fig.
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1.2a), which might lead to different noncovalent interactions with various proteins. The difference in molecular shapes resulted in a different number of conformers. As shown in Fig. 1.2b, we simulated and counted the number of PL conformers having less than 2 kcal/mol difference from its energy minimum. The numerical value of 2 kcal/mol was derived from our assumption that conformers of each PL within a range of 2 kcal/mol from energy minimum can freely exist in the cellular environment under the physiological condition. The calculation results showed that linear PLs (1 and 3) have more conformers than branched PLs (2, 4, and 5). The conformational flexibility of linear PLs might cause the nonspecific, indiscriminate interactions via induced fit with various proteins. Based on their molecular shapes, we hypothesized that linear PLs would show more nonspecific protein labeling than branched PLs.
1.3.2. Protein-labeling patterns of 5 photoaffinity linkers
To examine the actual protein-labeling patterns of each PL, we mixed the 5 different PLs with either cell lysates or living cells. The mixtures were then subjected to UV irradiation to fix their transient binding with proteins by generating covalent bonds with the bound proteins. Proteins crosslinked with each PL were labeled with Cy5-azide using copper-mediated bioorthogonal Click chemistry and visualized in a fluorescent scanner after
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1D-gel electrophoresis. Each PL showed different protein-labeling patterns in cell lysates and living cells (Fig. 1.3). As expected from calculated data, the protein-labeling patterns, particularly in living cells, were highly dependent on the molecular shapes of PLs. In living cells, branched PLs resulted in less nonspecific protein labeling than linear PLs. We also observed that the smaller branched PL 5 showed significantly reduced nonspecific labeling in comparison with the larger branched PL 4 (Fig. 1.3, right panel, 4 and 5), which was surprising given that these PL structures differ by only 4 methylene units (4: C21H35N5O4, 5: C17H27N5O4). This result is in accordance with the findings of a previous study reporting the importance of small PLs.6a In cell lysates, nonspecific protein labeling by PL 2 was significantly lower than that by PL 1, but the protein-labeling patterns of diazirine-based PLs (3, 4, and 5) were generally similar and showed a little difference in protein bands in accordance with changes of their molecular shapes. The overall differences in protein-labeling patterns by various diazirine-based PLs in cell lysates were not as marked as that in
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living cells, which might be due to the fact that the diazirine-based PL conformers are not as diverse as we expected. Considering the much higher protein concentrations and complex protein networks with various protein
protein interactions in living cells,10 protein complex surfaces inside living cells should be more diverse than those in cell lysate. We believe that the diverse protein surfaces in living cells delineate the difference in the protein-labeling patterns of diazirine-based PLs. This observation was confirmed again by 2D-gel electrophoresis. Examination of the nonspecific protein-labeling patterns of the 5 different PLs showed that in living cells, branched PLs tended to bind fewer proteins than linear PLs, which is in accordance with our original hypothesis regarding the molecular shape and conformational flexibility of PLs (Fig. 1.4).
1.3.3. Protein-labeling of benzophenone-based probes derived from a tubulin inhibitor
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Based on this observation, we predicted that photoaffinity probes with branched PLs might show reduced nonspecific labeling and increased specific binding of the target proteins. To test this assumption, we designed a series of photoaffinity probes with various PLs using compound 6, reported as a tubulin-targeting anticancer agent.11 We first synthesized photoaffinity probes 7 and 8 by attaching benzophenone-based PLs 1 and 2 to the derivatives of 6 (Fig. 1.5). Then, we compared the protein-labeling patterns of probes 7 and 8 in either cell lysates or living cells in the absence or presence of agent 6. After photo-crosslinking, the proteomes were labeled with Cy5-azide and analyzed using 1D-gel electrophoresis. Probe 7 (with a linear PL) showed more nonspecific protein labeling than probe 8 (with a branched PL) in both cell lysates and living cells (Fig. 1.6a). The treatment concentration of each probe and agent 6 was chosen according to our previous report11,13 and a dose-dependent study. Interestingly, these protein-labeling patterns were consistent with those of PLs 1 and 2 themselves. However, tubulin was not selectively labeled by either probe 7 or 8 in cell lysates. In contrast, the overall protein-labeling patterns in living
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cells by probe 7 were not perturbed by an excess amount of soluble ligand
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6 in the 1D gel. But, fewer proteins were labeled by 8 contained a branched PL than by 7. Moreover, the target protein tubulin (Fig. 1.6a: asterisk) was clearly labeled by 8; the labeling of target proteins by probe 8 was inhibited in the presence of soluble ligand 6, which was not observed for probe 7.
Therefore, we concluded that the protein-labeling patterns can be significantly affected by changing the molecular shapes of the PLs in the photoaffinity probes.
To improve the resolution of the protein analysis, we applied the dual- color system, running 2 different samples on a single and the largest 2D gel,11 which ruled out the gel-to-gel variation. We used Cy5-azide or Cy3- azide for bio-orthogonal reaction with proteins labeled with each photoaffinity probe in the absence or presence of 6, respectively. Proteins labeled in yellow (Cy3+Cy5) or green (Cy3) are likely to be nonspecific binders. In contrast, proteins showing as red (Cy5) are probably potential target proteins of soluble ligand 6. This substantially simplifies the target identification process. As observed in the 1D gel, the photoaffinity probe 7 showed more nonspecific binding than probe 8 in both cell lysates and living cells (Fig. 1.6b) and intensive protein labeling by probe 7, but not by 8, was observed in cell lysates. In terms of specificity/selectivity, both photoaffinity probes (7 and 8) failed to label the target protein tubulin in cell lysates (Fig. 1.6b and 1.6c), which demonstrates the importance of the cellular environment for the interaction between proteins and small molecules.12 In living cells, the protein-labeling intensities by probes 7 and 8 were not very different. However, the number of protein spots labeled by 7 was larger, as observed in 1D gel (Fig. 1.6a and 1.6b). The enlarged sections of 2D-gel images showed that not only the number of protein spots but also the labeling patterns depended on the molecular shapes of PLs (Fig.
1.6c). Protein a (vimentin) and protein b (tubulin) were labeled in yellow by 7, but in red by 8. To identify the protein, we excised the protein spot
from 2D gel and trypsinized for mass spectrometry (MS) analysis. The immunostaining results showed that 6 did not functionally modulate the vimentin filament. Although the probe 8 could label tubulin, its selectivity for target protein was good enough to be used as a tubulin probe.
1.3.4. Protein-labeling of diazirine-based probes derived from a tubulin inhibitor
To find the PL with the best selectivity, we moved our attention to diazirine-based PLs. Recent studies have reported the superior properties of diazirine-based PLs in comparison with benzophenone-based PLs.6a,14 Thus, we decided to reduce the nonspecific labeling of photoaffinity probes by using diazirine-based PLs (3, 4, and 5). We synthesized 3 different photoaffinity probes 9, 10, and 11 (Fig. 1.7). In cell lysates, an excess amount of soluble ligand 6 did not affect protein labeling by probes 9, 10, and 11. Protein labeling intensities of 9 and 11 were similar to each other, which was in accordance with the results observed for protein-labeling
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patterns of PLs 3 and 5 (Fig. 1.8a, right panel). Protein labeling of probe 10 is a little higher than that of probes 9 and 11. We suspect the ligand part of probe 10 increased the overall protein labeling. In living cells, the overall protein-labeling patterns created by probes 9, 10, and 11 were consistent with the patterns for PLs 3, 4, and 5 (Fig. 1.8a, left panel). Photoaffinity probes with diazirine-based PLs (9, 10, and 11) were more specific than probes with benzophenone-based PLs (7 and 8). The target protein tubulin was clearly labeled by probes 9, 10, and 11, which supports the superiority of diazirine-based probes over benzophenone-based ones.6a,14 The photoaffinity probe with a diazirine-based linear PL (9) labeled tubulin more selectively than the probe with a benzophenone-based linear PL (7).
Nonspecific protein labeling by branched probes (8 and 10) was also markedly reduced than that by linear probes (7 and 9). Most importantly, the smaller branched diazirine-based probe (11) eliminated more nonspecific binding than all other photoaffinity probes.
To analyze the protein-labeling patterns with higher resolution, we performed dual-color 2D-gel electrophoresis using 9, 10, and 11. In cell lysates, none of the protein-labeling patterns for all these probes was affected by the presence of 6. Most proteins were labeled in yellow, which agreed with the results of 1D-gel experiments (Fig 1.8a and 1.8b). In contrast, we observed clear differences among protein-labeling patterns by 9, 10, and 11 upon treatment with a large excess of soluble ligand 6 in living cells; several proteins were labeled in red (Fig. 1.8b). The number of protein spots in red was reduced as PLs were changed from linear (9) to branched (10) and to smaller branched PLs (11). Among proteins labeled by probe 9, one of the proteins located on the right-hand side of the 2D gel was lactate dehydrogenase, a specific binder of a linear diazirine-based PL, which is not specific to agent 6 as we previously reported.13 Other proteins labeled by 9, 10, and 11 were located in the upper left region of the 2D gel (dotted
rectangle). In the magnified images (Fig. 3d), the color-labeling patterns of three protein spots were determined by the molecular shape of PLs in the
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photoaffinity probes. Probe 9 labeled proteins a, b, and c in red. Probe 10 labeled protein a and b, and probe 11 labeled protein b in red. Protein a, b, and c were excised from 2D gel and trypsinized for MS analysis. We have previously demonstrated that vimentin (protein a) and HSP60 (protein c)11 are not the target proteins of agent 6. Only tubulin (protein b) is the target protein of agent 6, confirmed by functional biochemical assays.11 The photoaffinity probe 11 with a smaller branched PL selectively labeled the target protein tubulin. Probes 9 and 10 labeled tubulin, but they also bound other non-target proteins; such behavior increases the amount of time and efforts put in target validation. These results confirmed that photoaffinity probes with branched PLs are generally better than those with linear PLs.
Moreover, a smaller branched PL in this study shows lower nonspecific labeling and higher selectivity toward target protein, tubulin.
1.4. Conclusion
In conclusion, we systematically studied the molecular shape-dependent protein labeling of PLs. Direct comparison of 5 different PLs with various molecular shapes and photoactivatable moieties showed significantly reduced nonspecific protein labeling by branched PLs in comparison with linear PLs in living cells. This result is probably due to the high conformational flexibility of linear PLs. The application of these findings in the design of photoaffinity probes clearly confirmed the superiority of branched PLs in specific labeling procedures. Exploiting the molecular shape-dependent properties of PLs, we synthesized a tubulin-selective photoaffinity probe 11 and demonstrated that the photoaffinity-based approach with a well-designed probe can identify the target proteins in living cells effectively. Currently, we plan to apply our result to a series of bioactive compounds having known multiple target proteins to monitor the labeling efficiency of compactly branched PL toward each target protein.
1.5. Materials and methods
Cell culture procedure. HeLa (human cervical adenocarcinoma cell line) was obtained from American Type Culture Collection [ATCC, USA]. HeLa cells were cultured in RPMI 1640 [GIBCO, USA] supplemented with 10%
(v/v) fetal bovine serum [GIBCO, USA] and 1% (v/v) antibiotic- antimycotic solution [GIBCO, USA]. The cells were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 C, and cultured in 7 )ODVN >1DOJHQH 1XQF ,QWHUQDWLRQDO 86$@ DFFRUGLQJ WR PDQXIDFWXUHUV¶
instruction. The growth medium was changed every two to three days. Cells were grown to confluence prior to the experiment.
Antiproliferative activity assay. Cell viability was measured to determine the cytotoxicity of our probe using the Cell Counting Kit (CCK)-8 assay [Dojindo, Japan] and the experimental procedure is based on the PDQXIDFWXUHU¶V PDQXDO +H/D FHOOV ZHUH FXOWXUHG LQWR -well plates at a density of 2 × 103 cells/well for 24 h, followed by the treatment of 6, 7, 8, 9, 10, and 11. After 72 h incubation ZLWK HDFK FRPSRXQG ȝ/ RI :67-8 solution [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt] was added to each well, and plates were incubated for additional 1 h at 37 °C. The absorbance of each well at 450 nm was measured using Synergy HT [Bio-Tek, USA]. The percentage of cell viability was calculated by following formula: % cell viability = (mean absorbance in test wells) / (mean absorbance in control well) × 100. Each experiment was performed in triplicate.
1D Gel Fluorescence Imaging
Cell lysate labeling. HeLa cell (150 dish * 3) was scrapped with cold
Ca2+- and Mg2+-free phosphate buffered saline (PBS) [WelGENE, S. Korea]
and centrifuged. The supernatant was discarded, and cell pellet was kept at
±80 C until use. RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP- 40, 0.5% deoxycholate, protease inhibitor [Roche, Switzerland]) was added to the cells for lysis. The cells were incubated for 15 min on ice. The cell lysate was centrifuged at 4 °C, 13000 rpm for 15 min. Supernatant was taken, and the protein concentration was measured with BCA assay kit [Thermo, USA], and protein concentration was adjusted to 1 mg/mL. The protein and compounds was mixed. The mixture was incubated at RT for 30 min. 365-nm UV light from BLAK-Ray (B-100AP) UV lamp [UVP, USA] was irradiated to the mixture for 30 min on ice. The mixture was under click chemistry with Cy5-azide [Lumiprobe, USA] (40 M), tris[(1- benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) [Sigma, USA] (100
M), CuSO4 [Sigma, USA] (1 mM), tris(2-carboxyethyl)phosphine (TCEP) [TCI, Japan] (1 mM) and t-BuOH [Sigma, USA] (5%) for 1 h. 5X Laemmli buffer was added and incubated at 95 °C for 5 min. The protein samples were separated by 1DGE and scanned with Typhoon Trio.
Live cell labeling. HeLa cells were seeded on 6 well plates. Compounds were treated for 3 h. 356-nm UV light was irradiated to the mixture for 30 min on ice. The cells were wash with PBS and kept at ±80 C until use.
RIPA buffer was added to the cells for lysis. The cells were incubated for 15 min on ice. The cell lysate was scrapped and centrifuged at 4 °C, 13000 rpm for 15 min. Supernatant was taken and the protein concentration was measured with BCA assay, and protein concentration was adjusted to 1 mg/mL. The mixture was under click chemistry with Cy5-azide (40 M), TBTA (100 M), CuSO4 (1 mM), TCEP (1 mM) and t-BuOH (5%) for 1 h. 5X Laemmli sample buffer was added and incubated at 95 °C for 5 min.
The protein samples were separated by 1DGE and scanned with Typhoon
Trio.
Fluorescence detection and quantification. The in-gel fluorescence signal was visualized at the Cy3 (532 nm excitation) or Cy5 (633 nm excitation) channel by Typhoon Trio [Amersham Bioscience, USA] and analyzed by ImageQuant TL program [Amersham Bioscience, USA].
2D Gel Fluorescence Imaging
Cell lysate labeling. HeLa cell (150 dish * 3) was scrapped in cold PBS and centrifuged. The supernatant was discarded, and cell pellet was kept at
±80 °C until use. RIPA buffer was added to the cells for lysis. The cells were incubated for 15min on ice. The cell lysate was centrifuged at 4 °C, 13000 rpm for 15 min. Supernatant was taken, the protein concentration was measured with BCA assay, and protein concentration was adjusted to 1 mg/mL. The protein and compounds were mixed. The mixture was incubated at RT for 30 min. 356-nm UV light was irradiated to the mixture for 30 min on ice. The mixture was under click chemistry with Cy5-azide or Cy3-azide [Lumiprobe, USA] (40 M), TBTA (100 M), CuSO4 (1 mM), TCEP (1 mM) and t-BuOH (5%) for 1 h. Acetone was added to the mixture for precipitation, and the mixture was kept at ±20 C for 20 min.
The mixture was centrifuged at 4 C, 14,000 rpm for 10 min. Supernatant was discarded, and the pellet was washed with cold acetone two times. The pellet was resolved with rehydration buffer (7 M Urea, 2 M Thiourea, 2%
CHAPS (w/v), 40 mM DTT, IPG buffer (5 l/ml) in ddwater). The proteome labeled with photoaffinity probe in the absence (labeled with Cy5-azide) and presence of 6 (labeled with Cy3-azide) were mixed (1:1 ratio). The proteins were separated on 2DGE and scanned with Typhoon Trio.
Live cell labeling. HeLa cells were seeded on 6 well plate. The cells were treated with compounds for 3 h. 356-nm UV light was irradiated to the mixture for 30 min on ice. The cells were wash with PBS and kept at ±80 C until use. RIPA buffer was added to the cells for lysis. The cells were incubated for 15 min on ice. The cell lysate was scrapped and centrifuged at 4 °C, 13000 rpm for 15 min. Supernatant was taken and the protein concentration was measured with BCA assay and protein concentration was adjusted to 1 mg/mL. The mixture was under click chemistry with Cy5- azide or Cy3-azide (40 M), TBTA (100 M), CuSO4 (1 mM), TCEP (1 mM) and t-BuOH (5%) for 1 h. Acetone was added to the mixture for precipitation, and the mixture was kept at ±20 C for 20 min. The mixture was centrifuged at 4 C, 14000 rpm for 10 min. Supernatant was discarded, and the pellet was washed with cold acetone two times. The pellet was resolved with rehydration buffer. The proteome labeled with photoaffinity probe in the absence (labeled with Cy5-azide) and presence of 6 (labeled with Cy3-azide) were mixed (1:1 ratio). The proteins were separated on 2DGE and scanned with Typhoon Trio.
2-dimensional gel electrophoresis (2DGE). Isoelectric focusing (IEF) was performed with 24 cm pH 3 10 ImmobilineTM Drystrip gel [GE healthcare, USA] using Ettan IPGphor3 IEF system [GE healthcare, USA]. The Drystrip was loaded to 12% SDS-PAGE gel, and proteins were separated by Ettan Daltsix [GE healthcare, USA].
MS Analysis for Protein Identification
Protein spots were excised from 2D gel and dehydrated in acetonitrile for 10 min. Acetonitrile was removed and dried under reduced pressure. For mass analysis, the resulting gel pieces were re-swelled at 4 °C for 45 min
in buffer containing trypsin and 50 mM (NH4)2CO3 and incubated overnight at 37 °C for tryptic digestion. The samples with gel pieces were centrifuged, and the supernatant was collected for mass analysis. The residual peptides in gel pieces were further extracted with 50% acetonitrile containing 20 mM (NH4)2CO3 and 5% formic acid three times at room temperature. The combined peptide samples were condensed down in SpeedVac until the desired sample concentration was reached. MS analysis was performed with the instruments in NICEM at Seoul National University. LC-MS/MS experiments were performed using an integrated system consisting of HPLC [Dionex, Ultimate 3000 RSLCnano system, USA] interfaced to Q Exactive [Thermo Scientific, USA] equipped with a nano-electrospray ionization source. Peptides were reconstituted in solvent A [water/acetonitrile (98:2, v/v), 0.1% formic acid] and then injected into LC- nano ESI-MS/MS system. Samples were first trapped on a trap column [Acclaim pepmap100, 100 m i.d. 20 mm, 5 m, 10 nm, Thermo Scientific, USA] and washed for 10 min with 98% solvent A and 2%
solvent B [water/acetonitrile (2:98, v/v), 0.1% Formic acid] at a flow rate of 4 L/min, and then separated on a Zorbax 300SB-C18 capillary column (75 m i.d. 150 mm, 3.5 m, 10 nm) at a flow rate of 300 nL/min. The LC gradient was run at 2% to 45% solvent B over 30 min, then from 45%
to 70% over 10 min, followed by 70% solvent B for 5 min, and finally 5%
solvent B for 15 min. Resulting peptides were electrosprayed through a coated silica tip [FS360-20-10-N20-C12, PicoTip emitter, New Objective]
at an ion spray voltage of 2,000 eV. The raw data from Q Exactive was processed with Proteome Discoverer 1.3 software [Thermo Scientific, USA]. To process an MS/MS spectrum, a thorough search was performed against a NCBI database. All the protein MS data can be found in excel file of supporting information.
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