Identification of Protein Targets of Bioactive Small Molecules Using

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Identification of Protein Targets of Bioactive Small Molecules Using Randomly Photomodified Probes Petr Šimon, Tomáš Knedlík, Kristýna Blažková, Petra Dvo#áková, Anna B#ezinová, Libor Kostka, Vladimír Šubr, Jan Konvalinka, and Pavel #ácha ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00791 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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ACS Chemical Biology

Identification of Protein Targets of Bioactive Small Molecules Using Randomly Photomodified Probes Petr Šimon1‡, Tomáš Knedlík1,2‡, Kristýna Blažková1,3, Petra Dvořáková1,3, Anna Březinová1, Libor Kostka4, Vladimír Šubr4, Jan Konvalinka1,2* and Pavel Šácha1* 1 Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 16610, Prague 6, Czech Republic 2 Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 12843, Prague 2, Czech Republic 3 Department of Cell Biology, Faculty of Science, Charles University, Viničná 7, 12843, Prague 2, Czech Republic 4 Institute of Macromolecular Chemistry of the Czech Academy of Sciences, Heyrovského n. 2, 16206, Prague 6, Czech Republic

Supporting Information Placeholder ABSTRACT: Identifying protein targets of bioactive small molecules often requires complex, lengthy development of affinity probes. We present a method for stochastic modification of small molecules of interest with a photoactivatable phenyldiazirine linker. The resulting isomeric mixture is conjugated to a hydrophilic copolymer decorated with biotin and a fluorophore. We validated this approach using known inhibitors of several medicinally relevant enzymes. At least a portion of the stochastic derivatives retained their binding to the target, enabling target visualization, isolation, and identification. Moreover, the mix of stochastic probes could be separated into fractions and tested for binding affinity. The structure of the active probe could be determined and the probe re-synthesized to improve binding efficiency. Our approach can thus enable rapid target isolation, identification, and visualization, while providing information required for subsequent synthesis of an optimized probe.

Despite the considerable success of rational drug discovery and de novo drug design, phenotypic screening remains a popular tool in academia and industry for discovery of novel bioactive small molecules.1, 2 However, subsequent identification of the targets of lead compounds remains a challenge. Chemical proteomics3, 4, functional genomics5, bioinformatics, and other methods may be applied to this task.6 Most experimental approaches rely on the preparation of an affinity probe, which is often a laborious, trial-and-error process. The probe typically consists of a small molecule derivative, a linker, and a reporter tag (a fluorophore and/or affinity tag). The most crucial, and typically most challenging, step in probe synthesis is selection of an appropriate position for linker attachment (Figure 1a). In the absence of structural information about the interaction between the small molecule and its target, rational design of linker attachment is not possible. The optimal linker attachment is usually found (if at all) through time-consuming structure-activity-relationship studies. Furthermore, probe prepa-

ration often requires numerous synthetic steps (more than 20 steps have been reported7-10). To overcome these challenges, we set out to develop a new methodology for facile preparation of affinity probes for identification of unknown targets of biologically active small molecules (Figure 1b). This new approach utilizes our recently developed N(2-hydroxypropyl)methacrylamide (HPMA) conjugates called iBodies.11, 12 iBodies are rationally designed conjugates decorated with targeting ligands attached to the copolymer backbone via linkers positioned according to structural analysis of the proteininhibitor complexes. The multitude of inhibitor molecules present on the resulting conjugate considerably increases binding affinity for the target.12 In our new approach, the bioactive small molecule is first stochastically modified with a photoactivatable linker (containing the carbene-generating group 3-trifluoromethyl-3-phenyldiazirine). Upon UV irradiation, it forms a reactive carbene that reacts unselectively with the small molecule and yields a mixture of isomers with randomly attached linkers (Figure 1b). This mixture is subsequently purified, analyzed by mass spectrometry, and conjugated to HPMA copolymer via free amino groups. The resulting conjugate contains a stochastic mixture of isomers of the bioactive small molecule attached via a flexible linker to the hydrophilic HPMA copolymer backbone. The HPMA conjugate also contains an affinity tag, such as biotin, and/or a fluorophore, such as ATTO488, for efficient isolation of the target from complex matrices and visualization in cells, respectively (Figure 1b). RESULTS AND DISCUSSION Development of stochastic conjugates. To establish the practicality of our approach, we prepared conjugates targeting various enzyme classes: serine proteases (fibroblast activation protein), metallopeptidases (glutamate carboxypeptidase II), and aspartic proteases (pepsin, cathepsin D, HIV protease). As affinity probes (or “targeting ligands”), we selected known inhibitors of these enzymes (compounds 1, 2, 3; Figure 2a-c).

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Figure 1. Affinity probe-HPMA copolymer conjugates for target identification. a) Development of affinity probes with “rationally” attached linkers is laborious and time-consuming, since it requires optimal linker attachment. b) The stochastic photomodification approach employing a photoactivatable linker generates a mixture of affinity probe isomers with linkers attached at various positions. In the second step, the photomodified affinity probes are conjugated to hydrophilic HPMA copolymer decorated with biotin for subsequent isolation of the target and the fluorophore ATTO488 for target visualization. Fibroblast activation protein (FAP), expressed in stromal fibroblasts of epithelial cancers, is being evaluated as a potential target molecule for cancer diagnostics and therapy.13 Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is a promising therapeutic and diagnostic target for prostate cancer. The structural biology of GCPII and molecular recognition of its inhibitors are well-understood.14, 15 Aspartic proteases (represented by bovine pepsin, cathepsin D from Ixodes ricinus,16 and HIV protease17) are members of a family of hydrolases that includes many viral and intracellular enzymes implicated in cancer development and viral replication. All these enzymes are inhibited to different levels by the class-specific inhibitor pepstatin A. Figure 2. Small-molecule compounds used for preparation of affinity probe conjugates. a) FAP inhibitor. b) GCPII inhibitor. c) Pepstatin A, a class-specific inhibitor of aspartic proteases. d) Photoactivatable PEG linker, attaching the inhibitor molecule to the HPMA copolymer. The arrows show linker positions for conjugates prepared by structure-assisted design.11, 12 Using our novel methodology, we prepared “stochastic” conjugates targeting these model enzymes and compared them with previously developed “designed” conjugates (i.e., iBodies) targeting the same proteins.11, 12 Using enzyme-specific activity assays, we first verified that the inhibitors (1, 2, and 3) modified with a phenyldiazirine linker (Figure 2d; Supporting Figure 1-4) and the resulting stochastic conjugates I, II, and III were able to inhibit the corresponding enzymes (Supporting Table 1 and 3). The inhibition constants of the photomodified inhibitors were higher than those of the free inhibitors, which suggests that only a fraction of the inhibitors was modified with the phenyldiazirine linker in a way that did not compromise binding to the enzyme. Correctly modified inhibitor moieties present on the HPMA backbone enabled strong binding to the corresponding enzymes. We obtained the following inhibition constants (referring to the polymer as a whole): 26 nM for FAP (stochastic conjugate I), 0.3 nM for GCPII (stochastic conjugate II) and 11 nM for HIV protease (stochastic conjugate III) (Supporting Table 3). These affinities are comparable to those of monoclonal antibodies (mAbs) raised against these targets18 and thus should suffice for most in vitro and in vivo applications. Stochastic conjugates isolate the corresponding proteins. We used our stochastic conjugates to isolate FAP, GCPII, and aspartic proteases from complex protein matrices. All the model enzymes were readily pulled down from cell lysates (Figure 3). When compared with iBodies decorated with inhibitors containing linkers prepared using structure-assisted design (Figure 2),11, 12 the stochastic conjugates have slightly reduced binding. Nevertheless, the affinity remains sufficient for the isolation of target proteins from a complex mixture (Figure 3). This result demonstrates the ability to use these stochastic conjugates for pull-down and identification of protein targets of bioactive small molecules. The binding of the polymer conjugates to their protein targets can be quan-

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titatively outcompeted by small molecule inhibitors, as exemplified by anti-GCPII stochastic conjugate II and low-molecularweight GCPII inhibitor 2-(phosphonomethyl)pentanedioic acid (2PMPA) (Supporting Figure 5). Figure 3. Use of stochastic conjugates I, II and III to isolate model enzymes from cell lysates. The conjugates were pre-bound to streptavidin agarose beads and then mixed with the proteincontaining samples. As positive controls (PC), enzyme-specific iBodies were used.11, 12 Negative controls used include HPMA conjugate lacking inhibitor (NC) and blank streptavidin agarose [NC(SA)]. Ten microliter samples were loaded onto the gel. a) FAP was isolated from FAP stable transfectant cell lysate by stochastic conjugate I (silver stained gel). b) GCPII was isolated from LNCaP cell lysate by stochastic conjugate II (western blot). GCPII was visualized by the mouse GCPII selective mAb GCP-0419, followed by IRDye® 800CW goat anti-mouse secondary antibody conjugate. c, d, e) Pepsin, cathepsin D, and HIV protease were isolated from cell lysates spiked with the corresponding enzyme (“Load”) by stochastic conjugate III (silver stained gels). Stochastic conjugates visualize FAP and GCPII on cells. Additionally, we evaluated the ability of the conjugates to detect proteins expressed on the cell surface. Using flow cytometry (Figure 4) and confocal microscopy (Figure 5 and Supporting Figure 6), we analyzed cells incubated with the corresponding conjugates and positive and negative controls. The conjugates strongly stained cells expressing GCPII and FAP, whereas they did not bind to GCPII- and FAP-negative cells (Figure 4, 5 and Supporting Figure 6). Figure 4. Detection of FAP and GCPII on stably transfected U251 cells by flow cytometry. The cells were incubated with 200 nM conjugates (2×105 cells per well; 1 h at 37 °C). As positive controls (PC), enzyme-specific iBodies were used.11, 12 HPMA conjugate lacking inhibitor was used as a negative control (NC). a) FAP expressed on U251_FAP+ cells was detected by stochastic conjugate I; stochastic conjugate III targeting aspartic proteases was used as an additional negative control. b) GCPII expressed on U251_GCPII+ cells was detected by stochastic conjugate II. Stochastic conjugates can be optimized by HPLC purification of photomodified inhibitors. We reasoned that sometimes the affinity of prepared stochastic conjugates might not be sufficient for a firm target binding and its subsequent isolation, especially in the case of poor protein binders. To achieve tighter binding of the conjugates to the target, we semi-purified the pool of photomodified products by HPLC prior to conjugation to HPMA (Figure 6a). Specifically, we separated the mixture of GCPII inhibitor isomers into individual fractions and analyzed the inhibition activity of fractions (Supporting Figure 7, 8 and Supporting Table 2). Figure 5. Visualization of FAP and GCPII in transfected U251 cells by confocal microscopy. The cells were incubated with 250 nM conjugates (1 h at 37 °C). As positive controls (PC), enzyme-specific iBodies were used.11, 12 A conjugate lacking inhibitor was used as a negative control (NC). a) FAP was visualized by stochastic conjugate I (green). b) GCPII was visualized by stochastic conjugate II (green). Cell nuclei (blue) were stained by Hoechst H34580 dye (0.25 μg mL-1; 5 min, 37 °C). Moreover, using multi-tandem mass spectrometry, we were able to determine the linker position on the small molecule ex ante


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(Supporting Figure 9, 10). In this manner, we identified the fraction enriched with inhibitors containing productively bound linker not compromising the inhibitor-enzyme binding. The affinity of the resulting anti-GCPII stochastic conjugate IV increased significantly (Supporting Table 3), and it enabled substantially improved visualization of GCPII on cell membranes (Figure 6b). This approach led to a stochastic conjugate exhibiting similar properties as the rationally designed iBody (Supporting Figure 6b, c; and Supporting Figure 11). The fraction of photomodified GCPII inhibitors with compromised binding to GCPII yielded stochastic conjugate V, which was unable to visualize and isolate GCPII (Figure 6b, c). Figure 6. Improving stochastic anti-GCPII conjugates. a) After photomodification, the isomers were separated into fractions by HPLC and then conjugated to the polymer, yielding enriched (IV) and depleted (V) conjugate. b) GCPII was visualized by 250 nM stochastic conjugates II and IV (green); stochastic conjugate V did not enable visualization or isolation. As a positive control (PC), the published GCPII-specific iBody was used.12 Cell nuclei (blue) were stained by Hoechst H34580 dye (blue). (c) Western blot analysis of GCPII isolation from an LNCaP cell lysate by stochastic conjugates II, IV and V; GCPII was visualized by the mouse GCPII selective mAb GCP-0419, followed by IRDye® 800CW goat anti-mouse secondary antibody conjugate. As positive controls (PC), the previously described GCPII-specific iBody12 and mAb 2G720 were used. As negative controls, conjugate lacking inhibitor (NC), stochastic conjugate I targeting FAP and blank streptavidin agarose [NC(SA)] were used. Identification of protein targets using pull-downs followed by MS detection. Finally, to demonstrate the considerable potential of these affinity probe-HPMA conjugates for identification of protein targets, we analyzed GCPII pull-down samples by mass spectrometry to determine whether the methodology can identify the target enzyme in a complex biological matrix. The pull-down coupled with MS detection confirmed our Western blot data and identified GCPII as the target of the stochastic conjugates (Table 1). Importantly, all negative controls provided no signal for GCPII binding, suggesting that the interaction is highly specific and limited only to the inhibitor-GCPII active site. After MS analysis, we “extracted” the proteins not present in the negative controls. We achieved that by computational subtraction of the proteins found in the negative controls from the proteins found in the IP experiments (II, IV, PC, mAb 2G7). Afterwards, having excluded known common MS contaminants, GCPII was the top-ranking protein found (Supporting Table 5; Supporting Information, MS_protein_identification). When depleted stochastic conjugate V was used, GCPII corresponding peptides were not found at all (Supporting Table 6; Supporting Information, MS_protein_identification). Not surprisingly, most of the major contaminants were various keratins (see Supporting Information, MS_data_summary).

Table 1. Mass spectrometry results of GCPII isolation from LNCaP cells. Probe

Characteristics

Peptidesa ProtScoreb

stochastic conjugate II

GCPII-specific

4

10

stochastic conjugate IV

GCPII-specific

24

42

anti-GCPII iBody

PC

29

48

anti-GCPII mAb 2G7

PC

29

53

conjugate lacking inhibitor

NC

0

0

streptavidin agarose resin

NC

0

0

stochastic conjugate I

NC, FAP-specific

0

0

stochastic conjugate V

Contains inactive fractions of GCPII probe

0

0

aNumber of GCPII peptides identified. bUnused ProtScore (ProteinPilot™ software, Sciex) refers to a measure of the total, unique peptide amount of evidence for a given detected protein. The Unused ProtScore is calculated using all the peptides detected for the protein. GCPII was first isolated from an LNCaP cell lysate by stochastic conjugates II and IV, the whole elution fraction was then analyzed by mass spectrometry. As positive controls (PC), previously described GCPII-specific iBody12 and mAb 2G720 were used. HPMA conjugate lacking inhibitor was used as a negative control (NC), as was blank streptavidin agarose and stochastic conjugate I targeting FAP.

In this proof-of-principle study, we circumvented the lengthy trial-and-error probe preparation7-10 that often presents an obstacle in the protein target identification workflow. In fact, even in probe-dependent settings, time-consuming enzyme-inhibitor structural analysis is no longer needed for successful target identification. Recently, new methods of probe-independent ligandtarget identification based on thermal proteome profiling have been described.4 This powerful proteomic mass-spectrometry profiling yields valuable information if the target proteins are present in sufficient quantity, but eventually an affinity probe needs to be prepared to verify which of the candidate hits are actual targets. Compared to UV beads21, which also use random linking, our presented approach enables to identify the productive linker attachment position, which makes subsequent targeted synthesis possible. The method that we describe offers several advantages over potential alternatives for affinity pull-down. HPMA polymers provide a soluble, hydrophilic scaffold with a very low capacity for non-specific binding. Furthermore, multiple copies of the small molecule ligand potentially bound to the scaffold offer an avidity effect that can improve the binding of even a relatively weak ligand. The method thus enables identification of targets from hits with medium to high affinity obtained by phenotypic screening. It also enables the identification of rare or low-copy protein targets of bioactive ligands or binding partners. The resulting affinity probe-HPMA conjugates can be used both for pull-down and subsequent proteomics and for the visualization of target-expressing cells. Very recently, we applied this approach successfully for the identification of the protein target of MCC950/ CRID3, a small molecule inhibitor of the NLRP3 inflammasome pathway (Vande Walle et al., in preparation). Ultimately, in just a few steps, we can proceed from discovering bioactive small molecules to identifying and visualizing protein targets. METHODS Synthesis of low-molecular-weight compounds. Chemical reagents and solvents were of analytical grade and were used as received. All manipulations with diazirine derivatives were carried out in dark. All the progress of the reactions and separations were monitored by Waters UPLC/MS system using water-MeCN gradient (0.1% formic acid as modifier) on Waters BEH C18 1.7 μm, 2.1 × 100 mm, flow 0.5 mL min-1. Flash chromatography



separations were performed on C18 modified silica gel (300-400 mesh) using Isco Teledyne apparatus. Gel chromatography was performed on Sephadex LH-20 (28 × 1200 mm column) with isocratic elution (80% methanol/water), flow 10 mL/hour. Preparative HPLC was performed on Phenomenex C18 column (21.2 x 250 mm, 10 μm) with water-MeCN gradient (0.1% TFA as modifier), flow 10 mL min-1. The high-resolution mass spectra (HRMS) were measured on LC/MS TOF (Agilent 6230). Yields of UV-PEG15-compounds 2 – 4 are not calculated due to impurities (e.g. free ligand); last step of their “purification” was binding via amino group to HPMA copolymer. General procedure of UV irradiation. Appropriate small molecule derivative 1, 2, or 3 (5 eq.) and diazirine 4 (1 eq.) were dissolved in methanol and evaporated to dryness in vacuo. The solid residue was irradiated using LED diode (365 nm, 1 W) until starting diazirine was present (UPLC/MS analysis). The reaction mixture was dissolved in 80% methanol and subjected to Sephadex LH-20 gel chromatography. Fractions containing desired product (UPLC/MS analysis) were collected, evaporated to dryness, and purified using preparative HPLC. Fractions containing desired product (UPLC/MS analysis) were collected, evaporated to dryness, and used in reaction with an HPMA copolymer precursor. Preparation of N-(47-amino3,6,9,12,15,18,21,24,27,30,33,36,39,42,45pentadecaoxaheptatetracontyl)-4-(3-(trifluoromethyl)-3Hdiazirin-3-yl)benzamide (4). To solution of 4-(3trifluoromethyl)-3H-diazirin-3-yl)benzoic acid (80 mg, 0.348 mmol) in DMF (5 mL) and DIPEA (0.2 mL, 1.15 mmol) was added TSTU (105 mg, 0.349 mmol). The reaction mixture was stirred at r.t. for 1 hour, H2N-PEG15-NHBoc (287 mg, 0.350 mmol) was added and solution was stirred for additional 2 hours. The reaction mixture was directly injected on C18 column and product was purified by flash chromatography with water-MeOH gradient (0-100%) to give Boc-protected derivative (UPLC/MS (m/z) 1033.46 [M+H]+, Tr 4.47 min). Fractions with protected derivative were collected, evaporated, and dried in vacuo. Residue was dissolved in DCM (5 mL) and TFA (3 mL) was added. The solution was stirred at r.t. for 1hour, evaporated in vacuo, and purified by flash chromatography on C18 column with water (0.1 TFA as modifier) and MeCN gradient (0-100%). Fractions with desired derivative 4 (TFA salt) were collected, evaporated, and dried in vacuo. Yield 250 mg (69%). UPLC/MS (m/z) 933.38 [M+H]+, Tr 3.70 min; HRMS (ESI): Calculated for C41H72F3N4O16 [M+H]+: 933.4895, Found: 933.4817.

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Preparation of compound 2-UV-PEG15. GCPII inhibitor 2 (100 mg, 0.189 mmol) and diazirine 4 (40 mg, 0.038 mmol) were subjected to UV irradiation according to general procedure leading to mixture of isomers compound 2-UV-PEG15 (16 mg). UPLC/MS (m/z) 717.96 [M+2H]++, Tr 3.33-3.78 min; HRMS (ESI): Calculated for C62H100BrF3N5O24 [M+H]+: 1434.5894, Found: 1434.5803.

Preparation of compound 3-UV-PEG15. Pepstatin A 3 (100 mg, 0.146 mmol) and diazirine 4 (27 mg, 0.029 mmol) were subjected to UV irradiation according to general procedure leading to mixture of isomers compound 3-UV-PEG15 (6 mg). UPLC/MS (m/z) 796.28 [M+2H]++, Tr 3.81-4.15 min; HRMS (ESI): Calculated for C75H135F3N7O25 [M+H]+: 1590.9460, Found: 1590.9385.

Preparation of compound 1-UV-PEG15. FAP inhibitor 1 (100 mg, 0.146 mmol) and diazirine 4 (27 mg, 0.029 mmol) were subjected to UV irradiation according to general procedure leading to mixture of isomers compound 1-UV-PEG15 (7 mg). UPLC/MS (m/z) 625.49 [M+2H]++, Tr 3.16-3.90 min; HRMS (ESI): Calculated for C58H86F5N6O18 [M+H]+: 1249.5919, Found: 1249.5810.


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LC-ESI MS/MS. Samples were analyzed by a LC (Rheos 2200, FLUX instruments) coupled with ESI-FTMS (LTQ Orbitrap XL, Thermo Scientific). Chromatographic separation was performed with a Kinetex XB-C18 reversed-phase analytical column (50mm x 2.1mm, 2.6μm, Phenomenex®) in connection with BEH C18 Pre- column (VanGuard, 1.7 μm, 2.1mm x 5mm, Waters), maintained at the room temperature. LC-MS grade 0.1% formic acid solution in acetonitrile - B and 0.1% formic acid in water - A (Fisher chemical) were used as a mobile phase at flow rate 200 μL min-1. The elution was programmed by using the gradient (0min - 2% B, 0 - 1min 32%B, 1 - 11min 42%B, 11 14min 100%B, 14 - 16min 100%B, 16 - 17min 2%B, 17 – 20min 2%B). All injection were 2.5μL. MS spectra were measured in + ESI mode on the conditions: Source Voltage: 4.9kV, Source Current 7.7μA, Sheath Gas Flow Rate: 35arb, Aux Gas Flow Rate: 5.0arb, Sweep Gas Flow Rate: 0.02arb, Capillary Voltage: 9.0V, Capillary Temp: 275°C, Tube Lens Voltage: 149.8V. MS/MS spectra were obtained by He - collision induced dissociation of double charged parent ion m/z 717.8 following the settings (normalized collision energy 20.0%, isolation width - 3.0 m/z). GCPII enzyme activity inhibition assay. The inhibition constants of all compounds targeting GCPII were determined using HPLC-based assay with recombinant extracellular GCPII, as described previously22. Conjugates II, IV and V were purified on 100kDa molecular weight cutoff Amicon Ultra 0.5mL Centrifugal Filters (Merck) prior to the measurement. The data were processed and IC50 values were obtained using GraFit v.5.0.11 (Erithacus Software Ltd.). Assuming a competitive mode of inhibition, Ki value was calculated using the Cheng-Prusoff equation23 from the kinetic parameters of pteroyl-di-L-glutamate cleavage. Cell line U251-GCPII. U251 MG cell line with doxycyclinecontrollable expression of GCPII was prepared analogously to cell line HEK293-TetOff-A2, which was recently published24. U251 cells were grown on 35 mm dish in DMEM medium (Sigma-Aldrich) supplemented with 4 mM L-glutamine and 10% FBS and stably transfected with pTet-Off® Advanced vector (Clontech) using FuGENE® HD transfection reagent (Roche). Transfected cells were grown in the presence of Geneticin® (Invitrogen; final concentration 400 μg mL-1). Selected colonies were grown to confluence and then transiently transfected by pTreTight-GCPII using FuGENE® HD transfection reagent following manufacturer’s protocol. A day after transient transfection, the cells were harvested, lysed and analyzed for the level of expression and for the ability to regulate the expression by addition of doxycycline by western blotting. The best clone (U251-TetOff-26) was selected for further stable transfection. U251-TetOff-26 cells were stably co-transfected with pTRE-Tight-GCPII and pPUR vector (Clontech) using FuGENE® HD transfection reagent following manufacturer’s protocol. Selected colonies of both stable trans-

fectants were picked using cylinders and then were grown to confluence in presence or absence of 100 nM doxycycline hyclate (Sigma-Aldrich) in DMEM medium (supplemented with Lglutamine and FBS) containing puromycin (Invitrogen; final concentration 5 μg mL-1) and Geneticin® (final concentration 400 μg mL-1). Flow cytometry analysis of GCPII-expressing cells. Cells U251-GCPII were grown on a 100 mm dish to 90% confluence in DMEM medium with or without 100 nM doxycycline, supplemented with 10% FBS and 4 mM L-glutamine. The medium was removed, and cells were rinsed with PBS and subsequently incubated in 1.5 ml trypsin/EDTA solution for 3 min to release adherent cells from the dish surface. Cells were resuspended and transferred into 8 ml of DMEM medium, centrifuged at 250×g for 2 min and washed twice with 5 ml PBS. Then, 500 μl of 10% fetal bovine serum in PBS was added to block the cell surface (at 37 °C for 1 h). The final concentration of the cell suspension was 4×106 cells mL-1. Afterwards, 50 μl of cell suspension (containing 2×105 cells) was incubated with 200 nM solutions of anti-FAP or anti-GCPII conjugates for 1 h at 37 °C in a polypropylene 96-well plate (round bottom). Cells were then washed twice with 200 μl of 10% fetal bovine serum in TBS. Finally, the cell suspension was diluted with 200 μl of 10% fetal bovine serum in TBS, and a single cell suspension was analyzed with a BD LSR Fortessa™ cell analyzer (Becton, Dickinson and Company). The gates on the side scatter and forward scatter were set to ensure measurement of viable single cells; 20,000 events were measured for each sample. All experiments were performed in triplicates and their analysis was performed using BD FACSDiva™ Software. A histogram (created using FlowJo 10) showing a representative measurement for each sample is presented. Pulldown of GCPII. Lymph Node Carcinoma of the Prostate (LNCaP) cells endogenously expressing GCPII were lysed by sonication in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Tween 20. The obtained cell lysate was diluted in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20 (TBST) to a final protein concentration of 0.3 mg mL-1. Various anti-GCPII conjugates (200 nM in TBST) were bound to 25 μl Streptavidin Agarose Ultra Performance (Solulink) for 1 h at 4 °C. To compare the conjugates with antibodies, biotinylated monoclonal antibody 2G720 was used as well (200 nM in TBST). After washing with TBST (2×1 mL), the resin was mixed with 1 mL of the LNCaP lysate and incubated for 1 h at 4 °C. The resin was then washed with TBST (3×1 mL) and proteins were eluted from the resin with 30 μL of reducing SDS sample buffer and heating to 98 °C for 5 min. For the conjugate competition pull-down, the pre-bound conjugates were incubated with recombinant human GCPII25 in TBST (1 ng/μL; 1 h at 4 °C), in the absence or presence of 25 μM 2(phosphonomethyl)-pentanedioic acid (2-PMPA)26, the lowmolecular-weight inhibitor of GCPII. Western blotting of GCPII. Protein samples were resolved by SDS-PAGE and blotted to a nitrocellulose membrane (1 h, 100 V). The membrane was blocked with 0.55% (w/v) casein in TBS (Casein Buffer 20X-4X Concentrate, SDT; 1 h at RT). Afterwards, GCPII was probed with the primary antibody GCP-0419 for 12 h at 4 °C (200 ng mL-1; diluted in 0.55% casein solution). The membrane was washed with PBST´ (3×5 min) and incubated with IRDye® 800CW goat anti-mouse secondary antibody conjugate (LI-COR Biosciences; 1:15,000 in PBS with 0.05% Tween 20; 1 h at RT). The unbound conjugate was washed out



with PBST´ (3×5 min) and GCPII was visualized using an Odyssey CLx Imaging System (LI-COR). Confocal microscopy of cells expressing GCPII. Stably transfected U251-GCPII cells were grown in the presence or absence of 100 nM doxycycline in 4-Chamber 35 mm Glass Bottom Dishes (In Vitro Scientific) in DMEM supplemented with 10% FBS and 4 mM L-glutamine to approximately 70% confluence. The next day, the cell media were removed, and 250 nM anti-GCPII conjugates diluted in 10% FBS in TBS added to each chamber. Cells were incubated for 1 h at 37 °C. The medium was removed, cells were rinsed with 500 μl of PBS and Hoechst Stain Solution H34580 (Sigma) was added for 5 min (0.25 μg mL-1 final concentration) to stain cell nuclei. Cells were then washed twice with 500 μl of PBS and fresh 10% FBS in TBS solution was added. Confocal images (pinhole 1 Airy unit) were obtained at 37 °C using a Zeiss LSM 780 confocal microscope (Carl Zeiss Microscopy) with an oil-immersion objective (Plan-Apochromat 63×/1.40 Oil DIC M27). The fluorescent images were taken using 1.2% of the 405 nm diode laser (max. power 30 mW) for excitation with emission collected from 410 to 585 nm (voltage on detector: 730 V) for Hoechst 34580 and 2.1% of the 488 nm argon-ion laser (max. power 25 mW) for excitation with emission collected from 490 to 630 nm (voltage on detector: 750 V) for ATTO488. All images were taken using the same settings. The microscope was operated and images were processed with ZEN 2011 software (Carl Zeiss Microscopy). HIV-protease enzyme activity inhibition assay. The determination of the inhibition constant for HIV-protease was performed by spectrophotometric assay using the chromogenic peptide substrate KARVNle*NphEANle-NH2 as previously described27. The 1 mL reaction mixture contained 100 mM sodium acetate, 300 mM NaCl, pH 4.7, 6.8 pmol of HIV-1 protease27 and inhibitor in concentrations between 1 and 500 nM (for the HPMA-pepstatin conjugate) or 10 and 10,000 nM (for the pepstatin derivate). Substrate was added to a final concentration of 16 μM. Afterwards, the hydrolysis of substrate was followed as a decrease in absorbance at 305 nm using a UNICAM UV500 UV−VIS spectrophotometer (Thermo, Cambridge, MA). The data were analyzed using the equation for competitive inhibition according to Williams and Morrison28. The mechanism of inhibition was determined by analysis of Lineweaver-Burk plots. Pulldown of aspartic proteases (pepsin, cathepsin D and HIV-protease). As model aspartic proteases we used bovine pepsin (Worthington Biochemical Corporation; 33K865), cathepsin D from Ixodes ricinus16 (kindly provided from Dr. Michael Mareš, IOCB CAS in Prague) and recombinant HIV-1 protease27. Conjugates (200 nM in TBST) were bound to 25 μL Streptavidin Agarose Ultra Performance (Solulink) for 1 h at 4 °C. The resin was then washed twice with 1 mL of 50 mM sodium acetate, 150 mM NaCl, 0.05% Tween 20, pH 5.0. The resin was mixed with 1 mL of the LNCaP lysate (total protein concentration 300 μg mL-1) spiked with the corresponding enzymes (5 μg mL-1) and incubated for 1 h at 4 °C. The resin was then washed with 50 mM sodium acetate, 150 mM NaCl, 0.05% Tween 20, pH 5.0 (3×1 mL) and proteins were eluted from the resin with 30 μL of reducing SDS sample buffer and heating to 98 °C for 5 min. FAP enzyme activity inhibition assay. IC50 values for antiFAP compounds were determined using a fluorescence spectroscopy assay described recently11, employing a fluorogenic FAPspecific substrate29 that was prepared by standard Boc-peptide chemistry. Flow cytometry analysis of FAP-expressing cells. Flow cytometry analysis was performed analogously as described for

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GCPII, using the recently described cell line U251-FAP.11 Briefly, 90 μl of cell suspension (containing 1×105 cells) was incubated with 200 nM anti-FAP conjugates. Pulldown of FAP from a cell lysate. Stably transfected U251_FAP+ cells were used for preparation of FAP-containing cell lysate, which was prepared by sonication in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% TritonX-100, 0.1% SDS, 10% glycerol, containing 1 mM EDTA, 10 μM pepstatin A, 20 μM E-64. Next, 1 ml of 1 μM anti-FAP conjugate (and a negative control conjugate lacking any inhibitor) in TBST were incubated with 30 μL of Streptavidin Agarose Ultra Performance (Solulink) for 1 h at 4 °C. The resin was washed three times with 1 mL TBST and then incubated with 1.3 ml of the U251-FAP cell lysate diluted in TBST (0.4 mg mL-1 total protein concentration) for 1.5 h at 4 °C. Afterwards, flow-through fractions were collected and the resin was washed with TBST (5×1 mL). Finally, proteins were eluted with 30 μL of reducing SDS sample buffer and heated to 98 °C for 10 min. Proteins were resolved by SDS-PAGE and visualized by silver staining. Confocal microscopy of FAP-expressing cells. Microscopy with U251-FAP cell line was performed analogously as described for GCPII. The cells were incubated with 300 nM anti-FAP conjugates diluted in fresh DMEM medium. Mass spectrometry analysis. Proteins in the sample were reduced by dithiothreitol and alkylated by iodoacetamide prior trypsin digestion overnight at pH 8.5. Resulting peptides were analyzed on UltiMate 3000 RSLCnano system (Thermo Scientific) coupled to a TripleTOF 5600 mass spectrometer with a NanoSpray III source (Sciex). The peptides were trapped and desalted with 2% acetonitrile in 0.1% formic acid at flow rate of 5 μL min-1 on Acclaim PepMap100 column (5 μm, 2 cm×100 μm ID, Thermo Scientific). Eluted peptides were separated using Acclaim PepMap100 analytical column (3 μm, 25 cm×75 μm ID, Thermo Scientific). The 70 min elution gradient at constant flow of 300 nl min-1 was set to 5% of phase B (0.1% formic acid in 99.9% acetonitrile, phase A 0.1% formic acid) for first 10 min, then with gradient elution by increasing content of acetonitrile. TOF MS mass range was set to 350–1250 m/z, in MS/MS mode the instrument acquired fragmentation spectra with m/z ranging from 100 to 1600. Protein Pilot 4.5 (Sciex) was used for protein identification using database of Swiss-Prot human proteins (downloaded 15Feb2016) and common contaminants. Mass spectrometry data processing. The MS data processing was carried out in the Microsoft Office Access 2013. For the protein identification, we grouped proteins identified in two negative control experiments (conjugate lacking inhibitor, NC; blank streptavidin agarose, NC-SA). Afterwards, we subtracted the parameters (i.e. protein sequence coverage, number of peptides and “unused ProtScore”) of the proteins present in the negative control from those of proteins present in the IP samples. The proteins were then ordered according to the “unused ProtScore”; usually only proteins not present in the negative controls were filtered.

ASSOCIATED CONTENT Supporting Information Supporting Information Available: Synthesis of monomers, polymer precursors and polymer conjugates; HPLC isomer separation and fraction identification; characteristics of prepared compounds and conjugates (pdf); mass spectrometry data and analysis (xlsx). This material is available free of charge via the internet at http://pubs.acs.org.


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AUTHOR INFORMATION

Corresponding Author *For J.K., phone, +420 220 183 218; email: [email protected] *For P.Š., phone, +420 220 183 452; email: [email protected]

Author Contributions ‡These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We would like to thank J. Starková and K. Šrámková for their excellent technical support, S. Matějková and Š. Štanga for elemental analyses, M. Hubálek for protein mass spectrometry, M. Mareš and J. Srp for providing cathepsin D from Ixodes ricinus, R. Souček for HRMS measurement, J. Bařinková for help with preparative HPLC, and H. Hoffman for language editing. This work was supported by Grant No. GA16-02938S from the Grant Agency of the Czech Republic, InterBioMed Project LO 1302 from the Ministry of Education of the Czech Republic, and by the Charles University, project GA UK No. 1510-243-250045.

REFERENCES 1. Haasen, D., Schopfer, U., Antczak, C., Guy, C., Fuchs, F., and Selzer, P. (2017) How Phenotypic Screening Influenced Drug Discovery: Lessons from Five Years of Practice, Assay Drug Dev. Technol. 15, 239– 246. 2. Lee, J. A., Uhlik, M. T., Moxham, C. M., Tomandl, D., and Sall, D. J. (2012) Modern Phenotypic Drug Discovery Is a Viable, Neoclassic Pharma Strategy, J. Med. Chem. 55, 4527–4538. 3. Rix, U., and Superti-Furga, G. (2009) Target profiling of small molecules by chemical proteomics, Nat. Chem. Biol. 5, 616–624. 4. Savitski, M. M., Reinhard, F. B., Franken, H., Werner, T., Savitski, M. F., Eberhard, D., Martinez Molina, D., Jafari, R., Dovega, R. B., Klaeger, S., Kuster, B., Nordlund, P., Bantscheff, M., and Drewes, G. (2014) Tracking cancer drugs in living cells by thermal profiling of the proteome, Science 346, 1255784. 5. Nijman, S. M. (2015) Functional genomics to uncover drug mechanism of action, Nat. Chem. Biol. 11, 942–948. 6. Jung, H. J., and Kwon, H. J. (2015) Target deconvolution of bioactive small molecules: the heart of chemical biology and drug discovery, Arch. Pharm. Res. 38, 1627–1641. 7. Blex, C., Michaelis, S., Schrey, A. K., Furkert, J., Eichhorst, J., Bartho, K., Gyapon Quast, F., Marais, A., Hakelberg, M., Gruber, U., Niquet, S., Popp, O., Kroll, F., Sefkow, M., Schulein, R., Dreger, M., and Koster, H. (2017) Targeting G Protein-Coupled Receptors by Capture Compound Mass Spectrometry: A Case Study with Sertindole, Chembiochem 18, 1639–1649. 8. Knockaert, M., Gray, N., Damiens, E., Chang, Y. T., Grellier, P., Grant, K., Fergusson, D., Mottram, J., Soete, M., Dubremetz, J. F., Le Roch, K., Doerig, C., Schultz, P., and Meijer, L. (2000) Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors, Chem. Biol. 7, 411–422. 9. Sin, N., Meng, L., Auth, H., and Crews, C. M. (1998) Eponemycin analogues: syntheses and use as probes of angiogenesis, Bioorg. Med. Chem. 6, 1209–1217. 10. Taunton, J., Hassig, C. A., and Schreiber, S. L. (1996) A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p, Science 272, 408–411. 11. Dvorakova, P., Busek, P., Knedlik, T., Schimer, J., Etrych, T., Kostka, L., Stollinova Sromova, L., Subr, V., Sacha, P., Sedo, A., and Konvalinka, J. (2017) Inhibitor-Decorated Polymer Conjugates Targeting Fibroblast Activation Protein, J. Med. Chem. 60, 8385–8393. 12. Sacha, P., Knedlik, T., Schimer, J., Tykvart, J., Parolek, J., Navratil, V., Dvorakova, P., Sedlak, F., Ulbrich, K., Strohalm, J., Majer, P., Subr,

V., and Konvalinka, J. (2016) iBodies: modular synthetic antibody mimetics based on hydrophilic polymers decorated with functional moieties, Angew. Chem. Int. Ed. Engl. 55, 2356–2360. 13. Garin-Chesa, P., Old, L. J., and Rettig, W. J. (1990) Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers, Proc. Natl. Acad. Sci. U. S. A. 87, 7235– 7239. 14. Pavlicek, J., Ptacek, J., and Barinka, C. (2012) Glutamate carboxypeptidase II: an overview of structural studies and their importance for structure-based drug design and deciphering the reaction mechanism of the enzyme, Curr. Med. Chem. 19, 1300–1309. 15. Ferraris, D. V., Shukla, K., and Tsukamoto, T. (2012) Structureactivity relationships of glutamate carboxypeptidase II (GCPII) inhibitors, Curr. Med. Chem. 19, 1282–1294. 16. Hanova, I., Brynda, J., Houstecka, R., Alam, N., Sojka, D., Kopacek, P., Maresova, L., Vondrasek, J., Horn, M., Schueler-Furman, O., and Mares, M. (2018) Novel Structural Mechanism of Allosteric Regulation of Aspartic Peptidases via an Evolutionarily Conserved Exosite, Cell Chem. Biol. 25, 318–329 e314. 17. Krausslich, H. G., Ingraham, R. H., Skoog, M. T., Wimmer, E., Pallai, P. V., and Carter, C. A. (1989) Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides, Proc. Natl. Acad. Sci. U. S. A. 86, 807–811. 18. Tykvart, J., Navratil, V., Sedlak, F., Corey, E., Colombatti, M., Fracasso, G., Koukolik, F., Barinka, C., Sacha, P., and Konvalinka, J. (2014) Comparative analysis of monoclonal antibodies against prostatespecific membrane antigen (PSMA), Prostate 74, 1674–1690. 19. Sacha, P., Zamecnik, J., Barinka, C., Hlouchova, K., Vicha, A., Mlcochova, P., Hilgert, I., Eckschlager, T., and Konvalinka, J. (2007) Expression of glutamate carboxypeptidase II in human brain, Neuroscience 144, 1361–1372. 20. Knedlik, T., Navratil, V., Vik, V., Pacik, D., Sacha, P., and Konvalinka, J. (2014) Detection and quantitation of glutamate carboxypeptidase II in human blood, Prostate 74, 768–780. 21. Kawatani, M., Okumura, H., Honda, K., Kanoh, N., Muroi, M., Dohmae, N., Takami, M., Kitagawa, M., Futamura, Y., Imoto, M., and Osada, H. (2008) The identification of an osteoclastogenesis inhibitor through the inhibition of glyoxalase I, Proc. Natl. Acad. Sci. U. S. A. 105, 11691–11696. 22. Tykvart, J., Schimer, J., Barinkova, J., Pachl, P., PostovaSlavetinska, L., Majer, P., Konvalinka, J., and Sacha, P. (2014) Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery, Bioorg. Med. Chem. 22, 4099–4108. 23. Cheng, Y., and Prusoff, W. H. (1973) Relationship between Inhibition Constant (K1) and Concentration of Inhibitor Which Causes 50 Per Cent Inhibition (I50) of an Enzymatic-Reaction, Biochem. Pharmacol. 22, 3099–3108. 24. Tykvart, J., Schimer, J., Jancarik, A., Barinkova, J., Navratil, V., Starkova, J., Sramkova, K., Konvalinka, J., Majer, P., and Sacha, P. (2015) Design of highly potent urea-based, exosite-binding inhibitors selective for glutamate carboxypeptidase II, J. Med. Chem. 58, 4357– 4363. 25. Barinka, C., Rinnova, M., Sacha, P., Rojas, C., Majer, P., Slusher, B. S., and Konvalinka, J. (2002) Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II, J. Neurochem. 80, 477–487. 26. Jackson, P. F., Cole, D. C., Slusher, B. S., Stetz, S. L., Ross, L. E., Donzanti, B. A., and Trainor, D. A. (1996) Design, synthesis, and biological activity of a potent inhibitor of the neuropeptidase N-acetylated alpha-linked acidic dipeptidase, J. Med. Chem. 39, 619–622. 27. Kozisek, M., Bray, J., Rezacova, P., Saskova, K., Brynda, J., Pokorna, J., Mammano, F., Rulisek, L., and Konvalinka, J. (2007) Molecular analysis of the HIV-1 resistance development: enzymatic activities, crystal structures, and thermodynamics of nelfinavir-resistant HIV protease mutants, J. Mol. Biol. 374, 1005–1016. 28. Williams, J. W., and Morrison, J. F. (1979) The kinetics of reversible tight-binding inhibition, Methods Enzymol. 63, 437–467. 29. Keane, F. M., Yao, T. W., Seelk, S., Gall, M. G., Chowdhury, S., Poplawski, S. E., Lai, J. H., Li, Y. H., Wu, W. G., Farrell, P., de Ribeiro, A. J. V., Osborne, B., Yu, D. M. T., Seth, D., Rahman, K., Haber, P., Topaloglu, A. K., Wang, C. M., Thomson, S., Hennessy, A., Prins, J., Twigg, S. M., McLennan, S. V., McCaughan, G. W., Bachovchin, W. W.,



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and Gorrell, M. D. (2014) Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs, Febs Open Bio 4, 43–54.


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Figure 1. Affinity probe-HPMA copolymer conjugates for target identification. a) Development of affinity probes with “rationally” attached linkers is laborious and time-consuming, since it requires optimal linker attachment. b) The stochastic photomodification approach employing a photoactivatable linker generates a mixture of affinity probe isomers with linkers attached at various positions. In the second step, the photomodified affinity probes are conjugated to hydrophilic HPMA copolymer decorated with biotin for subsequent isolation of the target and the fluorophore ATTO488 for target visualization. 104x102mm (300 x 300 DPI)

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Figure 2. Small-molecule compounds used for preparation of affinity probe conjugates. a) FAP inhibitor. b) GCPII inhibitor. c) Pepstatin A, a class-specific inhibitor of aspartic proteases. d) Photoactivatable PEG linker. The arrows show linker positions for conjugates prepared by structure-assisted design.11, 12 202x84mm (300 x 300 DPI)


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Figure 3. Use of stochastic conjugates I, II and III to isolate model enzymes from cell lysates. The conjugates were pre-bound to streptavidin agarose beads and then mixed with the protein-containing samples. As positive controls (PC), enzyme-specific iBodies were used.11, 12 Negative controls used include HPMA conjugate lacking inhibitor (NC) and blank streptavidin agarose [NC(SA)]. Ten microliter samples were loaded onto the gel. a) FAP was isolated from FAP stable transfectant cell lysate by stochastic conjugate I (silver stained gel). b) GCPII was isolated from LNCaP cell lysate by stochastic conjugate II (western blot). GCPII was visualized by the mouse GCPII selective mAb GCP-0419, followed by IRDye® 800CW goat anti-mouse secondary antibody conjugate. c, d, e) Pepsin, cathepsin D, and HIV protease were isolated from cell lysates spiked with the corresponding enzyme (“Load”) by stochastic conjugate III (silver stained gels). 142x93mm (300 x 300 DPI)



Figure 4. Detection of FAP and GCPII on stably transfected U251 cells by flow cytometry; the cells were incubated with 200 nM conjugates (2×105 cells per well; 1 h at 37 °C). As positive controls (PC), enzymespecific iBodies were used.11, 12 HPMA conjugate lacking inhibitor was used as a negative control (NC). a) FAP expressed on U251_FAP+ cells was detected by stochastic conjugate I; stochastic conjugate III targeting aspartic proteases was used as an additional negative control. b) GCPII expressed on U251_GCPII+ cells was detected by stochastic conjugate II. 82x83mm (300 x 300 DPI)


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Figure 5. Visualization of FAP and GCPII in transfected U251 cells by confocal microscopy; the cells were incubated with 250 nM conjugates (1 h at 37 °C). As positive controls (PC), enzyme-specific iBodies were used.11, 12 A conjugate lacking inhibitor was used as a negative control (NC). a) FAP was visualized by stochastic conjugate I (green). b) GCPII was visualized by stochastic conjugate II (green). Cell nuclei (blue) were stained by Hoechst H34580 dye (0.25 μg mL-1; 5 min, 37 °C). 139x85mm (300 x 300 DPI)



Figure 6. Improving stochastic anti-GCPII conjugates. a) After photomodification, the isomers were separated into fractions by HPLC and then conjugated to the polymer, yielding enriched (IV) and depleted (V) conjugate. b) GCPII was visualized by 250 nM stochastic conjugates II and IV (green); stochastic conjugate V did not enable visualization or isolation. As a positive control (PC), the published GCPII-specific iBody was used.12 Cell nuclei (blue) were stained by Hoechst H34580 dye (blue). (c) Western blot analysis of GCPII isolation from an LNCaP cell lysate by stochastic conjugates II, IV and V; GCPII was visualized by the mouse GCPII selective mAb GCP-0419, followed by IRDye® 800CW goat anti-mouse secondary antibody conjugate. As positive controls (PC), the previously described GCPII-specific iBody12 and mAb 2G720 were used. As negative controls, conjugate lacking inhibitor (NC), stochastic conjugate I targeting FAP and blank streptavidin agarose [NC(SA)] were used. 98x106mm (300 x 300 DPI)


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Upon photomodification, an isomeric mixture of stochastically modified hit molecules is generated, which is then conjugated to a hydrophilic HPMA copolymer and decorated with biotin and a fluorophore. This approach is useful for rapid and efficient small molecule modification and subsequent identification of its protein target by immunoprecipitation followed by mass spectrometry analysis. 77x44mm (300 x 300 DPI)


Identification of Protein Targets of Bioactive Small Molecules Using

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