Edinburgh Research Explorer Drug ‘clicking’ on cell-penetrating fluorescent nanoparticles for in cellulo chemical proteomics

: Chemical proteomics approaches are widely used to identify molecular targets of existing or novel drugs. This manuscript describes the development of a straightforward approach to conjugate azide-labeled drugs via click chemistry to alkyne-tagged cell-penetrating ﬂ uorescent nanoparticles as a novel tool to study target engagement and/or identi ﬁ cation inside living cells. A modi ﬁ cation of the Baeyer test for alkynes allows monitoring the Cu(I)-catalyzed azide − alkyne cycloaddition (CuAAC) reaction, guaranteeing the presence of the drug on the solid support. As a proof of concept, the conjugation of the promiscuous kinase inhibitor dasatinib to Cy5-labeled nanoparticles is presented. Dasatinib-decorated ﬂ uorescent nanoparticles e ﬃ ciently inhibited its protein target SRC in vitro , entered cancer cells, and colocalized with SRC in cellulo . 1 H NMR shifts per (ppm) singlet, d t m coupling J


■ INTRODUCTION
Drug discovery efforts across industry and academia have generated extensive libraries of small molecule compounds that are currently used in high-throughput screening campaigns. One such test employs cell assays to search for compounds that elicit phenotypic responses in particular disease models in a target agnostic manner. This approach, so-called phenotypic screening, is aimed at accelerating the earlier stages of drug discovery and, potentially, favor the finding of first-in-class inhibitors. 1−3 After a phenotypic hit or lead is found, one of the main challenges is to elucidate the mechanism of action responsible for the observed pharmacological effect, 4 as the assays only provide information on compounds' activity in the cell model of choice without informing on the target. 5 Consequently, target identification (ID) studies are often performed to support subsequent lead optimization campaigns.
Among the strategies developed for target ID, chemical proteomics enable us to interrogate the full proteome for direct drug−target interactions. Most chemical proteomics approaches combine affinity chromatography with advanced mass spectrometry to enrich and facilitate target identification from complex biological samples. 6 An alternative approach has been proposed to directly visualize drug−target interactions in cells using drug-labeled nanodevices and fluorescent immunocytochemistry. 7 Regardless of the method, an essential step in these strategies is the conjugation of the drug to the solid support, which typically requires individual optimization of the coupling process for each drug. 8 Therefore, the development of versatile, robust conjugation methods is essential to facilitate the performance of target ID studies both in cell lysates and in native biological environments.
Herein, we present a straightforward procedure for the conjugation of azide-tagged drugs to miniaturized solid supports by click chemistry, along with an analytical method to rapidly determine whether the coupling reaction has been successful. As a proof of concept of the strategy, the synthesis, characterization, and biological validationin vitro and in celluloof a novel cell-penetrating fluorescent nanodevice decorated with the tyrosine kinase inhibitor (TKI) dasatinib is reported.

■ RESULTS AND DISCUSSION
Selection and Development of Azide-Tagged Dasatinib. Dasatinib is a potent promiscuous kinase inhibitor approved by the FDA for the treatment of chronic myeloid leukemia (CML) after acquired resistance to imatinib. 9 Dasatinib's therapeutic effect is mediated by inhibition of the constitutively active fusion protein BCR-ABL, which is the product of an aberrant chromosomal translocation and responsible for CML oncogenesis. Nevertheless, dasatinib also inhibits a wide range of kinases including the nonreceptor tyrosine kinase SRC and its family members (LCK, HCK, YES, FYN, FGR, BLK, LYN, FRK), receptor tyrosine kinases (KIT, PDGFR, DDR1/2, c-FMS, ephrin receptors), and TEC family kinases (TEC and BTK), 10 representing a good example of a promiscuous drug with potential use against numerous clinical disorders. The high potency of dasatinib, its interesting polypharmacological properties, and its easily functionalized chemical structure made it an optimal candidate to develop and test our strategy.
To introduce an azide "handle" in dasatinib for CuAAC reactions while retaining the essential kinase binding elements of its structure, the terminal hydroxyl group of the hydroxyethylpiperazinyl moiety was modified. The role of this moiety is to impart water solubility to the compound 7 and it is displayed outside the protein in the SRC-dasatinib cocrystal structure. 11 The substitution of the OH of dasatinib by N 3 was efficiently carried out using a reported procedure with minor modifications. 7,12 Briefly, chlorination of that position was achieved by treating dasatinib with mesyl chloride and triethylamine. The corresponding chloro derivative dasatinib-Cl (2) was then reacted with sodium azide to obtain derivative dasatinib-N 3 (3) (Scheme 1; see full synthetic details in the SI). Cell viability assays against breast cancer MDA-MB-231 cells demonstrated that 3 retained the antiproliferative properties of the parent drug 1 (see SI).
Synthesis of Dasatinib-Decorated Cy5-Labeled Cell-Penetrating Nanospheres. In cellulo chemical proteomics has the potential to provide more biologically relevant results by studying drug−target interactions in the native cellular environment. 13 It also provides a method to study target engagement, a relevant validation step in drug discovery programs. The development of facile and reliable strategies to conjugate drugs and fluorescent labels on devices with the capacity to penetrate cells without inducing toxicity is very attractive in this field. 500 nm cross-linked polystyrene nanospheres with demonstrated capacity to safely penetrate a wide variety of cell types 14−19 were redesigned for the goals of this project (Scheme 1). NH 2 -NP 500 (4) was synthesized and functionalized according to previous reports. 17,20 To increase biocompatibility, prevent unspecific protein binding, and maximize the freedom of the conjugated drug molecules, 4 was PEGylated before and after incorporation of the dye. By sequential coupling and orthogonal deprotection steps (Fmoc/ Dde deprotection strategy 21 ), bifuntionalized doubly-PEGylated nanoparticles (NPs) were labeled with the far-red fluorescent dye Cy5 (NH 2 -Cy5-PEG-NP 500 , 9). Then an alkyne handle was incorporated onto the NPs by conjugation of propiolic acid 22 to generate Alk-Cy5-PEG-NP 500 (10). Finally, reaction of 10 and 3 in the presence of CuI and Scheme 1. Synthesis of Dasatinib-Decorated Fluorescently-Labeled Cell-Penetrating Nanoparticles

Bioconjugate Chemistry
Article ascorbic acid 23 gave rise to dasatinib-decorated Cy5-tagged nanospheres, DAS-Cy5-PEG-NP 500 (11) (see synthesis and loadings in the SI). Number of NPs per unit of volume was calculated by spectrophotometry according to previous reports 24 (see calculations in the SI).
Analytical Determination of Dasatinib Incorporation onto the Solid Support. Although CuAAC reactions are typically described as virtually quantitative even under mild conditions, 25,26 click reactions on solid phase typically need optimized conditions to reach these standards. 27,28 To confirm the effective coupling of 3 to the alkyne-tagged NPs 10, a series of analytical methods were attempted: (i) detection of unreacted alkynes on the solid support by modification of the Baeyer test for alkynes; (ii) zeta potential characterization of NPs at different steps of the synthesis; (iii) validation by NMR and mass spectrometry of the functionalization strategy by solid phase synthesis using an amino-functionalized resin (Rink amide resin).
The Baeyer test is routinely used in chemistry laboratories to detect the presence of double or triple carbon−carbon bonds based on the capacity of KMnO 4 to oxidize alkenes and alkynes. Aqueous reduction of KMnO 4 (violet color) produces MnO 2 (blackish/brown color), a change that is detectable by spectrophotometry in the range of 250 to 500 nm. 24 Therefore, we anticipated that this method could be used to detect unreacted alkynes on the NPs and thereby assess the efficacy of the drug incorporation reaction onto the solid support. DAS-Cy5-PEG-NP 500 (11) were treated with an aqueous solution

Bioconjugate Chemistry
Article of KMnO 4 and absorbance spectra analyzed, with the test of NH 2 -Cy5-PEG-NP 500 (9) and Alk-Cy5-PEG-NP 500 (10) under the same conditions being used as negative and positive controls, respectively (see full details in the SI). After exploring different reaction conditions and careful spectroscopic analysis to improve the method, the measurement of the changes in the absorbance values at 420 nm after 5 min reaction in comparison to the unreacted KMnO 4 solution was found to be optimal. As shown in Figure 1a, analysis of the reaction of the positive control Alk-Cy5-PEG-NP 500 resulted in a significant increment of absorbance at 420 nm, a change that was undetectable for NH 2 -Cy5-PEG-NP 500 (which do not contain triple bonds). Importantly, the intensity of the signal was significantly reduced in DAS-Cy5-PEG-NP 500 , as it would be expected after conjugation with 3 by CuAAC. This simple analytical test served to verify that the click reaction took place at a relatively high yield, yet some unreacted alkynes were still present in the structure of DAS-Cy5-PEG-NP 500 , most probably being those less-accessible alkynes found in the interior of the polymeric framework of the NPs. An estimation of the CuAAC reaction yield can be performed with this spectrophotometric method, giving a value of 63.6%. The presence of unreacted alkynes suggests that sterically constrained environments may affect the reaction efficacy.
Chemical moieties displayed at the outer layer of NPs influence the surface charge and consequently the zeta potential of these colloidal structures. 29 To further validate the results obtained from the Baeyer test, the conjugation of dasatinib to the NPs was also assessed by measuring zeta potential of NH 2 -Cy5-PEG-NP 500 , Alk-Cy5-PEG-NP 500 , and DAS-Cy5-PEG-NP 500 . Figure 1b shows a displacement of zeta potential distribution to the negative range when amines were substituted by alkynes, whereas upon drug conjugation the presence of the drug on the NP displaces the zeta potential to less negative values, providing further qualitative evidence of the success of the CuAAC reaction.
As a final proof, rink amide resins (NH 2 -RINK, typically used in solid phase peptide synthesis) were treated with 3 using the same reaction conditions used for the functionalization of the NPs (see Scheme 1). Following release of the corresponding triazolo-modified dasatinib derivate from the solid support by linker cleavage in acidic conditions, NMR analysis confirmed the efficacy of this conjugation reaction (see SI).
Biological Assays. To assess if the biological activity of the drug is maintained after conjugation onto the NPs, DAS-Cy5-PEG-NP 500 were incubated with recombinant SRC and the activity of the kinase analyzed using a luminescence commercial kit. NPs without dasatinib (NH 2 -Cy5-PEG-NP 500 ) were used as negative control. As shown in Figure  2a, a concentration dependent reduction in SRC activity was observed in the presence of DAS-Cy5-PEG-NP 500 , with a 50% of inhibition observed at 2,041 × 10 6 NPs (51 × 10 3 NP/μL). By calculating the amount of drug per NP, the IC 50 value was estimated to be 33.4 nM.
Cross-linked polystyrene NPs have been previously shown to rapidly enter a wide range of cell types. [14][15][16][17][18][19]30 To prove that the presence of dasatinib does not interfere with the internalization process, cell uptake into MDA-MB-231 cells was analyzed by flow cytometry (Figure 2b and SI) and timelapse imaging (Movie S1). Cells exhibiting fluorescence in the Cy5 channel were considered "nanofected" and multiplicity of nanofection 50 (MNF50) values were calculated as the ratio of nanoparticles per cell necessary to "nanofect" 50% of the cell population. 24 As shown in Figure 2b, MNF50 values of DAS-Cy5-PEG-NP 500 only were slightly inferior when compared to the unclicked control NH 2 -Cy5-PEG-NP 500 , although no differences in internalization efficiency were observed at ≥300 NPs/cell. Target Engagement Study. To test the functionality of the nanodevices in cells, we next performed a target engagement assay. Triple negative breast cancer MDA-MB-231 cells were selected to this study as SRC protein is highly overexpressed in these cells. 31 MDA-MB-231 cells were treated with DAS-Cy5-PEG-NP 500 for 24 h and target-NP localization studied by confocal microscopy. The presence of a Cy5 fluorescent moiety on the NPs along with their size (500 nm) enabled the visualization of NPs by confocal microscopy into living cells. Immunocytochemistry analysis was carried out to visualize the SRC protein in the cells using an anti-SRC primary antibody and a fluorescent secondary antibody (Dylight 405). Z-stack images were acquired to image the NPs distributed throughout the cytoplasm (Figure 2c,d). Upon immunocytochemistry, image analysis showed colocalization of DAS-Cy5-PEG-NP 500 with SRC protein in MDA-MB-231 cells. In contrast, the control NPs not decorated with dasatinib (NH 2 -Cy5-PEG-NP 500 ) showed no colocalization with the target (see Supp. Inf.). These results were further confirmed by calculation of the Pearson's correlation coefficient (Figure 2e), demonstrating linear correlation (positive value) between the localization of DAS-Cy5-PEG-NP 500 and SRC. In contrast, a negative value was obtained by analysis the localization of NH 2 -Cy5-PEG-NP 500 and SRC. These results prove the capacity of DAS-Cy5-PEG-NP 500 to efficiently engage with a specific target in cellulo.

■ CONCLUSIONS
In conclusion, herein we have presented a novel drugdecorated nanoprobe with the capacity to penetrate mammalian cells and bind target proteins in the intracellular environment. The use of a conjugation strategy based on the efficient CuAAC reaction makes this approach accessible to many different drugs by simple incorporation of an azide handle. Caution, however, should be taken when assuming a quantitative yield for click reactions on miniaturized devices. To avoid false negatives, a rapid spectrophotometric test for click reactions on alkyne-tagged solid supports was developed based on the Baeyer test. This method can be used to estimate the yield of the coupling reaction by detecting unreacted alkynes on the solid support. As a proof of concept, fluorescent NPs were functionalized with dasatinib and the incorporation of the drug confirmed by different analytical methods. Dasatinib-decorated NPs induced a reduction in SRC activity in vitro, further demonstrating drug incorporation on the solid support, but also proving a correct spatial arrangement of the conjugate that maintained drug activity. These NPs were efficiently internalized into living MDA-MB-231 cells and, furthermore, demonstrated binding to the target protein (SRC kinase) in cellulo, proving the capacity of this novel nanodevice to detect drug-target interactions in complex living environments. The size of the nanoprobe (∼500 nm in ⌀) was selected based on the technical limitations of conventional fluorescence microscopy. 32

■ MATERIALS AND METHODS
General Chemistry Protocols. All commercially available chemicals were obtained from either Fisher Scientific, Matrix Scientific, Sigma-Aldrich, or VWR International Ltd. Reactions were performed under inert atmosphere (nitrogen) using anhydrous solvents. NMR spectra were recorded at ambient temperature on a 500 MHz Bruker Avance III spectrometer. Samples were dissolved in deuterated solvents commercially available from Sigma-Aldrich. 1 H NMR spectra: chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane. The data is presented as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants as a J value in Hertz (Hz) and interpretation. The number of protons (n) for a given resonance is indicated as nH, and is based on spectral integration values. TLCs were ran on Merck TLC Silica gel 60 F254 plates, typically 5 cm × 10 cm, and monitored using a 254 nm UV source. High resolution mass spectra were recorded by the MS Department of the University of Edinburgh on a Thermo MAT 900 XLP high resolution, double focusing mass spectrometer. All the couplings were carried out on the Eppendorf Thermomixer comfort and washed after centrifugation in an Eppendorf mini centrifuge.
Baeyer Test for Alkynes. 1 μL of stock KMnO 4 solution (2.5 mM) was added to 5 × 10 8 NPs or 0.5 mg Rink amide resins to a final volume of 10 μL in water. NPs and resins were then centrifuged at 13000 g for 5 min in an Eppendorf centrifuge and absorbance spectra were analyzed. 420 nm absorbance was represented as fold increase to control solution of KMnO 4 .
Dynamic Light Scattering and Zeta Potential. The hydrodynamic size and zeta potential of intermediate and final nanoparticle configurations were measured on a Malvern Zetasizer Nano-ZS (Malvern, Malvern Hills, UK) in molecular biology grade water in a disposable sizing cuvette for size measurements or clear disposable zeta cuvette for zeta potential measurements.
Kinase Activity Assay. Recombinant full-length human SRC activity was detected by luminescence using ADP-Glo Kinase Assay + SRC Kinase Enzyme System (Promega), according to manufacturer instructions in half area solid white 96-well plates at a total volume of 40 μL. The influence of a range of serial dilutions from 25 × 10 6 NPs, control NPs, or free drug on SRC activity (0.154 ng/μL) were analyzed by luminescence using a GloMax Multi (Promega) with an integration time of 0.5s and normalized to internal controls.
Uptake Efficiency. For flow cytometry assays, 5 × 10 5 MDA-MB-231 cells were seeded in 24-well plates and stabilized in an incubator for 18 h. Subsequently 3000, 1000, 300, 100, 30, 10, 3, or 0 of 9/cell or 11/cell were incubated in 500 μL of serum-free DMEM to evaluate cellular uptake. After 6 h, an equal volume of DMEM containing 2× FBS was added to all wells and cells were allowed to stabilize overnight. After washing with PBS, living cells were trypsinized for 5 min, Percentages of cells containing 9 or 11 were represented versus log (ratios NP/cell) and adjusted to a sigmoidal dose− response curve using GraphPad Prism Software. MNF50 values were calculated as the ratio NP/cell necessary to obtain a 50% of cells containing nanoparticles.
Confocal Microscopy Colocalization. MDA-MB-231 cells were seeded on 10 mm poly(L-lysine) precoated coverslips in 24 well plates (10 000 cells/well). In parallel, 6 × 10 7 of 9, 11, 5 or NP-free controls were incubated in 10 μL 5% BSA/H 2 O overnight at 4°C to block unspecific binding 34 (Supporting Information). NPs were thoroughly dispersed in a sonication bath for 15 min. Cells were then incubated in the presence or in the absence of 3000 NPs/cell in 500 μL of serum-free DMEM in duplicates. After 6 h, 500 μL of DMEM supplemented with 2× FBS were added to each of the wells and cells were kept in an incubator for another 18 h. NPs and cells were then fixed with paraformaldehyde (4% v/v) for 10 min and washed 3 times with PBS every 5 min. Cells were permeabilized for 15 min with 0.3% Tween/PBS and washed 3 times with PBS every 5 min. Coverslips were incubated in blocking buffer (0.3% BSA, 10% FBS, 5% nonfat dry milk, 0.1% Tween in PBS) for 30 min. Anti-Src Rabbit mAb (Cell Signaling Technology) was incubated in milk-free blocking buffer (0.3% BSA, 10% FBS, 0.1% Tween in PBS) for 2 h at a dilution of 1:400. After washing 3 times in 0.1% Tween/PBS, coverslips were incubated for 30 min with goat anti-rabbit IgG (H+L) secondary antibody, DyLight 405 conjugate (Thermofisher Scientific) in milk-free blocking buffer (0.3% BSA, 10% FBS, 0.1% Tween in PBS) at a dilution of 1:400. Coverslips were washed twice with 0.1% Tween/PBS, and mounted with Mowiol (Sigma-Aldrich) on a slide. NPs and SRC localization was imaged using a scanning confocal inverted microscope LSM 710 Axio Observer (Carl Zeiss, Jena, Germany). The

Bioconjugate Chemistry
Article images were acquired with a Plan-Apochromat 63×/1.4 OIL DIC M27 immersion objective and software ZEN 2010 (Carl Zeiss, Jena, Germany). Confocal images of 9, 11, 5, or NP-free controls in cell lysates and cells were obtained in a sequential mode using the following settings: (1) a 405 nm diode excitation laser (30 mW) at 2% power, an emission of in a range of 410 to 488 nm with a main beam splitter MBS-405, and a pinhole of 1.0 Airy Unit (0.6 μm optical section); and (2) a 633 nm HeNe excitation laser (5.0 mW) at 15% power, an emission wavelength range of 636−742 nm with a MBS-488/543/633, and a pinhole of 1.0 Airy Unit (0.7 μm optical section). Transmitted images were obtained with a T PMT. Zstack images were recorded in an average range of 6 μm, 13 slices, and a constant interval of 0.5 μm, according to NP size, to localize 500 nm nanoparticles throughout the cell volume. Images were processed with Software ZEN 2010 Black Edition (Carl Zeiss, Jena, Germany) to obtain overlap coefficient and Pearson's Correlation Coefficient as colocalization coefficients. Both coefficients, crosshair diagrams, and mask images were obtained after calibration according to negative controls and maintained for all the experiments (SI). Unpaired t test statistical analysis of four independent experiments was performed using Graph Pad Prism 5.0 for Windows GraphPad Software, La Jolla California USA, www.graphpad.com.

* S Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00481.
Full synthetic details, characterization spectra, results of control nanoparticles and resins, complementary biological data, confocal microscopy images (PDF) Time-lapse imaging of cell uptake into MDA-MB-231 cells (AVI)