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. 2019 Dec 27;5(1):588–596. doi: 10.1021/acsomega.9b03169

Inkjet Bioprinting on Parchment Paper for Hit Identification from Small Molecule Libraries

Jungmi Lee 1, Baskaran Purushothaman 1, Joon Myong Song 1,*
PMCID: PMC6964283  PMID: 31956806

Abstract

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In this study, an inkjet bioprinting-based high-throughput screening (HTS) system was designed and applied for the first time to a catecholpyrimidine-based small molecule library to find hit compounds that inhibit c-Jun NH2-terminal kinase1 (JNK1). JNK1 kinase, inactivated MAPKAPK2, and specific fluorescent peptides along with bioink were printed on parchment paper under optimized printing conditions that did not allow rapid evaporation of printed media based on Triton-X and glycerol. Subsequently, different small compounds were printed and tested against JNK1 kinase to evaluate their degree of phosphorylation inhibition. After printing and incubation, fluorescence intensities from the phosphorylated/nonphosphorylated peptide were acquired for the % phosphorylation analysis. The IM50 (inhibitory mole 50) value was determined as 1.55 × 10–15 mol for the hit compound, 22. Thus, this work demonstrated that inkjet bioprinting-based HTS can potentially be adopted for the drug discovery process using small molecule libraries, and cost-effective HTS can be expected to be established based on its low nano- to picoliter printing volume.

Introduction

High-throughput screening (HTS) of small molecule libraries is highly influential in the discovery of hit compounds because it is the starting point in the development of commercial drugs.1 Successful HTS techniques that are routinely used in the discovery process include time-resolved fluorescence resonance energy transfer (FRET),2,3 fluorescence polarization/anisotropy,4,5 alpha Screen,6 scintillation proximity assay,7 chemi-luminescence,8 and cell-free, enzyme-linked immunosorbent assay9 at the molecular and cellular levels. All of these biochemical-based HTS are targeted assays that include reaction components and target molecules that are incubated with synthetic or natural compounds in order to determine the activity of the compound (e.g., activator or inhibitor). Test compounds (small molecules or drugs) usually bind to known molecular targets or unintended off-targets. Such compounds have often led to unexplained reactions, resulting in side effects or drug repositioning opportunities. Drug repositioning is defined as finding new therapeutic activities for already existing drugs including marketed, discontinued, and even synthetic or natural product-based therapeutic agents.10,11 Recently, several studies have identified compounds from a pool of drugs approved for other applications.12,13 This is a profitable approach because the new applications of these compounds build on the available information regarding their pharmacokinetics, safety, and manufacturing.

Recently, we reported the in vitro and in vivo anti-inflammatory ability of 30 unique catechol-functionalized pyrimidine-based small molecules against phosphodiesterase-4B (PDE4B).14,15 Considering the cost-effectiveness of the drug repositioning approach, we intended to discover new potential targets of our synthetic catecholpyrimidine-based small molecule library. In this work, to validate this objective, an inkjet printing-based HTS platform was designed and applied to our 30 synthetic small molecule library. To date, inkjet printing technology has not been applied to screen small molecule libraries in order to find activities with respect to drug targets in the HT manner even though a few molecules have been tested to demonstrate the possibility of screening.16 First, inkjet bioprinting is very attractive for applications for screening of small molecule libraries because its ejection volume reaches the nano-to picoliter range. The use of such an ultrasmall ejection volume is inevitable due to enormous cost reduction expected for the drug discovery process as the number of molecules in the small molecule library increases. In addition, the ejection volume in inkjet bioprinting is highly reproducible, which is quite advantageous for obtaining quantitatively accurate drug efficacy data.

Currently, kinase activity assays depend on the use of expensive reagents and/or instrumentation. On the other hand, inkjet bioprinting can provide a straightforward, low-cost alternative to determine kinase activity through HTS.17,18 Over several decades, inkjet bioprinting technologies have been increasingly used as a tool for the synthesis of small molecules,19 thin film coatings,20 genomics validation,21 and biosensor fabrication.22−27 Moreover, bioprinting has been used for the direct printing of precise patterns to evaluate the activity of DNA,28 RNA,29 protein,30 enzymes,31−33 and cells34,35 on various substrates.

Herein, for the first time, we report an inkjet bioprinting-based HTS for screening a library of novel catecholpyrimidine-based small molecules (30 synthetic compounds) for discovering new hit compounds that inhibit kinase activity. The bioink composition consisting of Triton-X and glycerol was optimized to establish the matrix without any migration, diffusion, and absorption of printed reagents onto the substrate for the kinase-to-kinase phosphorylation. The phosphorylation reaction of two different kinase enzymes must be handled extremely carefully for finding a hit compound on the printing substrate. The optimized composition of bioink is essential for the successful HTS. The biomaterials (target enzymes: c-Jun NH2-terminal kinase1 (JNK1) and the mitogen-activated protein kinases (MAPKs) along with reaction components) and synthetic compounds (test compounds: catecholpyrimidine-based small molecules) were initially loaded into the printing cartridge and subsequently printed on the substrate. We show that the catecholpyrimidine-based small molecules and the enzymes (JNK1 and MAPK) interact synergistically to inhibit JNK1 activity. Taking the HTS approach as a starting point to assess hit kinase inhibitor compounds, 30 small molecules were tested by utilizing the bioprinting-based HTS platform. Our results demonstrate that the hit compound (compound 22) out of 30 small molecules was highly efficient at inhibiting kinase activity in the femtomolar range. Thus, the combination of the bioprinting technique and HTS led to the discovery of catecholpyrimidine small molecules as potential JNK1 inhibitors and represents a new alternative for the screening of synthetic and natural product-based small molecular libraries. These results suggest a bright future for inkjet bioprinting as a new platform for HTS of compound libraries. It should be noted that the present inkjet bioprinting system is still in the research stage and can be further developed in combination with an automated robotic system. The inkjet printing using cartridges can be adopted as a dispenser to provide a small ejection volume, ranging from nanoliter to picoliter. The number of HT printing reaction spots is potentially unlimited and can be simply adjusted using software as needed in HTS.

Results and Discussion

Small Molecules Library for Screening JNK1 Inhibition

JNK is a serine threonine protein kinase that phosphorylates c-Jun, a component of the transcription factor activator protein-1. JNK activity appears to be critical for both the immune response and for programmed cell death. Hence, therapeutically targeting JNK can provide clinical benefits in disease conditions such as arthritis, inflammatory disease, chronic obstructive pulmonary disease, and myocardial infarction. Recently, we reported on the anti-inflammatory activities of a catechol-functionalized pyrimidine-based small molecule library against PDE4B. Kwak et al.36 examined the mechanism by which roflumilast (PDE4B inhibitor) inhibits inflammatory mediators such as nuclear factor-κB, p38 mitogen-activated protein kinase, and c-Jun NH2-terminal kinase. Similarly, several other studies have reported the use of PDE4B inhibitors to suppress the activity of JNK/MAPKs. Considering these findings, the catecholpyrimidine-based small molecule library that showed selectivity to PDE4B was tested for multitargeting effect. The bioprinting-based HTS platform was designed and employed to screen the library of novel catecholpyrimidine-based small molecules against kinase.

The bioprinting-based dispersion technique is a promising approach for pharmaceutical applications. The bioprinting-based HTS has several advantages such as low cost due to the nano- to picoliter ejection volume, miniaturized automatic structure, biocompatibility and reproducibility and the ease of software-based HT pattern design. Despite these powerful advantages, with the exception of the present work, screening of library compounds for a particular target has not been performed through inkjet bioprinting to date.37,38Figure 1 represents the structure of the pyrimidine-based small molecules. Compounds 22, 26, and 27 were synthesized according to the previously reported procedure. The structure and molecular weight of the compounds were analyzed using NMR and ESI-QTOF mass spectrometry. The detailed synthetic procedures of compounds 22, 26, and 27 are described in the Supporting Information.

Figure 1.

Figure 1

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Schematic illustration of the JNK1-mediated phosphorylation cascade reaction using the inkjet printing method.

Schematic Representation of JNK1-dependent Phosphorylation Reaction

Figure 1 illustrates the scheme for performing the inkjet printing-based JNK1-dependent phosphorylation cascade reaction. To perform the JNK-1-dependent phosphorylation reaction, all of the reaction components were sequentially printed on the parchment paper surface and the reaction was carried out. The Ser/Thr 4 peptide was labeled with two fluorophores (donor-coumarin and acceptor-fluorescein) for the FRET pair. This labeled peptide was used to determine the JNK1-dependent phosphorylation cascade reaction on paper. As shown in Figure 1, reaction components, including the synthesized compounds, JNK1 + MAPKAPK2 + Ser/Thr 4 peptide mixture, adenosine triphosphate (ATP), and the development reagent were printed from CMYK cartridges. The reaction occurred on the parchment paper surface. JNK1 activates MAPKAPK2, and in the presence of ATP, the activated MAPKAPK2 transfers a γ-phosphate (γ-PO32–) group on ATP. γ-PO32– is transferred to a single serine or threonine residue in the Ser/Thr peptide substrate. The phosphorylation reaction is inhibited in the presence of the synthesized compound. After the phosphorylation reaction, a development reagent was printed to the reaction spot. The development reagent specifically recognized and cleaved the nonphosphorylated peptide. After cleavage, the fluorescence emission intensities of the phosphorylated and nonphosphorylated peptides were acquired. Based on the fluorescence emission intensity, % phosphorylation was calculated and IM50 (inhibitory mole 50) was determined.

Bioink Activity Optimization before the Printing-Based Assay

A FRET peptide (Ser/Thr 4 peptide), tagged with two fluorophores (coumarin and fluorescein), was used to determine the kinase activity on the substrate (parchment paper). The bioink composition based on Triton X-100 and glycerol was optimized for the best printing performance. In particular, the evaporation rate of the printed bioink on the parchment paper is highly important for dealing with consecutive enzymatic reactions such as kinase phosphorylation. The printing condition devoid of evaporation of printed bioink was optimized using glycerol and was utilized to print the assay components. To modify the viscosity, surface tension, and evaporation rate, Triton-X and glycerol were used for the preparation of bioink. Triton-X is used widely as a mild surfactant because it does not affect enzyme activity. Glycerol was used to modify the viscosity and evaporation rate. Nine different components of the 1.33× assay buffer with W/WO surfactant were prepared, and the JNK1 activity was investigated. The control group showed that the phosphorylation efficiency was 36.61%. Bioink solutions of JNK1 (7.5 × 10–7 g/mL) + MAPKAPK2 (5.0 × 10–6 g/mL) + Ser/Thr 4 peptide (4 μM) mixture, ATP (0.1 mM), and the development reagent in 1.33× assay buffer containing nine concentrations of the surfactant were prepared. Their activities in the prepared 1.33× assay buffer were measured. Figure 2 shows the effect of the surfactant dose on the JNK1 activity. Figure 3 illustrates the % phosphorylation change based on the increased addition (%) of glycerol. For the 0.1% Triton-X dose, there was a negligible decrease in the JNK1 activity. In the presence of the 0.1% Triton-X dose, glycerol was added (1–15%). Because of the viscosity of glycerol, the viscosity of bioink solution was also changed and the glycerol content impacted the JNK1 activity. When 1–4% of glycerol was added to the bioink solution, the JNK1 activity was not altered clearly. However, further addition of glycerol decreased the JNK1 activity, and when more than 5% glycerol was added to the assay buffer, the enzyme activity was decreased vertically as the amount of added glycerol increased.

Figure 2.

Figure 2

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Optimization of JNK1-dependent phosphorylation activity using inkjet printing in the presence of Triton-X and glycerol. Error bars represent the standard deviation of three independent measurements.

Figure 3.

Figure 3

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(a) Graphical illustration of 30 small compound-mediated JNK1 inhibition screening using inkjet printing. (b) Acquired fluorescence emission images of compounds 22, 26, and 27, and fluorescence emission images of 0% phosphorylation, 0% inhibition, and 100% phosphorylation conditions. The printed amount of small molecules is presented.

The printing ability of bioinks was measured prior to the printing-based JNK1 inhibition assay. The printing ability of each bioink composition was detected using an inkjet printer. Table 1 shows the printing ability of the bioinks. The printing was carried out in the presence of 0.1% Triton-X in the bioink. After adding 1–10% of glycerol to the 0.1% Triton-X-containing bioink, printing was performed well. As shown in Table 1, when 15% glycerol was added to the assay buffer, the bioink was not printed on the parchment paper surface because of its high viscosity. After printing, the spreading patterns of all of the bioink compositions (0–15% glycerol) were observed. At 0% Triton-X of only buffer solution, the printed pattern showed evaporation immediately with small spreading. At 0–10% glycerol with 0.1% Triton-X, there was no spreading or tailing of the printing pattern on the parchment paper. All of the printed patterns were printed clearly and remained without moisture absorption of parchment paper. Furthermore, these phenomena were sustained despite repeated printing on the parchment paper surface. Based on these results, 0.1% Triton-X with 2% glycerol was selected as the bioink media for the inkjet printing-based experiment.

Table 1. Evaluation of the Printability of Bioinks Based on Their Composition.

Triton-X 0% Triton-X 0.1% Triton-X
glycerol (%) 0 0 1 2 3 4 5 10 15
printability √ √ √ √ √ √ √ √ ×
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Determination of the JNK1 Inhibition Activity Using Inkjet Printing

The percentage of phosphorylation and inhibitory efficacy of synthesized compounds were acquired through the inkjet bioprinting HTS. As illustrated in Figure 3a, inkjet printing-based JNK1-dependent phosphorylation and JNK1 activity screening was performed. To investigate hit compounds as JNK1 inhibitors, four different synthesized small compounds were used. To check the inhibition activity of small compounds, two groups (control and test) of experiments were performed. To determine the JNK1-dependent phosphorylation efficacy, all of the bioinks were printed on parchment paper. As a control, the printing was carried out with (100% phosphorylation or 0% inhibition condition) and without ATP (0% phosphorylation or 100% inhibition condition) conditions, and phosphorylated-Ser/Thr 4 peptide was printed. The printing pattern was set to be 3 rows by 3 columns for reproducibility, and the pattern size was set by the spot diameter of 1 mm. After printing and incubation, the reaction spot was excited at 400 nm. As shown in Figure 4b, at the 0% inhibition condition, 35.27% of the Ser/Thr 4 peptide was phosphorylated and the emission ratio was 1.84. This result indicates that JNK1 (and MAPKAPK2) is active and can phosphorylate 35.27% of the Ser/Thr 4 peptide on the reaction spot, and 64.73% of the Ser/Thr 4 peptide was cleaved. In the 100% inhibition condition, 0% of the Ser/Thr 4 peptide was phosphorylated and the emission ratio was 3.86. For the phosphorylated-Ser/Thr 4 peptide, 100% of the peptide was phosphorylated and the emission ratio was 0.41%. These observed differences in the % phosphorylation indicate that the phosphorylation cascade reaction was successfully carried out on the parchment paper surface.

Figure 4.

Figure 4

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(a) Fluorescence emission of coumarin (445 nm) and fluorescein (525 nm) generated in the reaction spot in the presence of compound 22. (b) Fluorescence emission of coumarin (445 nm) and fluorescein (525 nm) generated in the reaction spot in the presence of compound 26. (c) Fluorescence emission of coumarin (445 nm) and fluorescein (525 nm) generated in the reaction spot in the presence of compound 27.

As illustrated in Figure 3a, the inkjet printing drug screening assay was performed in order to estimate the JNK1 inhibition ability of 30 small compounds. The compound cartridge (black color cartridge) was changed for the printing of each synthesized compound. After printing a compound, the black cartridge was changed to another black cartridge for the printing of another compound. All black cartridges were cleaned with ethanol before use. The printer head and dispensing part of the black cartridge were cleaned by printing of ethanol in the cartridge. Then, the black cartridge was filled with another synthesized compound along with the identical bioink. The cleaning process of the cartridge was executed automatically using a printer management software. The cleaned cartridge was reused after rapid cleaning. The bioink-synthesized compound → JNK1 + MAPKAPK2 + Ser/Thr 4 peptide mixture → ATP were sequentially printed on parchment paper at each reaction point of 8 rows by 20 columns. Table 2 shows the CMYK color values, density, and ejected volume per printing of each reagent, and the ejected amount of each reagent was calculated from these values. The present results verify that inkjet bioprinting HTS is a powerful screening tool of library compounds. Particularly, as shown in Table 2, the present inkjet bioprinting system provided the ejection volume to reach to ∼5 nL. After printing, 1.7 × 10–11 g of JNK1, 1.16 × 10–10 g of MAPKAPK2, 4.63 × 10–15 mol of Ser/Thr 4 peptide, and 1.04 × 10–13 mol of ATP were printed on each reaction spot.

Table 2. Summary of the Solution Densities of the Bioinks and Their Respective Ejection Volumes/Spot Areas.

  kinase-peptide mixture ATP development solution compound 22 compound 26 compound 27
density (g/mL) 1.05 1.12 1.11 1.05 1.08 1.02
CMYK value 100 50 100 50 50 50
ejected volume (×10–9 L/mm2) 7.37 ± 0.2 6.63 ± 0.17 8.15 ± 0.41 5.39 ± 0.25 5.47 ± 0.48 6.1 ± 0.31
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Figure 4b shows the acquired fluorescence emission images of compounds 22, 26, and 27 and the amounts of the printed small molecules. The amounts of printed small molecules were altered by the CMYK color codes. Table 3 shows the ejected amount of the small molecules (22, 26, and 27) after printing. As shown in Table 3, 0.23 × 10–15 to 2.25 × 10–15 mol of compound 22, 0.19 × 10–13 to 1.92 × 10–13 mol of compound 26, and 0.17 × 10–13 to 1.72 × 10–13 mol of compound 27 were printed on the parchment paper when the CMYK color codes were set in the range of 5–50. Then, the reagents printed on the parchment paper were incubated at room temperature for 1 h. Additionally, the development reagent printed on each reaction spot was incubated at room temperature for 1 h. After the printing-based drug screening reaction, the fluorescence emission from each reaction spot was acquired using a confocal microscope. In the presence of small molecules, a gradationally decreased emission signal of fluorescein (525 nm) and an increased emission signal of coumarin (445 nm) were recorded simultaneously.

Table 3. Number of Printed Moles and Grams of Small Molecules Ejected on Parchment Paper Based on K Value.

  K value
  
  5 10 15 20 30 40 45 50 IM50
printed moles of compound 22 (10–15 mol/10–13 g) 0.23/1.12 0.45/2.19 0.68/3.31 0.90/4.38 1.35/6.53 1.80/8.77 2.03/9.89 2.25/10.96 1.55 × 10–15
  ±0.05/±0.01 ±0.06/±0.03 ±0.06/±0.02 ±0.03/±0.04 ±0.04/±0.06 ±0.03/±0.06 ±0.08/±0.04 ± 0.02/±0.04  
printed moles of compound 26 (10–13 mol/10–11 g) 0.19/0.84 0.38/1.68 0.58/2.56 0.77/3.40 1.15/5.08 1.54/6.80 1.73/7.64 1.92/8.48 1.66 × 10–13
  ±0.04/±0.02 ±0.05/±0.03 ±0.04/±0.02 ±0.03/±0.05 ±0.02/±0.03 ±0.05/±0.04 ±0.03/±0.06 ±0.04/±0.02  
printed moles of compound 27 (10–13 mol/10–11 g) 0.17/0.79 0.34/1.58 0.52/2.72 0.69/3.21 1.03/4.79 1.38/6.42 1.55/7.21 1.72/8.00 1.09 × 10–13
  ±0.06/±0.03 ±0.07/±0.04 ±0.04/±0.02 ±0.05/±0.01 ±0.04/±0.03 ±0.06/±0.05 ±0.03/±0.01 ±0.03/±0.04  
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Figure 4 shows the decreased emission signal of fluorescein and an increased emission signal of coumarin. In the presence of compounds 22 (Figure 4a), 26 (Figure 4b), and 27 (Figure 4c), an increase in the coumarin emission intensity and a decrease in the fluorescein emission intensity were generated at each reaction spot. This result indicates that compounds 22, 26, and 27 inhibited JNK1, and thus, MAPKAPK2 (downstream cascade enzyme of JNK1) was not activated. Therefore, the amount of cascade reaction-mediated phosphorylation of the Ser/Thr 4 peptide was decreased. Figure 5 shows the decreases in the % phosphorylation by compound 22 (Figure 5a), compound 26 (Figure 5b), and compound 27 (Figure 5b) and the molecular structures of these small molecules. As shown in Table 3, the acquired IM50 of each compound was 1.55 × 10–15 mol (compound 22), 1.66 × 10–13 mol (compound 26), and 1.09 × 10–13 mol (compound 27). These results show that compound 22 has the lowest inhibitory activity of the three synthesized compounds. On the other hand, most of the small molecules showed IM50 values in the nanomolar range (Table S1). Although thirty small molecules were screened to find hit compounds through the present inkjet bioprinting system, more small molecules can be screened up to the number which is required in HTS in the present inkjet bioprinting system. This can be easily adjusted by designing printing spots of row-by-column using the software.

Figure 5.

Figure 5

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(a) Percentage of phosphorylation activity on each reaction spot in the presence of compound 22 and molecular structure of compound 22. (b) Percentage of phosphorylation activity on each reaction spot in the presence of compound 26 and molecular structure of compound 26. (c) Percentage of phosphorylation activity on each reaction spot in the presence of compound 27 and molecular structure of compound 27.

Generally, physicochemical properties of compounds can be varied according to their different chemical structures and functional groups. Because of the large compound number in libraries, it is unavoidable to face a variety of compounds having different physicochemical properties in a small molecule library. As the compound number of the library increases, the variety inherent to the compounds of the library is greatly developed. When the inkjet printing is applied to HTS of a small molecule library, conformance of a particular bioink has to be considered for a large number of library compounds with different physicochemical properties. It is interesting to prove whether a particular bioink can cover a large number of library compounds having different chemical structures and functional groups without the change of its composition for inkjet printing. In this work, the 30 small-molecules library is derivatives based on the catecholopyrimidine or morpholinopyrimidine core structure mainly. These chemical structures have different physicochemical properties such as solubility, lipophilicity, molecular weight, and so forth. The inkjet printing of all compounds was tested with a bioink in spite of the difference in core molecular structures and functional groups. Surprisingly, the inkjet printing of 30 synthetic molecules could be successfully performed using a bioink consisting of 0.1% Triton-X and 2% glycerol without the change of its composition. This is the first attempt and important demonstration to verify how a small molecule library can properly act on a bioink for inkjet HTS to identify a hit compound without the change of its composition.

Conclusions

In summary, a user-friendly bioprinting-based HTS was developed to screen a library of novel catecholpyrimidine-based small molecules, and hit compounds were found to target JNK1. The developed platform showed that a FRET peptide substrate was successfully utilized to determine the phosphorylation percentage, which in turn revealed the inhibitory activity of the synthetic molecules against JNK1. Collectively, the present result demonstrated that the hit compound (compound 22) identified out of 30 small molecules was highly efficient at inhibiting JNK1 activity in the femtomolar range, which was significantly more effective than the reported JNK inhibitors. Herein, bioprinting HTS led to the successful discovery of catecholpyrimidine small molecules as potential JNK1 inhibitors. Thus, the proposed bioprinting-based HTS represents a new option for screening synthetic and natural product-based small molecular libraries.

Materials and Methods

Chemicals and Materials

All of the assay components including JNK1, inactive MAPKAPK2, ATP (10 mM), Ser/Thr 4 peptide (1 mM), Ser/Thr 4 phosphopeptide (1 mM), phosphorylated-Ser/Thr 4 phosphopeptide (1 mM), and the developing reagent were purchased from Invitrogen (California, USA). Glycerol and Triton-X were purchased from Sigma-Aldrich (MO, USA). All of the chemicals and materials were used without further purification.

Synthesis of Small Molecules

The small molecules were synthesized according to a previously reported procedure as explained in detail in the Supporting Information.

Bioink Preparation

All of the reagents used in the experiment were freshly prepared prior to the experiment. The 5× assay buffer (250 mM of pH 7.5 N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid buffer, 50 mM MgCl2, 5 mM EGTA, 0.05% BRIG-35) was freshly prepared. JNK1 (7.5 × 10–7 g/mL), inactive MAPKAPK2 (5.0 × 10–6 g/mL), ATP (10 mM), Ser/Thr 4 peptide (4 μM), and Ser/Thr 4 phosphopeptide (4 μM) were prepared using the 1.33× assay buffer. The development solution was prepared using the 1× development buffer. The synthesized small molecule solutions were prepared using a mixture of water and dimethyl sulfoxide (DMSO).

Bioink Activity Optimization before the Printing-Based Assay

All reagents used in the experiment were freshly prepared and the amount of DMSO was kept below 2% in order not to affect the activities of the enzymes. All of the solutions were prepared just before the experiment and kept on ice at 0 °C, and the solutions remaining after the experiment were discarded without being reused. The synthesized compounds dissolved in DMSO could be stored at 4 °C for 3 weeks and used for printing. The powders of synthesized compounds were found to be stable up to 2 years at 4 °C. In this work, it was found that at least 1.5 mL solution composed of reagents along with the bioink media was needed in the cartridge for the successful inkjet printing. To modify the viscosity and evaporation velocity, Triton-X and glycerol were used for the bioink preparation. All bioinks were prepared in a 1.33× assay buffer containing 0.1% Triton-X and 1–15% glycerol. Prior to the printing, the phosphorylation activities and printing abilities were measured. JNK1 activity was measured in a 384-well plate. Initially, JNK1 + MAPKAPK2 + Ser/Thr 4 peptide mixtures (5 μL) containing different % of glycerol were transferred to a 384-well plate. ATP (2.5 μL) was added to each well, and the assay plate was placed on a shaker for 30 s to mix the reagents well. The plate was incubated at room temperature (25 °C) for 1.5 h for the cascade phosphorylation. During incubation, the well plate was covered with a transparent sheet to prevent evaporation and contamination of the solution. After incubation, a development solution (5 μL) was added to each well and the assay plate was placed on a shaker for 30 s to mix the reagents. The plate was incubated at room temperature (25 °C) for 1 h for enzymatic cleavage. Then, the well plate was covered with a transparent sheet. Finally, the stop solution (5 μL) was added, and after shaking for 30 s, the fluorescence intensities of coumarin (Ex/Em = 400/445 nm) and fluorescein (Ex/Em = 400/525 nm) were measured using a multiplate reader. Prior to the measurement, the total reaction volume was set to 20 μL after the addition of 1.33× assay buffer (2.5 μL). For the control, with and without ATP conditions experiments were performed, and the emission fluorescence intensity from the phosphorylated-Ser/Thr 4 peptide was acquired. The following two equations were used to calculate the percentage of phosphorylation for the assessment of the inhibition activity of the synthesized compounds.

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Bioink Printing Ability before the Printing-Based Assay

A set of four cartridges used for the printer (HP Officejet Pro 8100) was modified. The inks present inside the cartridges were removed, and the cartridges were washed 10 times with deionized water and 100% ethanol. To minimize the damage of the cartridge rubber pack during the clearing, the backside of the cartridge was cut and a hole was drilled and the inside was emptied. After the removal of their contents, the four cartridges were dried at room temperature (25 °C) overnight. These modified cartridges were used for the experiment.

To determine whether printing is possible based on the composition of all of the bioinks, 1.33× assay buffers containing 0.1% Triton-X and 1–15% of glycerol were prepared. The CMYK (cyan, magenta, yellow, and black) cartridges were filled with each bioink, and bioinks were printed onto the parchment paper surface. The CMYK color values that can verify the ejected volume from the printer head were adjusted using a graphic program (Adobe Photoshop CC), and the printing pattern was set using Microsoft Power Point software. The CMYK color codes were converted to RGB color codes, and the color of the printing pattern was set. The pattern was designed using the Microsoft PowerPoint software and each bioink was dispensed onto the printing pattern. All of the printing patterns were designed and were set to 8 rows by 20 columns. The individual printing spots of 8 row by 20 columns correspond to each well in the commercially available well-plate. The dimensions of the row-by-column printing spot can be freely adjusted using the software as required in HTS, for example, 1536 spots and more can be made. All of the color values were set as 100, and the shape of the printing pattern for the reaction was set as a spot with a diameter of 1 mm. All of the bioinks were printed on the each reaction spot, and their printing abilities were identified. The enzymatic stability was found to be stable up to 4 h after the preparation of enzymatic assay solution. Based on this result, the JNK1 inhibition assay was performed.

Determination of JNK1 Inhibition Activity Using Inkjet Printing

To investigate the JNK1-dependent phosphorylation and inhibition activity on parchment paper, all bioinks were printed on parchment paper. First, with and without ATP, 0% inhibition (100% phosphorylation) and 100% inhibition (0% phosphorylation) conditions were measured for the control. The printing patterns were designed as 3 rows by 3 columns for the control assay, 1 mm diameter spot, and 1 mm gap per each spot to spot. To evaluate the inhibitory activity of the synthesized compounds (as JNK1 inhibitors) using inkjet printing, the CMYK color codes settings were fixed at 100, 50, 100, and 100, respectively, in order to print constant amounts of JNK1 + MAPKAPK2 + Ser/Thr 4 phosphopeptide, ATP, phosphorylated-Ser/Thr 4 peptide, development reagent, and 1.33× assay buffer, respectively. The printing patterns were designed as 8 rows by 20 columns with a spot diameter of 1 mm and a gap of 1 mm for each spot. For the synthesized compounds, the color code settings were varied from 5 to 50 in order to vary the amount of the synthesized compounds. Herein, for this experiment, two printing heads and 2 sets of CMYK cartridges were prepared.

For the control assay, the inhibition activity of 100% was evaluated by printing the mixture of JNK1 + MAPKAPK2 + Ser/Thr 4 peptide without ATP (Figure 3b—first column), whereas the inhibition activity of 0% was assessed with printing the mixture of JNK1 + MAPKAPK2 + Ser/Thr 4 peptide along with ATP (Figure 3b—second column). The phosphorylation of 100% was evaluated by printing the phosphorylated-Ser/Thr 4 peptide (Figure 3b—third column).

After the control assay, inkjet printing-based phosphorylation cascade reactions were performed to determine the JNK1 inhibition activity of the synthesized small compounds. For this assay, the bioink-synthesized compound → JNK1 + MAPKAPK2 + Ser/Thr 4 peptide mixture → ATP was sequentially printed on parchment paper at each reaction spot. The assay component-printed papers were incubated for 1 h at room temperature (25 °C). The 1.33× assay buffer was printed once in 20 min to stimulate the phosphorylation cascade reaction on the parchment paper. After incubation, the developing solution was printed on the reaction spot, and the printed papers were incubated for 1 h at room temperature (25 °C). After incubation, fluorescence images [coumarin (Ex/Em = 400 nm/445 nm) and fluorescein (Ex/Em = 400 nm/525 nm)] were acquired using a confocal microscope (Leica, TCS SP8). The acquired fluorescence images were analyzed using ImageJ software. The amounts of the assay components printed on each spot were evaluated based on the volume printed per spot and their respective solution densities, as suggested by Song et al.39

Acknowledgments

This work was supported by National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (2018M3A7B4071235 and 2019R1A4A2001651). We are grateful to the Research Institute of Pharmaceutical Sciences at Seoul National University for providing experimental equipment and Brain Korea 21 plus (BK 21 plus).

Glossary

Abbreviations

HTS

high-throughput screening

JNK1

c-Jun NH2-terminal kinase1

IM50

inhibitory mole 50

PDE4B

phosphodiesterase-4B

CMYK

cyan, magenta, yellow, and black

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b03169.

  • Synthesis of compounds 27, 28, 29, and 30; operation stability of bioinks; and small molecules and their JNK1 IM50 values (PDF)

Author Contributions

† J.L. and B.P. contributed equally. All authors have given approval to the final version of the article.

The authors declare no competing financial interest.

Supplementary Material

ao9b03169_si_001.pdf (141.4KB, pdf)

References

  1. Macarron R.; Banks M. N.; Bojanic D.; Burns D. J.; Cirovic D. A.; Garyantes T.; Green D. V. S.; Hertzberg R. P.; Janzen W. P.; Paslay J. W.; Schopfer U.; Sittampalam G. S. Impact of high-throughput screening in biomedical research. Nat. Rev. Drug Discovery 2011, 10, 188–195. 10.1038/nrd3368. [DOI] [PubMed] [Google Scholar]
  2. Qian J.; Wang C.; Pan X.; Liu S. A high-throughput homogeneous immunoassay based on Forster resonance energy transfer between quantum dots and gold nanoparticles. Anal. Chim. Acta 2013, 763, 43–49. 10.1016/j.aca.2012.12.011. [DOI] [PubMed] [Google Scholar]
  3. Scholler P.; Moreno-Delgado D.; Lecat-Guillet N.; Doumazane E.; Monnier C.; Charrier-Savournin F.; Fabre L.; Chouvet C.; Soldevila S.; Lamarque L.; Donsimoni G.; Roux T.; Zwier J. M.; Trinquet E.; Rondard P.; Pin J.-P. HTS-compatible FRET-based conformational sensors clarify membrane receptor activation. Nat. Chem. Biol. 2017, 13, 372–380. 10.1038/nchembio.2286. [DOI] [PubMed] [Google Scholar]
  4. Talantsev E. F.; Strickland N. M.; Hoefakker P.; Xia J. A.; Long N. J. Critical current anisotropy for second generation HTS wires. Curr. Appl. Phys. 2008, 8, 388–390. 10.1016/j.cap.2007.10.036. [DOI] [Google Scholar]
  5. Zhang H. R.; Wu Q.; Berezin M. Y. Fluorescence anisotropy (polarization): from drug screening to precision medicine. Expert Opin. Drug Discovery 2015, 10, 1145–1161. 10.1517/17460441.2015.1075001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Marchand C.; Lea W. A.; Jadhav A.; Dexheimer T. S.; Austin C. P.; Inglese J.; Pommier Y.; Simeonov A. Identification of phosphotyrosine mimetic inhibitors of human tyrosyl-DNA phosphodiesterase I by a novel AlphaScreen high-throughput assay. Mol. Cancer Ther. 2009, 8, 240–248. 10.1158/1535-7163.mct-08-0878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cook N. D. Scintillation proximity assay: A versatile high-throughput screening technology. Drug Discovery Today 1996, 1, 287–294. 10.1016/1359-6446(96)10026-x. [DOI] [Google Scholar]
  8. Aljofan M.; Porotto M.; Moscona A.; Mungall B. A. Development and validation of a chemiluminescent immunodetection assay amenable to high throughput screening of antiviral drugs for Nipah and Hendra virus. J. Virol. Methods 2008, 149, 12–19. 10.1016/j.jviromet.2008.01.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lim M. J.; Foster G. J.; Gite S.; Ostendorff H. P.; Narod S.; Rothschild K. J. An ELISA-based high throughput protein truncation test for inherited breast cancer. Breast Cancer Res. 2010, 12, R78. 10.1186/bcr2722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Talevi A. Drug repositioning: current approaches and their implications in the precision medicine era. Expert Rev. Precis. Med. Drug Dev. 2018, 3, 49–61. 10.1080/23808993.2018.1424535. [DOI] [Google Scholar]
  11. Pushpakom S.; Iorio F.; Eyers P. A.; Escott K. J.; Hopper S.; Wells A.; Doig A.; Guilliams T.; Latimer J.; McNamee C.; Norris A.; Sanseau P.; Cavalla D.; Pirmohamed M. Drug repurposing: progress, challenges and recommendations. Nat. Rev. Drug Discovery 2019, 18, 41. 10.1038/nrd.2018.168. [DOI] [PubMed] [Google Scholar]
  12. Golebiowski A.; Klopfenstein S. R.; Portlock D. E. Lead compounds discovered from libraries. Curr. Opin. Chem. Biol. 2001, 5, 273–284. 10.1016/s1367-5931(00)00203-9. [DOI] [PubMed] [Google Scholar]
  13. Golebiowski A.; Klopfenstein S. R.; Portlock D. E. Lead compounds discovered from libraries: Part 2. Curr. Opin. Chem. Biol. 2003, 7, 308–325. 10.1016/s1367-5931(03)00059-0. [DOI] [PubMed] [Google Scholar]
  14. Purushothaman B.; Arumugam P.; Kulsi G.; Song J. M. Design, synthesis, and biological evaluation of novel catecholopyrimidine based PDE4 inhibitor for the treatment of atopic dermatitis. Eur. J. Med. Chem. 2018, 145, 673–690. 10.1016/j.ejmech.2017.12.069. [DOI] [PubMed] [Google Scholar]
  15. Purushothaman B.; Arumugam P.; Song J. M. A Novel Catecholopyrimidine Based Small Molecule PDE4B Inhibitor Suppresses Inflammatory Cytokines in Atopic Mice. Front. Pharmacol. 2018, 9, 485. 10.3389/fphar.2018.00485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee J.; Samson A. A. S.; Song J. M. Inkjet printing-based β-secretase fluorescence resonance energy transfer (FRET) assay for screening of potential β-secretase inhibitors of Alzheimer’s disease. Anal. Chim. Acta 2018, 1022, 89–95. 10.1016/j.aca.2018.03.033. [DOI] [PubMed] [Google Scholar]
  17. Rodríguez-Dévora J. I.; Zhang B. M.; Reyna D.; Shi Z. D.; Xu T. High throughput miniature drug-screening platform using bioprinting technology. Biofabrication 2012, 4, 035001. 10.1088/1758-5082/4/3/035001. [DOI] [PubMed] [Google Scholar]
  18. Setti L.; Fraleonimorgera A.; Mencarelli I.; Filippini A.; Ballarin B.; Dibiase M. An HRP-based amperometric biosensor fabricated by thermal inkjet printing. Sens. Actuators, B 2007, 126, 252–257. 10.1016/j.snb.2006.12.015. [DOI] [Google Scholar]
  19. Jeon S.; Lee J. P.; Kim J.-M. In situ synthesis of stimulus-responsive luminescent organic materials using a reactive inkjet printing approach. J. Mater. Chem. C 2015, 3, 2732–2736. 10.1039/c5tc00334b. [DOI] [Google Scholar]
  20. Steirer K. X.; Berry J. J.; Reese M. O.; van Hest M. F. A. M.; Miedaner A.; Liberatore M. W.; Collins R. T.; Ginley D. S. Ultrasonically sprayed and inkjet printed thin film electrodes for organic solar cells. Thin Solid Films 2009, 517, 2781–2786. 10.1016/j.tsf.2008.10.124. [DOI] [Google Scholar]
  21. Tian J.; Ma K.; Saaem I. Advancing high-throughput gene synthesis technology. Mol. BioSyst. 2009, 5, 714–722. 10.1039/b822268c. [DOI] [PubMed] [Google Scholar]
  22. Li B.; Santhanam S.; Schultz L.; Jeffries-EL M.; Iovu M. C.; Sauvé G.; Cooper J.; Zhang R.; Revelli J. C.; Kusne A. G.; Snyder J. L.; Kowalewski T.; Weiss L. E.; McCullough R. D.; Fedder G. K.; Lambeth D. N. Inkjet printed chemical sensor array based on polythiophene conductive polymers. Sens. Actuators, B 2007, 123, 651–660. 10.1016/j.snb.2006.09.064. [DOI] [Google Scholar]
  23. O’Toole M.; Shepherd R.; Wallace G. G.; Diamond D. Inkjet printed LED based pH chemical sensor for gas sensing. Anal. Chim. Acta 2009, 652, 308–314. 10.1016/j.aca.2009.07.019. [DOI] [PubMed] [Google Scholar]
  24. Elsharkawy M.; Schutzius T. M.; Megaridis C. M. Inkjet patterned superhydrophobic paper for open-air surface microfluidic devices. Lab Chip 2014, 14, 1168–1175. 10.1039/c3lc51248g. [DOI] [PubMed] [Google Scholar]
  25. Li J.; Rossignol F.; Macdonald J. Inkjet printing for biosensor fabrication: combining chemistry and technology for advanced manufacturing. Lab Chip 2015, 15, 2538–2558. 10.1039/c5lc00235d. [DOI] [PubMed] [Google Scholar]
  26. Lee J.; Samson A. A. S.; Yim Y.; Kim S. Y.; Jeon N. L.; Min D. H.; Song J. M. A FRET assay for the quantitation of inhibitors of exonuclease EcoRV by using parchment paper inkjet-printed with graphene oxide and FAM-labelled DNA. Microchim. Acta 2019, 186, 211. 10.1007/s00604-019-3317-9. [DOI] [PubMed] [Google Scholar]
  27. Zhang Y.; Ren T.; He J. Inkjet Printing Enabled Controllable Paper Superhydrophobization and Its Applications. ACS Appl. Mater. Interfaces 2018, 10, 11343–11349. 10.1021/acsami.8b01133. [DOI] [PubMed] [Google Scholar]
  28. Goldmann T.; Gonzalez J. S. DNA-printing: utilization of a standard inkjet printer for the transfer of nucleic acids to solid supports. J. Biochem. Biophys. Methods 2000, 42, 105–110. 10.1016/s0165-022x(99)00049-4. [DOI] [PubMed] [Google Scholar]
  29. Rozkiewicz D. I.; Brugman W.; Kerkhoven R. M.; Ravoo B. J.; Reinhoudt D. N. Dendrimer-mediated transfer printing of DNA and RNA microarrays. J. Am. Chem. Soc. 2007, 129, 11593–11599. 10.1021/ja073574d. [DOI] [PubMed] [Google Scholar]
  30. James C. D.; Davis R. C.; Kam L.; Craighead H. G.; Isaacson M.; Turner J. N.; Shain W. Patterned protein layers on solid substrates by thin stamp microcontact printing. Langmuir 1998, 14, 741–744. 10.1021/la9710482. [DOI] [Google Scholar]
  31. Apilux A.; Ukita Y.; Chikae M.; Chailapakul O.; Takamura Y. Development of automated paper-based devices for sequential multistep sandwich enzyme-linked immunosorbent assays using inkjet printing. Lab Chip 2013, 13, 126–135. 10.1039/c2lc40690j. [DOI] [PubMed] [Google Scholar]
  32. Zhang Y.; Lyu F.; Ge J.; Liu Z. Ink-jet printing an optimal multi-enzyme system. Chem. Commun. 2014, 50, 12919–12922. 10.1039/c4cc06158f. [DOI] [PubMed] [Google Scholar]
  33. Samson A. A. S.; Lee J.; Song J. M. Inkjet printing-based photo-induced electron transfer reaction on parchment paper using riboflavin as a photosensitizer. Anal. Chim. Acta 2018, 1012, 49–59. 10.1016/j.aca.2018.02.004. [DOI] [PubMed] [Google Scholar]
  34. Parsa S.; Gupta M.; Loizeau F.; Cheung K. C. Effects of surfactant and gentle agitation on inkjet dispensing of living cells. Biofabrication 2010, 2, 025003. 10.1088/1758-5082/2/2/025003. [DOI] [PubMed] [Google Scholar]
  35. Sun Y.; Song W.; Sun X.; Zhang S. Inkjet-Printing Patterned Chip on Sticky Superhydrophobic Surface for High-Efficiency Single-Cell Array Trapping and Real-Time Observation of Cellular Apoptosis. ACS Appl. Mater. Interfaces 2018, 10, 31054–31060. 10.1021/acsami.8b10703. [DOI] [PubMed] [Google Scholar]
  36. Kwak H. J.; Song J. S.; Heo J. Y.; Yang S. D.; Nam J.-Y.; Cheon H. G. Roflumilast inhibits lipopolysaccharide-induced inflammatory mediators via suppression of nuclear factor-kappa B, p38 mitogen-activated protein kinase, and c-Jun NH2-terminal kinase activation. J. Pharmacol. Exp. Ther. 2005, 315, 1188–1195. 10.1124/jpet.105.092056. [DOI] [PubMed] [Google Scholar]
  37. Arrabito G.; Pignataro B. Inkjet Printing Methodologies for Drug Screening. Anal. Chem. 2010, 82, 3104–3107. 10.1021/ac100169w. [DOI] [PubMed] [Google Scholar]
  38. Choi M.; Hwang J.; Choi J.; Hong J. Multicomponent High-throughput Drug Screening via Inkjet Printing to Verify the Effect of Immunosuppressive Drugs on Immune T Lymphocytes. Sci. Rep. 2017, 7, 6318. 10.1038/s41598-017-06690-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lee J.; Samson A. A. S.; Song J. M. Inkjet-Printing Enzyme Inhibitory Assay Based on Determination of Ejection Volume. Anal. Chem. 2017, 89, 2009–2016. 10.1021/acs.analchem.6b04585. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

ao9b03169_si_001.pdf (141.4KB, pdf)

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