Name: kinase inhibitor screening in self-assembled human protein microarrays
Uploaded: 2019-10-23T14:00:00
Duration: 13 min 22 s
Description: Watch this Scientific Journal Video about Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays at JoVE.com

The authors would like to thank everyone at the LaBaer&#8217;s lab for their help and criticism during the development of the project. This project was supported by the NIH grant U01CA117374, U01AI077883 and Virginia G. Piper Foundation.

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+study+of+Pseudomonas+aeruginosa+outer+membrane+protein+immunogenicity+using+self-assembling+protein+microarrays.">Genome-wide study of Pseudomonas aeruginosa outer membrane protein immunogenicity using self-assembling protein microarrays.</a> Infection and Immunity. 77 (11), 4877-4886 (2009).</li><li>Tang, Y., Qiu, J., Machner, M., LaBaer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+Protein-Protein+Interactions+Using+Nucleic+Acid+Programmable+Protein+Arrays.">Discovering Protein-Protein Interactions Using Nucleic Acid Programmable Protein Arrays.</a> Current Protocols in Cell Biology. 74, 11-15 (2017).</li><li>Yu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-catalyzed+azide-alkyne+cycloaddition+(click+chemistry)-based+detection+of+global+pathogen-host+AMPylation+on+self-assembled+human+protein+microarrays.">Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based detection of global pathogen-host AMPylation on self-assembled human protein microarrays.</a> Molecular and Cellular Proteomics. 13 (11), 3164-3176 (2014).</li><li>Anderson, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibody+signature+for+the+serologic+detection+of+ovarian+cancer.">Autoantibody signature for the serologic detection of ovarian cancer.</a> Journal of Proteome Research. 14 (1), 578-586 (2015).</li><li>Woyach, J. A., Johnson, A. J., Byrd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+B-cell+receptor+signaling+pathway+as+a+therapeutic+target+in+CLL.">The B-cell receptor signaling pathway as a therapeutic target in CLL.</a> Blood. 120 (6), 1175-1184 (2012).</li><li>Smith, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ibrutinib+in+B+lymphoid+malignancies.">Ibrutinib in B lymphoid malignancies.</a> Expert Opinion on Pharmacotherapy. 16 (12), 1879-1887 (2015).</li><li>Festa, F., Steel, J., Bian, X., Labaer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+cloning+and+expression+library+creation+for+functional+proteomics.">High-throughput cloning and expression library creation for functional proteomics.</a> Proteomics. 13 (9), 1381-1399 (2013).</li><li>Seiler, C. 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The authors declare no conflicts of interest.

Modifications and troubleshooting 
During the optimization phase of the study of kinase activity on NAPPA arrays, one of the main sources of background and low dynamic range observed was the BSA used on the printing mix. BSA was providing the primary amines necessary for crosslinking with the aminosilane surface and was trapping the DNA and the capture antibody on the spot. However, BSA is highly phosphorylated, making it difficult for the detection of the autophosphorylation signal on the array above the background noise. To solve this problem, several alternatives for BSA in the printing mix were tested, and poly-lysine was identified as good substitute. Poly-lysine lacks any phosphorylation site; therefore, the background from non-expressed arrays is very minimal. Moreover, microarrays printed with poly-lysine are reproducible and display good levels of proteins (Figure 2).
The next critical modification performed on the standard NAPPA assay was the addition of a Phosphatase/DNase treatment step. The treatment of the microarrays with phosphatase allows the removal of any phosphorylation that occurred in the IVTT mix during protein synthesis and capture (Figure 3). The source of this phosphorylation could be from intrinsic autophosphorylation activity or from the activity of kinases present in the IVTT mix. The removal of all phosphorylation post-expression allowed easy identification of the kinases that are active and can undergo autophosphorylation (Figure 3).
Critical steps within the protocol 
NAPPA is a robust technology, but as expected, there are several critical steps. The first is the acquisition of high quality DNA in the appropriate concentration. Using DNA of poor quality or in low concentrations will generate poor quality microarrays with several features not being expressed and displayed in the appropriate levels, decreasing the number of proteins analyzed on the array. The second critical step is the expression of proteins on the microarray. The use of an IVTT system that will express high levels of functional protein is vital for studying kinase activity on the array.
The next critical step on the TKI screening is how the microarrays are handled. The microarrays should not dry during any step of the protocol, and gentle handling is recommended to prevent scratches that can increase the background signal. Since the arrays from the entire experiment will be compared against each other, it is important to assure that every incubation step is even across all slides. For example, the time required to perform one step in a single array should be taken into consideration when a batch of 20 arrays is processed to prevent differences in the length of incubation across arrays.
Finally, design of the experiment and the inclusion of both positive and negative controls are critical for quality control and data analysis. The first set of controls are those printed in each array and includes negative controls [i.e., empty spots (without any material printed), water or empty vector (express only the tag)], as well as a positive control (i.e., purified IgG, that is detected by the secondary antibody and is inert to alteration in the phosphorylation levels). Collectively, they measure the background levels of the microarray, possible carryover during printing and the signal intensity of the detection method.
The next set of controls are the drug screening controls and include the dephosphorylated and autophosphorylated microarrays (in the presence or absence of DMSO). As mentioned earlier, the dephosphorylated microarray measures the level of phosphorylation after phosphatase treatment and therefore the baseline level for all other experiments. The lower the baseline level is, the higher the dynamic range of the assays. The autophosphorylated arrays present the maximum phosphorylation levels of all arrays and the signal should be strong and clear. It is used for data analysis, but also as a control that all reactions were performed successfully on the array.
Limitations of the technique 
As of now, one of the limitations of the drug screening presented here is its ability to screen only protein kinases that can be autophosphorylated. One possible way to overcome this is to print a kinase and known substrate in the same spot. Co-printing of DNA for two distinct proteins was successfully accomplished15, suggesting the feasibility of this approach. Moreover, the protein displayed on the array might not be folded correctly resulting in an inactive protein. The use of human-based expression system made a significant improvement in the kinase activity measured on the array; however, some proteins still cannot be analyzed on the array due to its inactivity.
A second limitation is the measurement of the phosphorylation using a pan anti-phospho-tyr antibody. Despite its non-specificity regarding the motif of the phosphorylation site, all measured phosphorylations occurred on tyrosine residues, leaving behind serines and threonines and their respective kinases. To date, more than 10 pan phospho-Ser/Thr antibodies have been tested without success, despite several attempts to optimize incubation and washing conditions. A new detection system that is independent of antibodies may be the best option to expand the number of protein kinases that can be screened for drug inhibition. In this context, a few options are available including radioactivity or chemical approaches like click conjugation. A series of optimizations are required to minimize the background signal and provide a good dynamic range for the assays.
The third limitation is the acquisition of cDNA clones to be printed on the array. The cDNA clones can be generated using any cloning technique including site-specific recombination systems, such as Creator or Gateway23. Another option is to purchase the clones from the DNAsu library, found at &#60;https://dnasu.org/DNASU/Home.do&#62;, where more than 17,000 cDNAs clones, including the entire human kinome, is readily available to be used for the construction of NAPPA arrays24.
The fourth limitation is that not every laboratory is equipped with appropriate equipment to fabricate and screen their own NAPPA arrays. This protocol provides alternative methods to generate the DNA to be printed on the microarray, without the need of high-throughput equipment, and protocols to manually perform all the hybridization steps. However, access to an arrayer and microarray scanner is still necessary. One option to overcome this issue is to use the NAPPA core service and facility, found at &#60;http://nappaproteinarray.org/&#62;, which distributes customized NAPPA microarrays at a non-profit academic price. Finally, as of any screening methodology, the data obtained on the arrays are susceptible to be artefacts (either positive or negative) and therefore should be validated using orthogonal assays.
Significance with respect to existing methods 
Several platforms are available commercially for the screening of protein kinases. One approach routinely used is binding assays, which can be performed with protein fragments, kinase domain, larger protein fragments with the kinase domain and some regulatory regions, and even full-length proteins. The proteins are usually expressed in bacterial systems due to the cost and simplicity in the expression and purification protocols. The interaction between the drug of interest and the protein is then measured with some type of report assay like fluorescence or presence of tags, for example. The main limitation of this set of approaches is the fact that the protein is not necessarily active during the interaction with the drug, which may result in the identification of false positive and false negative interactions. Protein fragments are particularly vulnerable to changes in the conformation and lack of activity and all the data obtained should be validated using active proteins, preferably in their full-length form. Another limitation of some of the platforms is the capability to screen only ATP analogs, limiting its overall use.
Most of the commercially available services for the screening of TKIs using enzymatic based approaches utilize only wild type versions of the kinase of interest, and sometimes only a selected few mutants. Knowing that drug resistance is very common in patients treated with TKI, it is important to be able to measure drug response in different mutants, for the selection of the most appropriate inhibitor. Due to NAPPA&#8217;s nature, the screening of kinase mutants is simple and can be easily accomplished, and the only required tool is the incorporation of the kinase mutant into the NAPPA cDNA collection, which can be done by site-specific mutagenesis, for instance.
Future applications 
One of the most common forms of treatment elapse in cancer therapy using kinase inhibitors is the acquisition of mutations in the drug target during a treatment course. The screening of these mutants regarding their response to kinase inhibitors is of vital importance for the selection of second/third generation of TKIs to achieve a personalized treatment for each patient. The drug screening approach presented here, provides an unbiased screening platform in which any tyrosine kinase inhibitor can be tested against a panel of tyrosine kinases present in the human genome. Since the proteins displayed on NAPPA arrays are expressed in vitro from the cDNA printed on the slide, any mutant variant can easily be incorporated in the cDNA collection to be displayed on the array. The facility in which the kinase mutants can be generated and expressed on the array, combined with the high-throughput power of the NAPPA technique, provides a unique environment for the study of kinase mutants and their response to drugs, making NAPPA suitable for personalized drug screening, one of the goals of precision medicine.

Protein kinases are responsible for the phosphorylation of their targets and can modulate complex molecular pathways that control many cellular functions (i.e., cell proliferation, differentiation, cell death and survival). Deregulation of kinase activity is associated with more than 400 diseases, making kinase inhibitors one of the main classes of drugs available for treatment of several diseases, including cancer, cardiovascular and neurological disorders as well as inflammatory and autoimmune diseases1,2,3.
With the advent of precision medicine, the identification of new therapies, especially kinase inhibitors, have great appeal pharmaceutically and clinically. Several approaches can be used for the identification of possible new pairs of kinase/kinase inhibitors, including the de novo design of kinase inhibitors and identification of new targets for existing FDA-approved drugs. The latter is especially attractive, since the time and money required to implement these drugs in clinics are drastically reduced due to the availability of previous clinical trial data. A canonical example of the repurposing of a kinase inhibitor is imatinib, initially designed for the treatment of chronic myelogenous leukemia (CML) through the inhibition of BCR-Abl, which can also be successfully used for the treatment of c-Kit over-expressing gastrointestinal stromal tumors (GISTs)4,5,6,7.
The screening of kinase inhibitors can be performed in binding assays or enzymatic-based assays. The first class of assays focuses on protein-drug interactions and can provide information such as ligation site and affinity. Since the activity of the kinase at the time of these assays is unknown, a number of interactions may be missed or falsely identified due to conformational changes in the protein. On the other hand, enzymatic-based assays require the protein kinases to be active and provide valuable information regarding the inhibitor&#8217;s effect on enzyme activity, however, this type of screening is generally more time consuming and expensive. Currently, both types of assays are commercially available from several sources. They represent a reliable option for the screening of kinase inhibitors with a few limitations, including: I) most of the methods involve testing of multiple kinases individually, which can make the screening of a large set of proteins costly; II) the set of kinases to be tested is limited to a list of preselected, wild-type kinases and several well-known mutated versions of some kinases, hindering the testing of many new mutated isoforms.
In this context, protein microarrays are a powerful platform capable of overcoming some of the limitations presented by commercially available techniques. It is suitable to perform enzymatic-based assays in high-throughput screening using full-length, active proteins of any sequence of interest. The microarrays can be generated by a self-assembled approach like NAPPA (nucleic acid programmable protein array), in which proteins are expressed just in time for the assays, increasing the likelihood that those displayed on the array are indeed active. The proteins displayed on NAPPA are produced using human-derived ribosomes and chaperone proteins in order to improve the likelihood of natural folding and activity.
The proteins are initially programmed by printing cDNAs coding for genes of interest fused with a capture tag, together with a capture agent, onto the microarray surface. The proteins are then produced on the microarrays using an in vitro transcription and translation (IVTT) system, and the freshly expressed proteins are immobilized on the microarray surface by the capture agent. Expressed NAPPA arrays can be used for the study of the proteins displayed on the array in an unbiased, high-throughput manner8,9.
Previously, it was shown that proteins displayed on NAPPA arrays are folded properly to interact with known partners10; furthermore, their enzymatic activity was first exploited in 2018, when it was shown that protein kinases displayed on the microarray autophosphorylate11. To date, NAPPA methodology has been used for many distinct applications, including biomarker discovery12,13,14,15,16,17, protein-protein interactions10,18, substrate identification19, and drug screening11. Its flexibility is one of the platform&#8217;s key characteristics that allows for adaptation to each application.
Here, a protocol for the screening of tyrosine kinase inhibitors in self-assembled NAPPA arrays is presented. The platform is optimized for the display of active human protein kinases and for the analysis of protein kinase activity, with low background and high dynamic range. Among the modifications implemented to use NAPPA for the screening of kinase inhibitors include: I) changes in the printing chemistry, II) de-phosphorylation of the protein microarray prior to the kinase inhibitor screening, and III) optimization of detection of phosphorylated proteins on the array. This protocol is the first of its kind and provides unique information about the kinase study in NAPPA microarrays.

<table>
	<tbody>
		<tr>
			<td>Reagent/Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>364 well plates (for arraying)</td>
			<td>Genetix</td>
			<td>x7020</td>
			<td></td>
		</tr>
		<tr>
			<td>800 &#181;L 96-well collection plate</td>
			<td>Abgene</td>
			<td>AB-0859</td>
			<td></td>
		</tr>
		<tr>
			<td>96-pin device</td>
			<td>Boekel</td>
			<td>140500</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Millipore-Sigma</td>
			<td>1.00066</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone 99.9%</td>
			<td>Millipore Sigma</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum seal for 96 well plates</td>
			<td>VWR</td>
			<td>76004-236</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminosilane (3-aminopropyltriethoxysilane)</td>
			<td>Pierce</td>
			<td>80370</td>
			<td></td>
		</tr>
		<tr>
			<td>ANTI-FLAG M2 antibody produced in mouse</td>
			<td>Millipore Sigma</td>
			<td>F3165</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Flag rabbit Antibody (polyclonal)</td>
			<td>Millipore Sigma</td>
			<td>F7425</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP 10 mM</td>
			<td>Cell Signaling</td>
			<td>9804S</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Glycerophosphate disodium salt hydrate</td>
			<td>Millipore-Sigma</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr>
			<td>bacteriological agar</td>
			<td>VWR</td>
			<td>97064-336</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td>ThermoFisher/Pierce</td>
			<td>37535</td>
			<td></td>
		</tr>
		<tr>
			<td>Brij 35</td>
			<td>ThermoFisher/Pierce</td>
			<td>BP345-500</td>
			<td></td>
		</tr>
		<tr>
			<td>BS3 (bis-sulfosuccinimidyl)</td>
			<td>ThermoFisher/Pierce</td>
			<td>21580</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumin)</td>
			<td>Millipore Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip 24 x 60 mm</td>
			<td>VWR</td>
			<td>48393-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-165-150</td>
			<td></td>
		</tr>
		<tr>
			<td>DeepWell Block, case of 50</td>
			<td>ThermoFisher/AbGene</td>
			<td>AB-0661</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC water</td>
			<td>Ambion</td>
			<td>9906</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO (Dimethyl Sulfoxide)</td>
			<td>Millipore-Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA-intercalating dye</td>
			<td>Invitrogen</td>
			<td>P11495</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Millipore-Sigma</td>
			<td>AMPD1-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Millipore-Sigma</td>
			<td>43816</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Millipore-Sigma</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Millipore-Sigma</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter plates</td>
			<td>Millipore-Sigma</td>
			<td>WHA77002804</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Permeable Seals, box of 50</td>
			<td>ThermoFisher/AbGene</td>
			<td>AB-0718</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass box</td>
			<td>Wheaton</td>
			<td>900201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>VWR</td>
			<td>48300-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Millipore-Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl (Hydrochloric acid)</td>
			<td>Millipore-Sigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution</td>
			<td>Millipore-Sigma</td>
			<td>83264</td>
			<td></td>
		</tr>
		<tr>
			<td>Human-based IVTT system</td>
			<td>Thermo Scientific</td>
			<td>88882</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmunoPure Mouse IgG whole molecule</td>
			<td>ThermoFisher/Pierce</td>
			<td>31202</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Millipore-Sigma</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl (Potassium chloride)</td>
			<td>Millipore-Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4(Potassium phosphate monobasic)</td>
			<td>Millipore-Sigma</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Kinase buffer</td>
			<td>Cell Signaling</td>
			<td>9802</td>
			<td></td>
		</tr>
		<tr>
			<td>KOAc (Potassium acetate)</td>
			<td>Millipore-Sigma</td>
			<td>P1190</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda Protein Phosphatase</td>
			<td>new england biolabs</td>
			<td>P0753</td>
			<td></td>
		</tr>
		<tr>
			<td>Lifterslips, 24 x 60 mm</td>
			<td>ThermoFisher Scientific</td>
			<td>25X60I24789001LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal 30-slide rack with no handles</td>
			<td>Wheaton</td>
			<td>900234</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCL2 (Magnesium chloride)</td>
			<td>Millipore-Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3VO4 (Sodium orthovanadate)</td>
			<td>Millipore-Sigma</td>
			<td>S6508</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (Sodium Chloride)</td>
			<td>Millipore-Sigma</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOAc (Sodium acetate)</td>
			<td>Millipore-Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH (Sodium hydroxide)</td>
			<td>Millipore-Sigma</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Midi / Maxi</td>
			<td>Macherey-Nagel</td>
			<td>740410.10 / 740414.10</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleoprep Anion II</td>
			<td>Macherey Nagel</td>
			<td>740503.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric Acid</td>
			<td>Millipore-Sigma</td>
			<td>79617</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine Solution (0.01%)</td>
			<td>Millipore-Sigma</td>
			<td>A-005-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Phosphatase (Lambda)</td>
			<td>New England Biolabs</td>
			<td>P0753</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse</td>
			<td>Invitrogen</td>
			<td>12091021</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS (Sodium dodecyl sulfate)</td>
			<td>Millipore-Sigma</td>
			<td>L6026</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS (Sodium dodecyl sulfate)</td>
			<td>Millipore-Sigma</td>
			<td>05030</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealing gasket</td>
			<td>Grace Bio-Labs, Inc</td>
			<td>44904</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica packets</td>
			<td>VWR</td>
			<td>100489-246</td>
			<td></td>
		</tr>
		<tr>
			<td>Single well plate</td>
			<td>ThermoFisher/Nalge Nunc</td>
			<td>242811</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate (3M, pH 5.5)</td>
			<td>Millipore-Sigma</td>
			<td>71196</td>
			<td></td>
		</tr>
		<tr>
			<td>TB media (Terrific Broth)</td>
			<td>Millipore-Sigma</td>
			<td>T0918</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>IBI scientific</td>
			<td>IB70144</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Millipore-Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Millipore-Sigma</td>
			<td>T7293</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Millipore-Sigma</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Millipore-Sigma</td>
			<td>Y1625</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td>Maker/model</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Programmable chilling incubator</td>
			<td>Torrey Pines IN30 Incubator with Cooling</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker for bacterial growth</td>
			<td>ATR Multitron shaker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum manifold with liquid waste trap</td>
			<td>MultiScreenVacuum Manifold 96 well</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well autopippetor/liquid handler</td>
			<td>Genmate or Biomek FX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid dispenser</td>
			<td>Wellmate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA microarrayer</td>
			<td>Genetix QArray2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic hybridization station</td>
			<td>Tecan HS4800 Pro Hybridization Station</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microarray scanner</td>
			<td>Tecan PowerScanner</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microarray data quantification</td>
			<td>Tecan Array-ProAnalyzer 6.3</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

kinase inhibitor screening in self-assembled human protein microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

Self-assembled NAPPA microarrays provide a solid platform that can been used for many distinct applications, including biomarker discovery, protein-protein interactions, substrate identification, and drug screening10,11,12,13,14,15,16,17,18,19,20.
The overall methodology adopted for the study of kinase activity and screening of tyrosine kinases inhibitors on NAPPA microarrays is schematically represented in Figure 1. First, NAPPA microarrays are generated by the immobilization of cDNA and capture agent onto the coated microarrays. The cDNAs are then used as a template for the transcription and translation of proteins, using a human-based IVTT system, and the newly synthesized proteins are immobilized by the capture agent9. The quality of the printed microarray can be monitored by measuring levels of DNA (confirming consistent printing) or protein displayed on the array (confirming protein expression and capture; Figure 1). To decrease the background signal and increase the dynamic range of the experiment, the microarrays are treated with 1) lambda phosphatase to remove phosphorylation from Ser/Thr/Tyr residues, then with 2) DNase to simplify the chemistry on the spot and decrease background (Figure 1).
The next step is the autophosphorylation reaction, in which microarrays are incubated with kinase buffer in the absence of ATP (negative control array, referred to as dephosphorylated microarrays), and kinase buffer is supplemented with ATP (positive control, referred to as autophosphorylated arrays) or ATP + DMSO (vehicle control). It should be emphasized that during this step, no kinase is added; therefore, the intrinsic activity of each kinase displayed on the microarray is quantified through measurement of its phosphorylation levels using a pan anti-phospho-tyrosine antibody followed by a cy3-labbeled secondary antibody (Figure 1).
Quality control of the NAPPA-kinase arrays displaying a panel of human protein kinases printed in quadruplicate is shown in Figure 2. The levels of immobilized DNA were measured by DNA staining and it showed an even signal across the microarray, suggesting the amount of DNA printed on the array was uniform. It is also possible to observe several features without any DNA staining. These features correspond to some controls in which DNA was omitted from the printing mix [i.e., empty spots (nothing was printed), water spots, purified IgG spot (poly-lysine, crosslinker and purified IgG), printing mix only (complete printing mix: poly-lysine plus crosslinker and anti-flag antibody, without any DNA)]. The levels of protein displayed on the NAPPA-kinase microarrays were assessed after the IVTT reaction using anti-tag antibody.
For the kinase screening, Flag was used as the tag of choice and the level of protein displayed on the microarray was measured using an anti-flag antibody. As shown, the majority of spots containing cDNA successfully displayed detectable levels of protein. Some of the control spots without cDNA also revealed signal with the anti-flag antibody: IgG spot (used to detect the activity of the secondary antibody) and empty vector spots (cDNA codes for the tag only) (Figure 2). NAPPA-kinase microarrays showed good reproducibility among slides, with the correlation of the levels of protein display among distinct printing batches higher than 0.88 (Figure 2). Within the same batch the correlation was even higher, close to 0.92 (data not shown).
Next, the kinase autophosphorylation activity of the proteins displayed on the array was measured using anti-phospho-tyrosine antibody (Figure 3). Protein displayed on the array showed high levels of protein phosphorylation after expression (Figure 3, left), which may be caused by intrinsic kinase activity of the protein displayed on the array or by active kinases present in the IVTT mix. This phosphorylation was completely removed with lambda phosphatase treatment and these microarrays were used for the kinase assays. After dephosphorylation, autophosphorylation reactions performed without ATP showed no significant levels of phosphorylation, as expected, while microarrays incubated with kinase buffer in the presence of ATP showed protein phosphorylation as fast as 15 min (Figure 3). For the drug screening, the kinase activity was measured after 60 min of autophosphorylation reaction to maximize the number of kinases tested.
The comparison between microarrays in which the phosphorylation levels were measured right after protein expression (Figure 3, left) and after 60 min of autophosphorylation reaction (Figure 3, right) showed: i) proteins phosphorylated only after expression, suggesting they can be exogenously phosphorylated by proteins present on the IVTT mix, but cannot be autophosphorylated; ii) protein phosphorylated only after the autophosphorylation reaction, suggesting these proteins were not active after protein expression and required co-factors present in the kinase buffer to be active; or iii) protein phosphorylated on both arrays, suggesting they were active in both settings (Figure 3).
As an example of the results obtained for the screening of tyrosine kinase inhibitors on NAPPA-kinase arrays three kinases inhibitors with distinct selectivity across protein kinases were used: staurosporine, imatinib and ibrutinib. For all screenings, dephosphorylated NAPPA microarrays were incubated with increasing concentrations of TKI (ranging from 100 nM to 10 uM) during the autophosphorylation reaction. The first TKI tested was staurosporine, a global protein kinase inhibitor, that showed potent kinase inhibition on the microarray across virtually all kinases tested11.
Next, imatinib was tested, an ABL and c-Kit inhibitor used for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors4,5,6,7. On NAPPA-kinase arrays imatinib showed a significant reduction in Abl1 and BCR-Abl1 activity whereas other kinases remained mostly unaffected (Figure 4A). The data quantification for the kinase activity was normalized against the dephosphorylated array and represented as a percentage of the positive control microarray (vehicle only). Data for TNK2 (non-relevant kinase), Abl1 and BCR-Abl1 is shown in Figure 4B. As expected, imatinib showed selective inhibition towards Abl1 and BCR-ABl1. The data for c-Kit was inconclusive due to lack of activity on the positive control arrays.
Finally, ibrutinib, an FDA-approved covalent inhibitor of Bruton&#8217;s tyrosine kinase (BTK), was tested. Ibrutinib is currently used in the treatment of several blood-related cancers with overactive BTK, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma, and Waldenstr&#246;m&#8217;s macroglobulinemia21,22. Figure 4C, is representative of typical results obtained for the ibrutinib screening. The kinase activity of ABL1 (non-relevant kinase) and BTK (canonical target) and ERBB4 (potential new target) is shown in Figure 4D. The data suggests ERBB4 can be inhibited by ibrutinib in a dose specific fashion. This inhibition was confirmed in vitro and in cell-based assays11, demonstrating the power of this platform.
Taken together, the data suggest that NAPPA-kinase microarray platform could be used for the unbiased screening of TK inhibitors. Moreover, the screening is fast and can be easily customized to include any variation of the protein kinases of interest.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59886/59886fig1.jpg" /> 
Figure 1: Schematic representation of quality control and screening of tyrosine kinase inhibitors in NAPPA arrays. NAPPA arrays are printed with cDNA coding for the protein of interest fused with a tag and a capture antibody. During the in vitro transcription and translation reaction (IVTT) the synthesized proteins are captured on the microarray surface through the tag by the capture antibody. The quality control (QC) of the arrays is performed by the measurement of the levels of DNA printed on the slide, using a fluorescent DNA-intercalating dye, and the levels of protein displayed on the array using tag-specific antibodies. For the kinase screening, the microarrays are treated with DNase and phosphatase, after the IVTT reaction, to remove the printed DNA and all phosphorylation that may have occurred during protein synthesis. The dephosphorylated arrays are now ready to be used for the drug screen. For each assay, three sets of controls are routinely used: (I) dephosphorylated arrays, in which the autophosphorylation reaction is performed without ATP; (II) autophosphorylated microarrays, in which the autophosphorylation reaction is performed in the presence of ATP; and (III) DMSO treated array (vehicle), in which the autophosphorylation reaction is performed with ATP and DMSO. The slides treated with different concentration of kinase inhibitors follow the exact same protocol used for the DMSO treated arrays. <a href="https://www.jove.com/files/ftp_upload/59886/59886fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59886/59886fig2.jpg" /> 
Figure 2: Representative results of quality control for self-assembled NAPPA-kinase arrays. DNA content measured by a fluorescent DNA-intercalating dye (left) and levels of protein displayed on the microarray measured by anti-Flag antibody (middle) are shown. On the right side is a correlation plot of the levels of protein displayed on two NAPPA-kinase arrays printed in separate batches. <a href="https://www.jove.com/files/ftp_upload/59886/59886fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59886/59886fig3.jpg" /> 
Figure 3: Representative results of kinase activity in NAPPA-kinase arrays. Microarrays displaying protein kinases in quadruplicate were used to study protein kinase activity on the array through the measurement of protein phosphorylation using anti-pTyr antibody, followed by cy3-labeled anti-mouse antibody. Control arrays without phosphatase/DNase treatment and without ATP during the autophosphorylation reaction were used to measure the background phosphorylation after protein expression (post-expression). The remaining microarrays were treated with phosphatase/DNA, and the autophosphorylation reaction was performed without ATP (dephosphorylated microarray, negative control) or with ATP (autophosphorylated microarrays). For the autophosphorylated microarrays the autophosphorylation reaction was performed for 15 min, 30 min, 45 min, or 60 min, as shown. <a href="https://www.jove.com/files/ftp_upload/59886/59886fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59886/59886fig4.jpg" /> 
Figure 4: Representative data from tyrosine kinase screen on NAPPA-kinase arrays. (A) Phosphatase/DNase treated NAPPA-kinase arrays were incubated in increasing concentrations of imatinib during the autophosphorylation reaction and the kinase activity was measure with anti-phospho-tyr antibody. (B) Quantification of kinase activity observed on NAPPA-kinase arrays exposed to imatinib. Data was normalized against the signal of the negative control arrays (dephosphorylated) and it is shown as a percentage of the positive control arrays (autophosporylation reaction performed in the presence of DMSO). Similar data is shown for the screening of ibrutinib (C,D). This figure has been modified from Rauf et al.11. <a href="https://www.jove.com/files/ftp_upload/59886/59886fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1. Alternative protocol for screening of tyrosine kinase inhibitors in NAPPA arrays using an automated hybridization station. <a href="https://www.jove.com/files/ftp_upload/59886/Supplementary_File_1_Festa_and_Labaer.docx">Please click here to download this file.</a>

Watch this Scientific Journal Video about Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays at JoVE.com

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

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