This work was supported by National Institutes of Health Grant 1R21CA195422 (to L. T.), Epstein Family Foundation Award (to N. D. P. C.), and NCI Cancer Center Support Grant P30CA030199. Additionally, this project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Chemical Biology Consortium Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Preparation of cell culture and reagents
<ol>
	<li>Formulate a 500 mL bottle of growth media with 10% fetal bovine serum, 1x antibiotic/antimicotic, 20 mM HEPES, and 1 mM sodium pyruvate. Store at 4 &#176;C.</li>
	<li>Thaw cellular thermal shift reagents (EA reagent, lysis buffer, and substrate) from frozen original stock bottles.</li>
	<li>Dispense reagents and buffer as 2 mL aliquots and store at -20 &#176;C. 
	NOTE: Avoid freeze/thaw for reproducibility and use only that volume of reagent required for the assay procedure.</li>
</ol>
2. Growth and maintenance of HEK293T cells
<ol>
	<li>Obtain low passage adherent HEK293T cells from cryo storage.</li>
	<li>Maintain HEK293T cells in growth media at 37 &#176;C, 5% CO2.</li>
	<li>Split cells every 4 days in a 1:14 ratio. 
	NOTE: For best performance, HEK293T cells are not allowed to passage greater than 25 times.</li>
</ol>
3. HEK293T cell preparation for transient transfection
<ol>
	<li>Detach HEK293T cells from 16 mL plates using 3 mL of the cell detachment reagent.</li>
	<li>Dilute with 12 mL of growth media. Collect cells by centrifugation at 1,400 x g for 4 min.</li>
	<li>Resuspend the pellet in 10 mL growth media. Measure the concentration and viability of cells using trypan blue and a cell counter.</li>
	<li>Plate 7.0 x 105 exponentially growing HEK293T cells per well in a 6 well cell culture plate approximately 24 h before transfection. 
	NOTE: Reserve one well for each plasmid to be transfected.</li>
	<li>Incubate for 24 h at 37 &#176;C, 5% CO2.</li>
</ol>
4. Transfection of HEK293T cells
<ol>
	<li>From a purified plasmid stock of pICP-ePL-N-SHP2-WT or pICP-ePL-N-SHP2-E76K (~200 ng/&#181;L) dilute 2 &#181;g of plasmid DNA into 200 &#181;L of transfection buffer.</li>
	<li>Vortex for 10 s and centrifuge at 1,400 x g for 4 min.</li>
	<li>Add 4 &#181;L of transfection reagent to the diluted DNA. Vortex for 10 s and centrifuge at 1,400 x g for 4 min.</li>
	<li>Incubate at 23 &#176;C for 10 min.</li>
	<li>Remove the 6 well plate containing growing HEK293T cells from incubator. Add the transfection mix to the attached cells in the 6-well plate. Incubate for 24 h at 37 &#176;C, 5% CO2.</li>
</ol>
5. Preparation of assay plates
<ol>
	<li>Prepare 10 mM stock solutions in DMSO of compounds to be tested.</li>
	<li>Dispense inhibitor solutions into a 384-well low dead volume source plate for immediate use.</li>
	<li>Spot the desired volume of inhibitors or vehicle (DMSO) using a liquid handler into 384-well real-time PCR plates at a target final volume of less than 0.5% DMSO (v/v). Seal the plate using a plate sealer with inert gas purging. 
	NOTE: For inhibitor dose-response assays, make sure to backfill DMSO accordingly so that equal amounts of DMSO are used for each inhibitor concentration. For best results, store plates at 23 &#176;C and use plates within 24 h.</li>
</ol>
6. Transfected cell detachment and preparation
<ol>
	<li>Preincubate growth media and cell detachment reagent in a 37 &#176;C water bath. Remove transfected cells from the incubator. Gently aspirate media from the wells of the plate. 
	NOTE: Use a new aspirator for every well that contains a different transfected plasmid.</li>
	<li>Add 0.3 mL of the cell detachment reagent. Gently rock the plate back and forth to thoroughly cover the surface of the plate bottom. Incubate at 23 &#176;C for 2 min.</li>
	<li>Add 1 mL of growth media to each well. Gently pipette media and cells in the well and transfer to a 15 mL Falcon centrifuge tube. Collect cells by centrifugation at 1,400 x g for 4 min.</li>
	<li>Gently but thoroughly aspirate off the media. 
	NOTE: Residual phenol red in the cell detachment reagent can interfere with the assay.</li>
	<li>Carefully resuspend cell pellet in 2 mL of growth media. Measure the concentration and viability of cells using trypan blue and a cell counter. 
	NOTE: Cell viability should be &#62; 90% for best results.</li>
	<li>Dilute cells to a concentration of 125 cells/&#181;L. For example, for a 5 &#181;L assay this is ~625 cells/&#181;L. For optimal viability keep cells in suspension for no more than 2 h.</li>
</ol>
7. Incubation of cells with SHP2 inhibitors
<ol>
	<li>Dispense cells into a sterile single channel solution trough.</li>
	<li>Centrifuge 384-well real-time PCR plate that has been pre-prepared by SHP2 inhibitor deposition at 2,500 x g for 5 min at 23 &#176;C.</li>
	<li>Remove seal from the compound plate. Using a 125 &#181;L multichannel pipette, add 5 &#181;L of the diluted cells to desired wells.</li>
	<li>Centrifuge the plate at 42 x g for 30 s without a lid on the plate. Attach a lid seal to the plate after the plate is spun down and incubate the assay plate at 37 &#176;C, 5% CO2 for 1 h.</li>
</ol>
8. Preparation of chemiluminescent reagent master mix
<ol>
	<li>Remove the necessary quantity of detection reagents (EA reagent, lysis buffer, and substrate) from -20 &#176;C storage after plating cells. Thaw reagents at 23 &#176;C. 
	NOTE: For convenience in transfer, prepare a volume that is 1.5x the total volume of cells that have been deposited to the plate. Do not reuse reagents after use.</li>
	<li>Prepare a master mix of the reagents using condition EA-10, which consists of component and volume fraction as follows: EA reagent (0.17), lysis buffer (0.17), substrate (0.67). 
	NOTE: Reagent ratios are based on optimization experiments performed as specified in the supplier&#39;s manual.</li>
</ol>
9. Isothermal or thermal profile gradient heat pulse
<ol>
	<li>Program the gradient capable thermocycler for the delivery of heat pulse.
	<ol>
		<li>For thermal profile gradient experiments, preprogram the thermocycler with the following example specifications:</li>
		<li>Heat pulse: 3 min desired melting temperature (vertical or horizontal gradient +/- 15 &#176;C; example 38-68 &#176;C spread across 24 wells yields temperature increments of 1.25 &#176;C).</li>
	</ol>
	</li>
	<li>Equilibration recovery step: 3 min 20 &#176;C with ramp speed = 1 &#176;C/s
	<ol>
		<li>For isothermal experiments set up the protocol set up as follows:</li>
		<li>Heat pulse: 3 min 55 &#176;C. Equilibration recovery step: 3 min 20 &#176;C with ramp speed = 1&#176;C/s 
		NOTE: For increased reproducibility, as it can be difficult to place the plate precisely at the time the thermocycler reaches its desired temperature, set up the program to count down 15 s before beginning the 3 min pulse. For the thermocycler used in this experiment see Table of Materials, the lid is kept up during the heat pulse.</li>
	</ol>
	</li>
</ol>
10. Lysis detection and measurement
<ol>
	<li>Supplement assay plates with lysis detection master mix by addition of equal volumes (5 &#181;L) to each well to be analyzed using a multichannel pipette.</li>
	<li>Centrifuge the plate 42 x g for 30 s. Store at 23 &#176;C in darkness for 30-60 min.</li>
	<li>Measure chemiluminescence using a microplate reader capable of detecting luminescence in 384-well format. Perform luminescence measurement with the plate type and integration time optimized (integration time 1000 ms, settle time 0 s). Measure values as counts/s and output for further analysis.</li>
</ol>
11. Data analysis
<ol>
	<li>Analyze the luminescence data using a scientific 2D graphing and statistics software.</li>
	<li>Generate thermal profiles by analyzing the normalized luminescence data (normalized to the vehicle control) using nonlinear regression and a Boltzmann sigmoidal model. 
	NOTE: In thermal profile experiments, the calculated V50, the temperature that is the halfway point between bottom and top of the curve, defines the melting temperature of SHP2. In isothermal experiments, EC50 defines the concentration of the inhibitor that gives half-maximal response.</li>
</ol>

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<li>Tartaglia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutations+in+PTPN11%2C+encoding+the+protein+tyrosine+phosphatase+SHP-2%2C+cause+Noonan+syndrome%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome.</a> Nature Genetics. 29 (4), 465-468 (2001).</li>
<li>Yokosuka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programmed+cell+death+1+forms+negative+costimulatory+microclusters+that+directly+inhibit+T+cell+receptor+signaling+by+recruiting+phosphatase+SHP2%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2.</a> Journal of Experimental Medicine. 209 (6), 1201-1217 (2012).</li>
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<li>Watanabe, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BTLA+is+a+lymphocyte+inhibitory+receptor+with+similarities+to+CTLA-4+and+PD-1%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">BTLA is a lymphocyte inhibitory receptor with similarities to CTLA-4 and PD-1.</a> Nature Immunology. 4 (7), 670-679 (2003).</li>
<li>Bialy, L., Waldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibitors+of+protein+tyrosine+phosphatases%3A+next-generation+drugs%5BTitle%5D)+AND+%22Angewandte+Chemie+International+Edition+England%22%5BJournal%5D)">Inhibitors of protein tyrosine phosphatases: next-generation drugs.</a> Angewandte Chemie International Edition England. 44 (25), 3814-3839 (2005).</li>
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<li>Tautz, L., Pellecchia, M., Mustelin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+PTPome+in+human+disease%5BTitle%5D)+AND+%22Expert+Opinion+in+Thereapeutics+Targets%22%5BJournal%5D)">Targeting the PTPome in human disease.</a> Expert Opinion in Thereapeutics Targets. 10 (1), 157-177 (2006).</li>
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</ol>

The authors declare that they have no conflicts of interest with the contents of this article.

We have presented a target engagement assay that can confirm direct binding of small molecules to the SHP2 phosphatase in cells. The assay can discriminate between low and high affinity inhibitors and, importantly, confirm a lack of potency by the allosteric inhibitors of the SHP099-type for the GOF oncogenic SHP2-E76K mutant. A strength of this miniaturized assay is its ability to be integrated into a SHP2 inhibitor screening campaign. The ability of the assay to confirm intracellular binding to SHP2 by unknown chemical...

Tyrosine phosphorylation plays an important role in signal transduction in cells1,2. This post-translational modification is catalyzed by protein tyrosine kinases (PTKs) and reversed by protein tyrosine phosphatases (PTPs). Therefore, aberrant PTK or PTP function leads to many inherited or acquired human diseases3,4,5,6. The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2) is a widely expressed non-receptor type PTP encoded by the proto-oncogene PTPN117 and is a key regulator of numerous physiological processes that involve signal transduction by activation of the Ras/Raf/ERK, PI3K/Akt, or JAK/STAT signaling pathways8. Normally, SHP2 activity is tightly regulated in order to prevent aberrant signaling. Under basal conditions SHP2 is autoinhibited by its N-terminal SH2 domain, which blocks access to the active site within the catalytic phosphatase domain (Figure 1A)9,10. Upon cell activation, tyrosine phosphorylated binding proteins recruit SHP2, causing it to adopt its active conformation, in which the active site is now accessible to its substrates. In many cancers SHP2 activity is elevated. Somatic gain-of-function (GOF) mutations in PTPN11 have been identified mainly in leukemias and prevent binding of the N-SH2 domain to the phosphatase domain, resulting in constitutively active SHP2 (Figure 1B)11. Germline GOF mutations in PTPN11 are responsible for ~50% of cases of Noonan syndrome, a developmental disorder with an increased risk of malignancy12. In solid tumors, where PTPN11 mutations are rare, greater levels of phosphorylated binding proteins lead to enhanced SHP2 activity (Figure 1C). SHP2 is also important for immune checkpoint signaling, as checkpoint receptors such as BTLA or PD-1 recruit SHP2 to dephosphorylate key signaling molecules, preventing immune cell activation13,14,15.
Targeting PTPs with small molecules has been a challenge, because the active site of PTPs is highly conserved and highly charged; inhibitors that target the active site are often potent, but exhibit poor selectivity and oral bioavailability16,17,18,19,20,21,22. Indeed, many reported SHP2 inhibitors suffer from poor selectivity and lack of efficacy in cells23. Recently, allosteric inhibitors of SHP2 with good potency and excellent selectivity have been reported (e.g., SHP09924 and RMC-455025) and have sparked renewed interest in SHP2 inhibitors. Compounds based on SHP099 and RMC-4550 are currently in phase I clinical trials to treat solid tumors with receptor tyrosine kinase (RTK) pathway activation26,27. While groundbreaking, these compounds are ineffective against many of the SHP2 oncogenic mutants that drive leukemogenesis in a significant number of blood cancer patients28,29,30. This lack of potency of SHP099-like compounds toward the SHP2 oncogenic variants stems from their unique allosteric mechanism, as they inhibit SHP2 activity by binding and stabilizing the inactive, closed conformation, which is disrupted in SHP2 mutants. Further, based on a recent report31, adaptive resistance mechanisms in patients treated with SHP099-like inhibitors are quite conceivable. Consequently, the development of next generation SHP2 inhibitors that target its active, open state is a subject of intense research.
The characterization of novel SHP2 inhibitors in cells is an essential aspect of the lead optimization process. Critically, proven target engagement of the inhibitor under physiological conditions provides an additional level of confidence that resources for medicinal chemistry are efficiently deployed on compounds with promising cellular efficacy. In the past, several methods to assess the binding of small molecule inhibitors to their targets have been developed, primarily for protein kinases32. To develop a SHP2 cellular target engagement assay, we utilized a cellular thermal shift assay33. This assay, similar to the in vitro thermal shift (PTS) assay for proteins34, monitors the target protein thermal stability, which is typically altered by the binding of small molecules. The original assay is a low throughput assay that utilizes antibodies to quantify target protein levels. Alternatively, we chose a recently reported variant of the thermal shift assay that utilizes a &#946;-galactosidase enzyme fragment complementation (EFC) assay (Figure 2)35. For these experiments, the protein of interest is expressed in cells as an N- or C-terminal fusion protein carrying an enhanced ProLabel tag (ePL, a 42 amino acid fragment of &#946;-galactosidase). Cells are then transferred to 384-well PCR compatible plates and incubated with compounds of interest. A thermocycler is utilized to apply a temperature gradient to the cells, whose proteins will denature and aggregate as the temperature increases based upon their thermal stability. The ability of a candidate compound to bind and stabilize the protein of interest will result in an increased thermal stability of that protein. Therefore, following the lysis of the cells, those tagged proteins that have been stabilized by a candidate compound will remain in solution at higher temperatures than tagged proteins of cells incubated with vehicle control. Reporter enzyme acceptor (EA) is able to complement the soluble ePL-tagged proteins, resulting in detectable &#946;-galactosidase activity using a luminescence substrate.
We recently developed a robust cellular thermal shift assay for wild-type SHP2 (SHP2-WT) and a frequent SHP2 oncogenic variant (SHP2-E76K) in a miniaturized 384-well format36. Here, we report a detailed protocol of this assay, which reliably detects target engagement by SHP2 inhibitors in cells and demonstrates a high degree of correlation between inhibitor potency and cellular thermal shift data. The general assay workflow is illustrated in Figure 3. Our platform uses N-terminally tagged full-length ePL-SHP2 fusion proteins. For the generation of the corresponding pICP-ePL-N-SHP2-WT and pICP-ePL-N-SHP2-E76K expression plasmids, please refer to our recent publication36. This assay can be performed using a thermal gradient to establish SHP2 thermal profiles and determine SHP2 melting temperatures in the presence or absence of inhibitor. Once thermal profiles have been established, it can also be performed under isothermal conditions, allowing inhibitor dose-response assessment. Both types of experiments are described below.

<table><tbody><tr><td>384-well gradient equipped thermocycler</td><td>Eppendorf AG</td><td>X50h</td><td></td></tr><tr><td>384-well low dead volume microplate Echo qualified</td><td>Beckman Coulter, Inc.</td><td>LP-0200</td><td></td></tr><tr><td>6-well cell culture plates</td><td>Greiner Bio-One</td><td>657 160</td><td>Sterile with lid</td></tr><tr><td>Antibiotic-Antimycotic (Anti Anti) 100 X</td><td>Thermo Fisher Scientific</td><td>15240-062</td><td></td></tr><tr><td>Cell counter</td><td>Thermo Fisher Scientific</td><td>Countess II FL</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium 1X + GlutaMAX</td><td>Thermo Fisher Scientific</td><td>10566-016</td><td>500 mL</td></tr><tr><td>Echo acoustic liquid handler</td><td>Beckman Coulter, Inc.</td><td>Echo 550</td><td></td></tr><tr><td>Electronic multichannel pipette</td><td>Thermo Fisher Scientific</td><td>E1 ClipTip</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>26140-079</td><td>500 mL</td></tr><tr><td>HEPES buffer</td><td>Thermo Fisher Scientific</td><td>15630-680</td><td>100 mL</td></tr><tr><td>InCell Pulse starter kit</td><td>Eurofins DiscoverX Corp.</td><td>94-4007</td><td>Components include EA buffer, lysis buffer, and substrate</td></tr><tr><td>Microplate reader</td><td>Tecan Trading AG</td><td>Spark</td><td></td></tr><tr><td>Single channel solution trough</td><td>Thermo Fisher Scientific</td><td>S253012005</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Thermo Fisher Scientific</td><td>11360-010</td><td>100 mM</td></tr><tr><td>Thermal microplate sealer</td><td>Agilent Technologies, Inc.</td><td>PlateLoc</td><td></td></tr><tr><td>Transfection reagents</td><td>Polyplus Transfection</td><td>jetPRIME</td><td></td></tr><tr><td>Trypan blue</td><td>Thermo Fisher Scientific</td><td>T10282</td><td></td></tr><tr><td>TrypLE Express reagent</td><td>Thermo Fisher Scientific</td><td>12605-010</td><td></td></tr><tr><td>Twin.tec 384 real-time PCR plates</td><td>Eppendorf AG</td><td>30132734</td><td></td></tr></tbody></table>

assessing cellular target engagement by shp2 (ptpn11) phosphatase inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2&#160;is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.

The thermal gradient experiment for SHP2-WT resulted in a sigmoidal cellular thermal profile with a narrow melting transition that is typical and consistent for a folded protein (Figure 4A). SHP2 consists of three independent domains: two SH2 domains and the catalytic domain (Figure 1). In the autoinhibited closed conformation these domains self-associate; the melting transition that was observed in the thermal profile experiment presumably reflected this state ...

Watch this Scientific Journal Video about Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors at JoVE.com

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...

assessing-cellular-target-engagement-shp2-ptpn11-phosphatase

Research

JoVE Journal

Cancer Research

6.2K Views.  Sanford Burnham Prebys Medical Discovery Institute. The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

Video: Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

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.

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 ...

Biochemistry

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein phosphatases (PPPs) coordinate over 90% of all cellular serine/threonine dephosphorylation. Deregulation of protein phosphorylation has been implicated in the pathophysiology of various diseases, including cancer and neurodegeneration. Despite their widespread activity, the molecular mechanisms controlling PPPs and those controlled by PPPs are poorly characterized. Here, a proteomic approach termed phosphatase inhibitor beads and mass spectrometry (PIB-MS) is described to identify and quantify PPPs, their posttranslational modifications, and their interactors in as little as 12 h using any cell line or tissue. PIB-MS utilizes a non-selective PPP inhibitor, microcystin-LR (MCLR), immobilized on sepharose beads to capture and enrich endogenous PPPs and their associated proteins (termed the PPPome). This method does not require the exogenous expression of tagged versions of PPPs or the use of specific antibodies. PIB-MS offers an innovative way to study the evolutionarily conserved PPPs and expand our current understanding of dephosphorylation signaling.

Here, we present a protocol for the enrichment of endogenous phosphoprotein phosphatases and their interacting proteins from cells and tissues and their identification and quantification by mass spectrometry-based proteomics.

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Medicine

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Immunology and Infection

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Temporal Regulation of Phosphatase Activity with the Rapamycin-Regulated System

Development and Application of Rapamycin-regulated Tyrosine Phosphatases

Tyrosine phosphatases are an important family of enzymes that regulate critical physiological functions. They are often dysregulated in human diseases, making them key targets of biological studies. Tools that enable the regulation of phosphatase activity are instrumental in the dissection of their function. Traditional approaches, such as overexpression of constitutively active or dominant negative mutants, or downregulation using siRNA, lack temporal control. Phosphatase inhibitors often have poor specificity, and they only allow researchers to determine what processes are affected by the inhibition of the phosphatase. 
We developed a chemogenetic approach, the Rapamycin-regulated (RapR) system, which allows for allosteric regulation of a phosphatase catalytic domain that enables tight temporal control of phosphatase activation. The RapR system consists of an iFKBP domain inserted into an allosteric site in the phosphatase. The intrinsic structural dynamics of the RapR domain disrupt the catalytic domain, leading to the inactivation of the enzyme. The addition of rapamycin mediates the formation of a complex between iFKBP and a co-expressed FRB protein, which stabilizes iFKBP and restores activity to the phosphatase's catalytic domain. 
This system provides high specificity and tight temporal control of phosphatase activation in living cells. The unique capabilities of this system enable the identification of transient events and interrogation of individual signaling pathways downstream of a phosphatase. This protocol describes guidelines for the development of a RapR-phosphatase, its biochemical characterization, and the analysis of its effects on downstream signaling and regulation of cell morphodynamics. It also provides a detailed description of a protein engineering strategy, in vitro assays analyzing phosphatase activity, and live cell imaging experiments identifying changes in cell morphology.

Author Spotlight: Developing Tools to Tune the Activity of Tyrosine Phosphatases

This protocol describes the design, creation, and application of rapamycin-regulated phosphatases. This method provides high specificity and tight temporal control of phosphatase activation in living cells.

Tyrosine phosphatases are an important family of enzymes that regulate critical physiological functions. They are often dysregulated in human diseases, ...

Biology

Quantitative PCR-based Assay to Measure Sonic Hedgehog Signaling in Cellular Model of Ciliogenesis

Ciliopathy refers to a collection of multisystemic and polygenic human diseases and disorders that arise due to aberration of structure or function of motile or non-motile (primary) cilia that are microtubule-based cell protrusions. Primary cilia (PC) are present in most human epithelial and endothelial tissues and serve as a hub for physiological signal transduction, including Sonic Hedgehog (SHH) signaling. Various situations demand examining the function of PC, which may be achieved by measuring the canonical SHH signaling pathway that is almost exclusively mediated by PC. Here, a quantitative PCR (qPCR) based technique is developed to measure the transcriptional level of SHH pathway genes in quiescent hTERT-RPE1 (or RPE1) cells that mostly contain PC. Quiescence in RPE1 cells is achieved by serum starvation for 48 h, while activation of the SHH pathway is promoted by a specific agonist. This cell culture-based assay is easy to follow and sensitive. Successful demonstration of this assay in ciliated RPE1 cells indicates its immense potential as an in vitro assay to examine the proper function of PC upon genetic or epigenetic alteration of one or multiple genes, which may be associated with various ciliopathies.

The study demonstrates a quantitative PCR-based assay for primary cilia-mediated Sonic Hedgehog (SHH) signaling pathway activation using SHH agonist in a cellular model of ciliogenesis.

Ciliopathy refers to a collection of multisystemic and polygenic human diseases and disorders that arise due to aberration of structure or function of ...

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

Summary

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Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

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Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

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Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

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Quantitative PCR-based Assay to Measure Sonic Hedgehog Signaling in Cellular Model of Ciliogenesis