Name: assessing cellular target engagement by shp2 (ptpn11) phosphatase inhibitors
Uploaded: 2020-07-17T15:00:00
Description: Watch this Scientific Journal Video about Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors at JoVE.com

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 as...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off-target+inhibition+by+active+site-targeting+SHP2+inhibitors.">Off-target inhibition by active site-targeting SHP2 inhibitors.</a> FEBS Open Biology. 8 (9), 1405-1411 (2018).</li><li>Chen, Y. N., 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=Allosteric+inhibition+of+SHP2+phosphatase+inhibits+cancers+driven+by+receptor+tyrosine+kinases.">Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.</a> Nature. 535 (7610), 148-152 (2016).</li><li>Nichols, R. J., 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=RAS+nucleotide+cycling+underlies+the+SHP2+phosphatase+dependence+of+mutant+BRAF-,+NF1-+and+RAS-driven+cancers.">RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers.</a> Nature Cell Biology. 20 (9), 1064-1073 (2018).</li><li>. Clinical Trial NCT03114319 Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT03114319?term=NCT03114319_rank=1">https://clinicaltrials.gov/ct2/show/NCT03114319?term=NCT03114319_rank=1</a> (2019)</li><li>. Clinical Trial NCT03634982 Available from: <a target="_blank" href="https://clinicaltrials.gov/ct2/show/NCT03114319?term=NCT03634982_rank=1">https://clinicaltrials.gov/ct2/show/NCT03114319?term=NCT03634982_rank=1</a> (2019)</li><li>Sun, 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=Selective+inhibition+of+leukemia-associated+SHP2(E69K)+mutant+by+the+allosteric+SHP2+inhibitor+SHP099.">Selective inhibition of leukemia-associated SHP2(E69K) mutant by the allosteric SHP2 inhibitor SHP099.</a> Leukemia. 32 (5), 1246-1249 (2018).</li><li>LaRochelle, J. 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P., 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=Mechanism+of+activating+mutations+and+allosteric+drug+inhibition+of+the+phosphatase+SHP2.">Mechanism of activating mutations and allosteric drug inhibition of the phosphatase SHP2.</a> Nature Communication. 9 (1), 4507 (2018).</li><li>Lu, H., 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=Resistance+to+allosteric+SHP2+inhibition+in+FGFR-driven+cancers+through+rapid+feedback+activation+of+FGFR.">Resistance to allosteric SHP2 inhibition in FGFR-driven cancers through rapid feedback activation of FGFR.</a> Oncotarget. 11 (3), 265-281 (2020).</li><li>Smyth, L. A., Collins, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+and+interpreting+the+selectivity+of+protein+kinase+inhibitors.">Measuring and interpreting the selectivity of protein kinase inhibitors.</a> Journal of Chemical Biology. 2 (3), 131-151 (2009).</li><li>Martinez Molina, D., 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=Monitoring+drug+target+engagement+in+cells+and+tissues+using+the+cellular+thermal+shift+assay.">Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay.</a> Science. 341 (6141), 84-87 (2013).</li><li>Ericsson, U., Hallberg, B., Detitta, G., Dekker, N., Nordlund, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermofluor-based+high-throughput+stability+optimization+of+proteins+for+structural+studies.">Thermofluor-based high-throughput stability optimization of proteins for structural studies.</a> Analytical Biochemistry. 357 (2), 289-298 (2006).</li><li>McNulty, D. E., 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=A+High-Throughput+Dose-Response+Cellular+Thermal+Shift+Assay+for+Rapid+Screening+of+Drug+Target+Engagement+in+Living+Cells,+Exemplified+Using+SMYD3+and+IDO1.">A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1.</a> SLAS Discovery. 23 (1), 34-46 (2018).</li><li>Romero, C., 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=A+cellular+target+engagement+assay+for+the+characterization+of+SHP2+(PTPN11)+phosphatase+inhibitors.">A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors.</a> Journal of Biological Chemistry. 295 (9), 2601-2613 (2020).</li><li>Ran, H., Tsutsumi, R., Araki, T., Neel, B. 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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&#39;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 ...

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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 ...

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