Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

Summary

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.

Abstract

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 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.

Introduction

Tyrosine phosphorylation plays an important role in signal transduction in cells^1,2. 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 diseases^3,4,5,6. 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 activation^13,14,15.

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 bioavailability^{16,17,18,19,20,21,22}. 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 activation^26,27. While groundbreaking, these compounds are ineffective against many of the SHP2 oncogenic mutants that drive leukemogenesis in a significant number of blood cancer patients^28,29,30. 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 β-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 β-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 β-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.

Protocol

1. Preparation of cell culture and reagents

1. 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 °C.
2. Thaw cellular thermal shift reagents (EA reagent, lysis buffer, and substrate) from frozen original stock bottles.
3. Dispense reagents and buffer as 2 mL aliquots and store at -20 °C.
   NOTE: Avoid freeze/thaw for reproducibility and use only that volume of reagent required for the assay procedure.

2. Growth and maintenance of HEK293T cells

1. Obtain low passage adherent HEK293T cells from cryo storage.
2. Maintain HEK293T cells in growth media at 37 °C, 5% CO₂.
3. 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.

3. HEK293T cell preparation for transient transfection

1. Detach HEK293T cells from 16 mL plates using 3 mL of the cell detachment reagent.
2. Dilute with 12 mL of growth media. Collect cells by centrifugation at 1,400 x g for 4 min.
3. Resuspend the pellet in 10 mL growth media. Measure the concentration and viability of cells using trypan blue and a cell counter.
4. 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.
5. Incubate for 24 h at 37 °C, 5% CO₂.

4. Transfection of HEK293T cells

1. From a purified plasmid stock of pICP-ePL-N-SHP2-WT or pICP-ePL-N-SHP2-E76K (~200 ng/µL) dilute 2 µg of plasmid DNA into 200 µL of transfection buffer.
2. Vortex for 10 s and centrifuge at 1,400 x g for 4 min.
3. Add 4 µL of transfection reagent to the diluted DNA. Vortex for 10 s and centrifuge at 1,400 x g for 4 min.
4. Incubate at 23 °C for 10 min.
5. 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 °C, 5% CO_2.

5. Preparation of assay plates

1. Prepare 10 mM stock solutions in DMSO of compounds to be tested.
2. Dispense inhibitor solutions into a 384-well low dead volume source plate for immediate use.
3. 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 °C and use plates within 24 h.

6. Transfected cell detachment and preparation

1. Preincubate growth media and cell detachment reagent in a 37 °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.
2. 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 °C for 2 min.
3. 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.
4. Gently but thoroughly aspirate off the media.
   NOTE: Residual phenol red in the cell detachment reagent can interfere with the assay.
5. 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 > 90% for best results.
6. Dilute cells to a concentration of 125 cells/µL. For example, for a 5 µL assay this is ~625 cells/µL. For optimal viability keep cells in suspension for no more than 2 h.

7. Incubation of cells with SHP2 inhibitors

1. Dispense cells into a sterile single channel solution trough.
2. 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 °C.
3. Remove seal from the compound plate. Using a 125 µL multichannel pipette, add 5 µL of the diluted cells to desired wells.
4. 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 °C, 5% CO₂ for 1 h.

8. Preparation of chemiluminescent reagent master mix

1. Remove the necessary quantity of detection reagents (EA reagent, lysis buffer, and substrate) from -20 °C storage after plating cells. Thaw reagents at 23 °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.
2. 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's manual.

9. Isothermal or thermal profile gradient heat pulse

1. Program the gradient capable thermocycler for the delivery of heat pulse.
   1. For thermal profile gradient experiments, preprogram the thermocycler with the following example specifications:
   2. Heat pulse: 3 min desired melting temperature (vertical or horizontal gradient +/- 15 °C; example 38-68 °C spread across 24 wells yields temperature increments of 1.25 °C).
2. Equilibration recovery step: 3 min 20 °C with ramp speed = 1 °C/s
   1. For isothermal experiments set up the protocol set up as follows:
   2. Heat pulse: 3 min 55 °C. Equilibration recovery step: 3 min 20 °C with ramp speed = 1°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.

10. Lysis detection and measurement

1. Supplement assay plates with lysis detection master mix by addition of equal volumes (5 µL) to each well to be analyzed using a multichannel pipette.
2. Centrifuge the plate 42 x g for 30 s. Store at 23 °C in darkness for 30-60 min.
3. 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.

11. Data analysis

1. Analyze the luminescence data using a scientific 2D graphing and statistics software.
2. 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.

Results

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 ...

Discussion

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...

Materials

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