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 PTPN11

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 fro....

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

List of materials used in this article

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