A High-Throughput Microfluidic Assay for SH2 Domain-Containing Inositol 5-Phosphatase 2

Abstract

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) is a potential drug target for the treatment of type 2 diabetes. This enzyme serves as a negative regulator of insulin-mediated signal transduction by catalyzing the dephosphorylation of the second messenger lipid molecule phosphatidylinositol 3,4,5-triphosphate. Traditionally, assays for phosphoinositide phosphatases such as SHIP2 have relied on radiolabeled phosphatidylinositol-containing lipid membranes and chromatographic separation of labeled phospholipid substrate from product by thin-layer chromatography. We have expressed and purified catalytically active phosphatase domain constructs of SHIP2 from Escherichia coli and developed a sensitive and antibody- or binding protein-independent assay for SHIP2 amenable to high-throughput screening of phosphoinositide phosphatases or phosphoinositide kinases. This microfluidic assay, with Z' values approximately 0.8, is based upon the difference in mobility within an electric field between a fluorophore-labeled phosphatidylinositol 3,4,5-triphosphate substrate and the corresponding 3,4-bisphosphate product. High-throughput screening of a 91,060-member compound library in 384-well format resulted in the identification of SHIP2 inhibitors.

Assaying PTEN catalysis in vitro

“…This allows the robust analysis of activity using small quantities of PTEN in the low nanomolar enzyme concentration range, allowing for example, analysis of protein immunoprecipitated from 100 lg to 500 lg soluble protein extracted from cells or tissues. Attempts to develop assays with more complex detection platforms, but with high throughput, specifically suited to screening for inhibitors of PTEN (and other lipid metabolising enzymes) has also been successful [20,21]. Progressing on this front, most recently, a novel gold nanoparticle based assay format presenting PtdInsP 3 in a membrane-like lipid surface with good sensitivity have also been described [22].…”

Inhibitors of the Lipid Phosphatase SHIP2 Discovered by High Throughput Affinity Selection-Mass Spectrometry Screening of Combinatorial Libraries

“…The 35 kDa catalytic domain of SHIP2 was prepared as described previously [3]. This protein target was screened in ALIS against a collection of mixture-based combinatorial libraries, representing nearly three million compounds in total, using 5 M protein plus 2.5 mM total library (1 M per component for a 2500-member library) in each binding reaction [16].…”

“…Also, by using the same substrate in both assays, the binding competition results from ALIS can be directly compared to those from the Caliper system [3]. This compound is not MS-sensitive and therefore not suitable as a titrant, so we tested its competitive binding profile by titrating NGD-61338 against SHIP2 with a fixed concentration of the PIP3 reagent.…”

“…A high throughput screening (HTS) technique using LabChip ® microfluidic technology from Caliper was recently reported that used a fluorophore-labeled substrate BODIPY-PIP3 mobility shift assay to screen a large and diverse compound *Address correspondence to this author at Aileron Therapeutics, Inc., 840 Memorial Drive, Cambridge, MA 02139, USA; E-mail: allen@allenannis.com library for inhibitors of SHIP2 [3]. The Caliper assay was further used to characterize the hits that arose from these HTS efforts.…”

Kinetic Mechanism and Inhibitor Characterization of WNK1 Kinase

“…It also allows us to determine the IC 50 value while maintaining a high ATP concentration, like that found under cellular conditions (in the millimolar range). The unique feature of this technology was formerly applied to assays for the lipid-modifying enzyme (35), protein kinase B (PKB/AKT) from cell lysate (29), DNA polymerase (36), and phosphatase (30).…”