Cellular and isoform-specific G-protein receptor kinase assay kits for medium- to high-throughput inhibitor testing

Abstract

Utility model-eligible assay kit for medium- to high-throughput inhibitor testing of G-protein receptor kinases (GRKs), characterized in that the assay kit comprises: a) genome-edited GRK knockout cells, wherein the cells - a knockout of GRK2 and GRK3 or - a knockout of GRK5 and GRK6 or - exhibit a quadruple knockout of GRK2, GRK3, GRK5 and GRK6, b) wherein the genome-edited GRK knockout cells have heterologous expression of exactly one GRK isoform selected from GRK2, GRK3, GRK5 or GRK6, c) wherein the genome-edited GRK knockout cells additionally exhibit heterologous expression of a G protein-coupled receptor (GPCR), d) wherein phosphorus site-specific antibodies are provided for the expressed GPCR which are suitable for the detection of GPCR phosphorylation, e) and a bead-based GPCR phosphorylation assay designed to be performed in a 96-well or 384-well plate format, enabling medium to high throughput inhibitor testing.

Description

Technical field

[0001] The present invention relates to cellular assay kits for the inhibitor testing of G-protein receptor kinases (GRKs). In particular, the invention relates to isoform-specific, cell-based assays with medium to high throughput, based on genome-edited GRK knockout cells and a bead-based GPCR phosphorylation assay. State of the art

[0002] G protein-coupled receptors (GPCRs) represent one of the largest receptor families in the human body and are the target of numerous pharmacological agents. The activity of these receptors is regulated, among other things, by GPCR kinases (GRKs), particularly by the ubiquitously expressed isoforms GRK2, GRK3, GRK5, and GRK6. While established in vitro and in silico methods for characterizing GRK inhibitors allow for statements regarding binding affinity or enzyme inhibition, they do not provide sufficient information about efficacy and isoform specificity in living cells. Furthermore, current cell-based systems suffer from the disadvantage that multiple GRK isoforms are expressed simultaneously, making it impossible to clearly attribute the inhibitory effect to a single GRK isoform. Object of the invention

[0003] The object of the present invention is therefore to provide an assay kit that: • Isoform-specific inhibitor testing of GRKs enables, • can be performed in living cells • suitable for medium to high throughput • and allows a clear functional assignment of the inhibitory effect to individual GRK isoforms. Solution to the task

[0004] This problem is solved by an assay kit according to claim 1, comprising a combination of genome-edited GRK knockout cells and a bead-based GPCR phosphorylation assay.

[0005] The genome-edited cells exhibit either: • a GRK2 / 3 knockout, • a GRK5 / 6 knockout, or • a quadruple knockout of GRK2, GRK3, GRK5 and GRK6 and simultaneously heterologously express exactly one GRK isoform selected from GRK2, GRK3, GRK5 or GRK6.

[0006] In addition, the cells express a G protein-coupled receptor (GPCR) for which phosphorus site-specific antibodies are available that can be used in a bead-based phosphorylation assay. Advantages of the invention

[0007] The assay kits according to the invention offer in particular the following advantages: • Unambiguous isoform specificity through defined genetic background • Functional cellular readout instead of purely biochemical measurements • High reproducibility through standardized knockout cell lines • Compatibility with 96-well and 384-well formats • Suitable for screening, lead optimization and SAR analysis Preferred embodiments

[0008] In a preferred embodiment, the genome-edited cells express the β2-adrenergic receptor (β2AR). Particularly preferably, a phosphorylation site-specific antibody against the phosphorylation sites T360 and S364 of the β2-adrenergic receptor is used, enabling the selective detection of GRK-dependent receptor phosphorylation. In a further preferred embodiment, the assay is performed as an immunoprecipitation-based bead assay, in which the GPCR is bound via an affinity tag and the phosphorylation is subsequently quantitatively determined. Brief description of the drawings Figure 1 shows the agonist-induced phosphorylation of the β2-adrenergic receptor in GRK knockout cell lines. Figure 2 shows a schematic flowchart of the bead-based phosphorylation assay. Figure 3 shows receptor phosphorylation in quadruple GRK knockout cells with single GRK overexpression. Figure 4 shows the inhibition of receptor phosphorylation by GRK2 / 3 inhibitors. Figure 4 shows the inhibition of receptor phosphorylation by GRK5 / 6 inhibitors. Example of implementation

[0009] In one embodiment, HEK293 cells with a quadruple GRK2 / 3 / 5 / 6 knockout are used, stably expressing the β2-adrenergic receptor as well as a single GRK isoform. The cells are stimulated with an agonist and subsequently incubated with a test inhibitor. Phosphorylation of the receptor at positions T360 / S364 is quantified using a bead-based immunoassay and used as a measure of isoform-specific inhibitory activity.

Claims

[1] Utility model-ready assay kit for medium to high throughput inhibitor testing of G-protein receptor kinases (GRKs), characterized by , that the assay kit includes: a) genome-edited GRK knockout cells, wherein the cells - a knockout of GRK2 and GRK3 or - a knockout of GRK5 and GRK6 or - exhibit a quadruple knockout of GRK2, GRK3, GRK5 and GRK6, b) wherein the genome-edited GRK knockout cells have heterologous expression of exactly one GRK isoform selected from GRK2, GRK3, GRK5 or GRK6, c) wherein the genome-edited GRK knockout cells additionally exhibit heterologous expression of a G protein-coupled receptor (GPCR), d) wherein phosphorus site-specific antibodies are provided for the expressed GPCR which are suitable for the detection of GPCR phosphorylation, e) and a bead-based GPCR phosphorylation assay designed to be performed in a 96-well or 384-well plate format, enabling medium to high throughput inhibitor testing. [2] Assay kit according to claim 1, for use in isoform-specific inhibitor testing of G protein receptor kinases (GRKs), wherein the inhibitory effect of at least one test inhibitor is determined separately and comparatively in genome-edited GRK knockout cells, each of which has exactly one heterologously expressed GRK isoform selected from GRK2, GRK3, GRK5 or GRK6. [3] Assay kit according to claim 2, wherein the isoform-specific inhibitor testing comprises a direct quantitative comparison of GPCR phosphorylation between at least two cell populations with identical genetic background and different single GRK isoform expression. [4] Assay kit according to claim 1 or 3, for use in cellular inhibitor screening in 96-well or 384-well plate format, wherein GRK-dependent GPCR phosphorylation serves as a functional readout for isoform-specific inhibitor action. [5] Assay kit according to any one of claims 1 to 4, for use in lead identification, lead optimization or structure-activity relationship (SAR) analysis of GRK inhibitors, wherein the isoform specificity of an inhibitor is determined based on different inhibition profiles for GRK2, GRK3, GRK5 and GRK6. [6] • Assay kit according to any one of claims 1 to 5, wherein the expressed GPCR is the β2-adrenergic receptor and the GRK-dependent phosphorylation is detected by means of a pT360 / pS364-specific antibody. [7] Assay kit according to any one of claims 1 to 6, for use in pharmaceutical drug discovery, in particular for the identification and characterization of isoform-specific GRK inhibitors with a reduced off-target profile.