A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

Summary

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

Abstract

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.

The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.

The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca²⁺ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a "dual-addition" approach can detect functional agonist and antagonist "hit" molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z' factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 µM final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca²⁺ signaling cascade.

Introduction

G protein-coupled receptors (GPCRs), which are present from yeast to humans, represent the largest superfamily of receptors in many organisms¹. They play critical roles in regulating nearly all biological processes in animals. There are 50-200 GPCRs in the genome of arthropods, meaning they represent the largest membrane receptor superfamily². They are classified into six major classes, A-F, based on their sequence similarity and functions³. GPCRs transduce various extracellular signals, such as those of hormones, neuropeptides, biogenic amines, glutamate, proton, lipoglycoproteins, and photons

Protocol

1. Cell maintenance

NOTE: A CHO-K1 cell line that stably expresses the kinin receptor from R. microplus, named BMLK₃, was developed by Holmes et al.¹⁶. The details of the development of the cell line are presented elsewhere¹⁴. All the following steps are performed under sterile conditions in a class II biosafety cabinet.

1. Grow the recombinant cell line in selective media (F-12K medium containing 10% fetal bovine serum [FBS] and 800 µg/mL G418 sulfate) to ensure the expression of the target receptor. Store the cells stably expressing the rece....

Results

An in-house drug plate (SAC2-34-6170) composed of 320 random small molecules was used for demonstrating this HTS assay as an example. The HTS had excellent assay quality with a Z' factor of 0.7 (Table 1). This Z' factor reflects the assay quality independent of the tested compounds³⁴. A Z′-factor of 0.5 or greater indicates a good assay signal dynamic range between the RFUs of the positive controls and the negative controls. Assays with a Z.......

Discussion

The goal of HTS is to identify hit molecules through screening massive numbers of small molecules. Therefore, the results from this example only represent a small part of a conventional HTS experiment. Furthermore, the hit molecules identified need to be validated in downstream assays such as a dose-dependent assay on the same recombinant cell line and on a CHO-K1 cell line carrying only the empty vector, which can be performed simultaneously to save small molecules. Cytotoxicity assays will help demonstrate that a lack .......

Materials

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA	Gibco Invitrogen	15050-065	with phenol red
0.4% trypan blue	MilliporeSigma	T8154	liquid, sterile
1.5 mL microcentrifuge tubes	Thermo Fisher	AM12400	RNase-free Microfuge Tubes
5 mL serological pipette	Corning	29443-045	Corning Costar Stripette individually wrapped
10 mL serological pipette	Corning	29443-047	Corning Costar Stripette individually wrapped
15 mL conical tubes	Falcon	352196	sterile
20 µL filter tips	USA Scientifc Inc.	P1121	sterile, barrier
25 mL serological pipette	Corning	29443-049	Corning Costar Stripette individually wrapped
50 mL conical tubes	Corning	430828	graduated, sterile
150 mL auto-friendly reservior	Integra Bioscience	6317	sterile, individually wrapped for cell seeding in day 1
150 mL auto-friendly reservior	Integra Bioscience	6318	sterile, stacked, for loading dye in day 2
384/ 12.5 µL low retention tips	Integra Bioscience	6405	long, sterile filter
384/ 12.5 µL tips	Integra Bioscience	6404	long, sterile filter
384-well plate	Greiner	781091	CELLSTAR, clear polystyrene, µClear, Black/Flat
Aluminum plate seals	Axygen Scientific	PCR-AS-200	polyester-based
Aluminum foil wrap	Walmart
Biosafty cabinet II	NuAire	NU-540-300
Cell counter	Nexcelom	AutoT4
cell counting slides	Nexcelom	SD-100	20 µL chamber
CO2 humidified incubator	Thermo Fisher	Forma Series II
Desk Lamp	SunvaleeyTEK	RS1000B
Dimethyl sulfoxide	MilliporeSigma	276855	anhydous, >99.9%
Drug plate	Corning	3680
Dulbecco's phosphate-buffered saline	Corning	21-031-CV	DPBS, 1x without calcium amd magnesium
Ethanol	Koptec	2000
F-12K Nutrient Mixture	Corning	45000-354	(Kaighn's Mod.) with L-glutamine
Fetal bovine serum	Equitech-Bio	SFBU30
Fluorescent calcium assay kit	ENZO Lifescience	ENZ-51017	10x96 tests
G418 sulfate	Gibco Invitrogen	10131-027	Geneticin selective antibiotic 50 mg/mL
Hank's buffer	MilliporeSigma	55037C	HBSS modified, with calcium, with magnesium, without phenol read
HEPES buffer	Gibco Invitrogen	15630-080	1 Molar
HTS data storage plateform	CDD vault		https://www.collaborativedrug.com/
Liquid handling system	Integra Bioscience	Viaflo	384/12.5 µL
Plate centrifuge	Thermo Fisher	Sorvall ST8
Plate reader	BMG technology	Clariostar
Poly-D-lysine	MilliporeSigma	P6407
Rhimi-K-1 agonist peptide	Genscript	custom order	QFSPWGamide
T-75 flask	Falcon	353136