Activation of Gq protein-coupled receptors can be monitored by measuring the increase in intracellular calcium with fluorescent dyes. Recent advances in fluorescent kinetic plate readers and liquid-handling technology have made it possible to follow these transient changes in intracellular calcium in a 1,536-well plate format for high-throughput screening (HTS). Here, we have applied the latest generation of fluorescence kinetic plate readers to multiplex the agonist and antagonist screens of a G protein-coupled receptor (GPCR). This multiplexed assay format provides an efficient and cost-effective method for HTS of Gq-coupled GPCR targets.
HopkinsAL, GroomCR. The druggable genome. Nat Rev Drug Discov, 2002; 1:727–730.
3.
EglenRM, BosseR, ReisineT. Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening. Assay Drug Dev Technol, 2007; 5:425–451.
4.
MayLT, LeachK, SextonPM, ChristopoulosA. Allosteric modulation of G protein-coupled receptors. Annu Rev Pharmacol Toxicol, 2007; 47:1–51.
5.
StrangePG. Antipsychotic drug action: antagonism, inverse agonism or partial agonism. Trends Pharmacol Sci, 2008; 29:314–321.
6.
TsienRY, RinkTJ, PoenieM. Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium, 1985; 6:145–157.
7.
GrynkiewiczG, PoenieM, TsienRY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem, 1985; 260:3440–3450.
8.
GeeKR, BrownKA, ChenWN, Bishop-StewartJ, GrayD, JohnsonI. Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes. Cell Calcium, 2000; 27:97–106.
9.
MintaA, KaoJP, TsienRY. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem, 1989; 264:8171–8178.
10.
KaoJP, HarootunianAT, TsienRY. Photochemically generated cytosolic calcium pulses and their detection by fluo-3. J Biol Chem, 1989; 264:8179–8184.
11.
LiX, LlorenteI, BraschM. Improvements in live cell analysis of G protein coupled receptors using second generation BD calcium assay kits. Current Chemical Genomics, 2008; 2:10–15.
CassuttKJ, OrsiniMJ, AbousleimanM, ColoneD, TangW. Identifying nonselective hits from a homogeneous calcium assay screen. J Biomol Screen, 2007; 12:285–287.
14.
ZhengW, SpencerRH, KissL. High throughput assay technologies for ion channel drug discovery. Assay Drug Dev Technol, 2004; 2:543–552.
15.
SullivanE, TuckerEM, DaleIL. Measurement of [Ca2+] using the Fluorometric Imaging Plate Reader (FLIPR)Methods Mol Biol, 1999; 114:125–133.
16.
HodderP, MullR, CassadayJ, BerryK, StruloviciB. Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader. J Biomol Screen, 2004; 9:417–426.
17.
XuYL, ReinscheidRK, Huitron-ResendizS, ClarkSD, WangZ, LinSHet al.Neuropeptide S: a neuropeptide promoting arousal and anxiolytic-like effects. Neuron, 2004; 43:487–497.
18.
SouthallNT, JadhavA, HuangR, NguyenT, WangY. Enabling the large scale analysis of quantitative high throughput screening data. In: Handbook of Drug Screening. SeethalaR, ZhangL2nd. Informa Healthcare: New york, 2009; 442–464.
19.
OkamuraN, ReinscheidRK. Neuropeptide S: a novel modulator of stress and arousal. Stress, 2007; 10:221–226.
IngleseJ, AuldDS, JadhavA, JohnsonRL, SimeonovA, YasgarAet al.Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci USA, 2006; 103:11473–11478.
22.
ClevelandPH, KoutzPJ. Nanoliter dispensing for uHTS using pin tools. Assay Drug Dev Technol, 2005; 3:213–225.
23.
YasgarA, ShinnP, JadhavA, AuldD, MichaelS, ZhengWet al.Compound management for quantitative high-throughput screening. JALA Charlottesv Va, 2008; 13:79–89.
24.
NiswenderCM, JohnsonKA, WeaverCD, JonesCK, XiangZ, LuoQet al.Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4. Mol Pharmacol, 2008; 74:1345–1358.
