Tuesday, April 12, 2005

Lindy Kauffman, Keith J. Olson and Richard M. Eglen

G protein coupled receptors (GPCRs) are a major class of targets for drug discovery and frequently are targets within high throughput screening (HTS) laboratories.⁽¹⁾ Although GPCRs are a large, diverse and highly conserved class of membrane-bound proteins, signaling principally involves modulation of only two membrane-bound enzymes, adenylyl cyclase and phospholipase C. GPCR coupling to Gα_s and Gα_i/o proteins activate or inhibit, respectively, adenylate cyclase, the enzyme responsible for converting adenosine triphosphate (ATP) to 3’ 5’ cAMP and inorganic pyrophosphate. cAMP acts at several downstream targets including ion channels, kinases that modulate gene transcription, and cell metabolism. GPCRs coupling to Gα_q/o proteins, alternatively, activate phosphoinositol phospholipase Cβ, which hydrolyzes phosphatidylinositol 4,5 bisphosphate (PIP₂) forming sn 1,2 diacylglycerol and inositol 1,4,5 trisphosphate (Ins P₃). Ins P₃ binds and opens endoplasmic Ins P₃ gated calcium channel, causing release of bound calcium into the cytosol.^(1,2)

click the image to enlarge Figure 1. DiscoveRx HitHunter technology. In a competitive immunoassay format, as used in the HitHunter cAMP HS assay, cAMP is chemically conjugated to a small peptide fragment (ED) derived from the -gal peptide. This conjugate complements to an inactive enzyme acceptor peptide (EA) forming active enzyme. Steric hindrance of EFC occurs when an antibody is added and sequesters ED-conjugated cAMP such that little or no active -gal is formed. However, free cAMP generated from the cell competitively displaces ED-conjugated cAMP from the antibody, allowing it to freely complement EA and thus generate a signal, directly proportional to the amount of cellular cAMP.

Measurement of the effects of ligand-bound GPCR on cell function, as opposed to measurement of the binding per se, permits characterization of compounds that modulate receptor activity in the cell environment. Thus, novel agonists are easily distinguished from antagonists. Moreover, agents acting on GPCRs by other mechanisms, such as allosteric regulators or G protein activators, can be easily identified. These functional modulators, collectively, are difficult to characterize using binding methods alone. Functional GPCR assays are also a prerequisite for the identification of endogenous or synthetic ligands for orphan GPCRs, including those compounds acting as inverse agonists to reduce constitutive G protein coupling.⁽³⁾

GPCR second messenger assays

Measurement of GPCR second messengers usually involves assays to determine accumulation of cAMP or calcium. Intracellular changes in these second messengers are undertaken either in broken cells following lysis or in intact cells using cell-penetrating dyes, respectively. Although spatial information is lost in such ‘end point’ protocols (in contrast to imaging based assays), assays of this nature provide highly quantitative pharmacological information on ligands that modulate the GPCR function, which is important in defining the action of the novel compound and its structure activity relationships.⁽⁴⁾

Cyclic AMP, specifically 3’ 5’ cyclic AMP, plays an important role in many signal transduction pathways. Consequently, intracellular levels of cAMP are tightly regulated via the activity of the adenylyl cyclase enzyme family, some of which can be activated or inhibited by G proteins subunits mobilized by GPCR activation. Changes in intracellular cAMP levels correlate with GPCR activation and measurement of cAMP level offers a simple functional assay for GPCR screening. There are several assays for the measurement of cAMP, including those in which the accumulation of cAMP is measured in a homogenous assay format. These have been reviewed in detail.^(4,5) The one discussed below is based upon β-galactosidase (β-gal) enzyme fragment complementation (EFC).⁽⁵⁾

Galactosidase is one of several enzymes to undergo complementation, i.e. biological activity is restored by non-covalent interaction of different polypeptides. A major advantage of using β-galactosidase is that the signal is generated by enzymatic turnover, which amplifies the readout and provides the basis for highly sensitive bioassays. β-galactosidase complementation is compatible with solutions frequently encountered in screening, including crude cellular lysates, or the relatively high concentrations of dimethyl sulphoxide (DMSO) frequently used to dissolve compounds. Collectively, these attributes enable one to configure sensitive screening assays in a homogeneous fashion. β-gal is an enzyme extensively used in cellular biology as a biological reporter. EFC occurs when fragments of β-gal complement in trans by intracistronic combination to form active enzyme. The commercial availability of numerous β-gal substrates enables colorimetric, fluorescent or chemilumescent signals to be generated. In a competitive immunoassay approach, β-gal EFC is now widely used for high throughput detection of cAMP (Figure 1). Here, cAMP is chemically conjugated to a small (~5 Kd) peptide fragment derived from the β-gal αpeptide. This conjugate complements to an inactive β-gal αpeptide forming active enzyme. Steric hindrance of EFC occurs when an antibody is added to the assay that binds conjugated cAMP such that little or no active β-gal is formed. However, free cAMP generated by the cell competitively displaces the conjugated nucleotide from the antibody, allowing it to freely complement and thus generating a signal.^(4,5)

The HitHunter assay for cAMP is deployed for the detection of compounds interacting at Gα_s or Gα_i coupled GPCRs,⁽⁶⁾ and has been miniaturized to high density 384 and 1536 well plate formats.⁽⁷⁾ Signal to background ratios are routinely produced of at least 30 fold, resulting in assays of both high precision (Z’ factors of >0.7) and, due to β-gal turnover, employing protocols using low analyte concentrations. Indeed, DiscoveRx has recently adapted this approach for measurement in cell lysates of very low levels (10 nmoles/well) of cAMP (Figure 2). This format, termed cAMP HS, has an EC₅₀ of 6 nM cAMP, with a two log dynamic range and a signal to background ratio of approximately 10. The typical applications of this assay employ very low numbers of cells per well (2500 – 5000), with Z’ factors that are robust. As is the case with all DiscoveRx cAMP assays, there are very low levels of interference from library compounds or other assay excipients. The cAMP HS assay thus provides a useful tool, for the researcher or the HTS laboratory, for the measurement of low cAMP levels as may occur during orphan receptor screening, or when characterizing the action of ligands at endogenously expressed GPCRs.

click the image to enlarge Figure 2. DiscoveRx HitHunter cAMP HS assay. CHO K1 cells were harvested and resuspended in phospho buffered saline to cell densities equivalent to 5000 and 2500 cells per well. The cells were treated with increasing concentrations of forskolin and incubated for 30 minutes at 37 C. After adenylate cyclase induction, or addition of cAMP standard, the reagents were added and a chemiluminescent signal read.

Measuring changes in intracellular calcium mobilized by GPCR activation provides a highly sensitive assay technique for the measurement of ligand function. Although measuring calcium transients is an important HTS technique, the protocols can be time consuming, particularly, when the experimental parameters must be varied from cell to cell. Furthermore, calcium is a second messenger downstream from Gα_q coupled GPCR induced activation of PLC. Some compounds in the screening library may modulate intracellular calcium levels by means other than binding to the receptor; or may alter dye fluorescence, resulting in false positive or negative hits. The measurement of the second messenger, Ins P₃ specifically, is undertaken differently using mass assays such as GLC (gas liquid chromatography), anion exchange chromatography or HPLC, which are not easily adaptable to assays requiring high throughput. An assay developed at DiscoveRx Corp. uses fluorescent polarization (FP) to measure Ins P₃. The Ins P₃ assay is a competitive binding assay, in which cellular Ins P₃ displaces a fluorescent derivative of Ins P₃ from a specific binding protein. The assay measures changes in FP, a single wavelength ratiometric technique, in which a fluorescent derivative of Ins P₃ is used as a tracer. The FP Ins P₃ assay is performed in crude cell lysates, thereby avoiding laborious separation and filtration steps. It is therefore important that the Ins P₃ binding protein exhibits high affinity and selectivity for the D-myo-1, 4, 5-inositol-Ins P₃ isomer over other inositol polyphosphates. The buffer used in the Ins P₃ assay is optimized to ensure high affinity binding, and competition binding studies with several substituted inositol phosphates demonstrate that the Ins P₃ binding protein is specific for the D-myo inositol 1,4,5 Ins P₃ isomer.

Summary

Functional analysis of GPCR response is a common approach for high throughput screening of libraries of compounds. A series of assays developed at DiscoveRx Corp. allows for measurement of key second messengers such as cAMP or Ins P₃ in a homogeneous fashion. These assays can be automated for robotic fluid dispensing in HTS systems, and are also adaptable to the lower throughput needs of basic research laboratories. Collectively, the assays facilitate the use of cell-based testing to characterize the actions of GPCR ligands.

About the authors

Lindy Kauffman is Director of Development, Keith R. Olson is Director of Strategic Marketing and Richard M. Eglen is Chief Scientific Officer, all with DiscoveRx Corp., a privately held company developing a series of technologies for use in basic research and high throughput screening. Additional information on GPCR assays from DiscoveRx is available at www.discoverx.com.

Reference
1. Dunlop, J., Eglen, R.M. Drug Discovery Today: Technologies, 4:61-68 (2004).
2. Kenakin, T.P. Pharmacol Revs Comm., 11:93-111 (2000)
3. Kenakin T.P. Receptors and Channels. 10:51-60 (2004)
4. Williams, C. Nature Reviews: Drug Discovery, 3:125.
5. Eglen, R.M , Singh, R, Combin Chem & HTS, 6:313-318 (2003).
6. Golla, R., Seethala, R., J Biomol. Screening, 7:515-525 (2002).
7. Weber, M., Ferrer, M., Zheng, W., Inglese, J., Strulovici, B., Kunapuli, P. Assay and Drug Devel. Techs. 2:39-49 (2004).

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