Principles and Role in Psychopharmacology


Drugs may exert their effects by specifically interacting with receptors, enzymes, or transporters, by interfering with DNA, RNA, or ribosomal processes, by exerting physico-chemical effects, or in the case of monoclonal antibodies, by targeting a variety of different proteins. With regard to receptors, this classification includes GPCRs, ion channels, and nuclear receptors. Since the latter are primarily the therapeutic focus for obesity, diabetes, and cancer, the GPCR and ion-channel family represent the main receptor targets in psychopharmacology. As a therapeutic class, GPCRs are more successful than ion channels as measured in terms of FDA-approved drugs and hence, GPCRs appear to be more "drugable" targets. However, there is little doubt that ion channels have been generally underexploit-ed, especially as targets for CNS disorders, especially when one considers that most of the neuropsychopharmacologi-cal ion channel drugs are ► GABAA modulators that were discovered prior to the revolution in molecular biology and genomics.

In the search for new psychopharmacological drugs, the identification of novel chemical structures that interact with the target of interest is arguably the most crucial step in the drug discovery process. Such compounds are usually identified in the screening of large chemical collections (ranging from hundreds of thousands to up to several million) in high-throughput screening (HTS) campaigns. Accordingly, the focus of this article will be the measurement of the functional effects of compounds at GPCRs and ion channels in such HTS formats.

Functional Assays: What to Measure?

The design of an HTS-compatible functional assay is determined not only by the functional response being measured (i.e., change in ► second messenger or ion concentration or a downstream consequence of such changes), but also by the pharmacological profile - agonist, antagonist, and modulator - of the desired compound. The simplest type of assay is one in which the test compound is being evaluated for its agonist response. The ► potency of this effect is generally characterized in terms of the concentration at which a response that is 50% of the maximum (EC50) is observed as well as the size of the maximum response. For instance, the response may either be of a magnitude comparable to the reference agonist or may produce a reduced response relative to the reference agonist (full and partial agonist, respectively; Fig. 1a). Compounds may also bind to the receptor and produce allosteric modulatory changes that might reduce or increase the EC50 (PAM or NAM, respectively) (Fig. 1b), as well as the maximal response (Fig. 1c). If an assay is being established in order to detect modulatory compounds, then the choice of agonist concentration should be picked according to whether a PAM or NAM is desired, in which case EC20- or EC80-equivalent agonist concentrations are usually employed (Fig. 1d). Although ► benzodiazepines represent a well-established class of ion-channel (GABAa receptor) PAMs, more recently allosteric modulators of GPCRs have attracted considerable attention as potential therapeutics (Conn et al. 2009).

As regards the shift toward the identification of allosteric modulators as drug targets, one of the attractive features of certain types of instrumentation is the ability to record data in real-time rather than producing just single, end-point readouts. This permits compounds to be assessed using two-addition, or sometimes even three-addition protocols. For example, in the absence of exogenous agonist, an initial addition of compound will detect direct agonist effects. If there is no direct agonist effect, then a second addition, this time of agonist, can detect a PAM (Fig. 2).

As regards antagonists, the ability of a compound to block an agonist-stimulated response is related to the functional affinity of the agonist as well as the affinity of the antagonist. For example, a low affinity antagonist will have difficulty in blocking the effects of a high affinity agonist. Although the potency of a competitive antagonist can be quantified in terms of the pA2 derived from a Schild plot of the rightward shift of an agonist concentration-effect curve at increasing antagonist concentrations, such analyses are not suitable for an HTS screen. Accordingly, the choice of the reference agonist and the concentration and preincubation time of test compound, should be given consideration. In certain expression systems, some receptors demonstrate activity in the absence of an agonist and are therefore described as possessing ► constitutive activity. Compounds that block this constitutive activity are described as inverse agonists.

General Methodologies

The general principle of functional assays for receptors is that they should demonstrate that a compound binds

Receptors: Functional Assays. Fig. 1. Schematic representation of agonist-related responses. (a) Compounds may directly activate the receptor and produce a response that is comparable to or submaximal when compared with the endogenous ligand (full agonist and partial agonist, respectively). (b) Compound might produce a leftward or rightward shift in the agonist concentration-effect curve. Such compounds are designated positive or negative allosteric modulators (PAMs and NAMs, respectively). (c) In addition to shifting the EC50, a compound may also have an effect on the maximal response (in this case increasing the maximum response). (d) An expanded view of parts of the concentration effect curves shown in Panel B illustrate how the absolute modulatory effects of PAMs and NAMs are a function of the agonist concentration. Hence, PAMs produce a proportionately greater potentiation of an EC20 when compared with EC80 response, whereas NAMs give a greater attenuation of an EC80 when compared with EC20 response.

to a receptor and "does something.'' Whether that "something'' is direct activation, inhibition, or modulation ofan agonist response, the key is being able to reliably demonstrate and quantitate a response. These methods most frequently rely upon a biosensor to detect that there has been an increase or a decrease in some component of an intracellular signaling pathway.

Some methods are unique to the class of receptor, for example [35S]GTPgS is an hydrolysis-resistant analog of GTP (guanosine triphosphate). Its binding is specific for GPCRs in comparison to, for example, the measurement of ion flux which is specific for ion channels. However, some techniques are more generally applicable to the measurement of changes in intracellular molecules.

These include, but are not limited to, techniques such as fluorescence resonance energy transfer (FRET) and the related technique of homogenous time-resolved fluorescence (HTRF), and bioluminescence resonance energy transfer (BRET).

Fluorescence and Bioluminescence Resonance Energy Transfer (FRET and BRET)

FRET refers to the process whereby energy is transferred from a fluorescent donor to a suitable energy acceptor, with the absorption spectrum of the acceptor overlapping the emission spectrum of the donor. Since the efficiency of this process is inversely proportional to the sixth power of the distance between the two molecules, then the acceptor

Receptors: Functional Assays. Fig. 2. Raw data traces from a real-time Ca2+ kinetic readout, showing the effect of a compound with (a) Agonist activity or (b) PAM activity. Ca2+ flux assays can be designed to identify compounds exhibiting agonist or PAM activity in one and the same assay well. Test compounds are added to cells loaded with a Ca2+ sensitive dye and incubated for 2.5 min. Thereafter, a submaximal (approximately EC20) concentration of agonist is added. A compound displaying intrinsic agonist activity will increase the Ca2+ response in the first read (see left-hand figure), while PAMs will only show activity (i.e., a potentiation of the EC20 agonist effect) in the second read (see right-hand figure). The EC20 agonist effect after vehicle treatment, which was measured in a separate well of a 96-well plate, is indicated in the right panel in gray.

and donor molecules need to be in close proximity, in the region of 1-10 nm, for significant FRET to occur. The readout is a change in fluorescence emission of the acceptor, which may either increase or decrease as the proximity of the donor and acceptor also increases or decreases. The most common FRET acceptor-donor pair is the cyan fluorescent protein and yellow fluorescent protein, both of which are variants of green fluorescent protein. However, these proteins are not suitable for detecting ionchannel-mediated changes in membrane potential and therefore, special dyes are employed for this purpose (see below).

BRET is based on the same donor-acceptor energy transfer principle as FRET, with the difference being that in BRET the donor is a luminescent molecule, generally coelanterazine, which is excited by the enzyme Renilla lucif-erase (Rluc) rather than a fluorescent molecule; this avoids the need for the external illumination source required for FRET. The BRET-acceptor molecule can be a fluorescent protein such as green or yellow fluorescent protein.

Ca2+ Sensors

Fluorescent Ca2+-sensitive probes linked to a fluorescence plate reader, such as the Fluorometric Imaging Plate Reader (FLIPR™, Molecular Devices, Corp.) or Functional Drug Screening System (FDSS, Hamamatsu), have become probably the gold standard for HTS of GPCRs. This is in part related to the combined liquid-handling and imaging capabilities of machines such as the FLIPR and FDSS as well as the properties of Ca2+-sensitive dyes, such as organic fluorophores, fura-2, indo-1, fluo-3, fluo-4, and Calcium Green. In addition, Ca2+-sensitive proteins may be used as Ca2+ sensors. Fluorescent proteins are generally engineered to contain the calcium-binding protein calmodulin as a molecular Ca2+-sensing switch, which may be associated with either a single or two different fluorescent proteins, with the latter producing a FRET signal due to Ca2+ binding to calmodulin-producing conformational changes in the sensor.

Aequorin is a Ca2+-activated bioluminescent photoprotein derived from the jellyfish Aequoria victoria, which emits luminescence as a result of the irreversible binding of Ca2+ ions. It comprises two components, the 22 kDa apoaequorin protein and a coelenterazine cofactor, and cells can be engineered to express apoaequorin, whereas coelenterazine needs to be added exogenously. When Ca2+ binds to the apoprotein, coelenterazine is oxidized to coelenteramide and this results in the emission of a blue light that is detected as luminescence.

Functional Assays for GPCRs

GPCRs activate intracellular signal transduction mechanism via either G-protein- or b-arrestin-mediated pathways, with examples of the various methodologies used to measure components of these pathways being presented in Table 1. With respect to the G-protein-mediated pathway, the binding of an agonist produces conformational changes in the GPCR which permit interaction between the C-terminal, intracellular receptor domain, and the heterotrimeric G-protein, which comprises an a, b, and g subunit. This

Receptors: Functional Assays. Table 1. Summary of methods available for assessing functional effects at G-protein-coupled receptors (GPCRs).

Assay type


[35S]GTPgS binding

Generic assay for studying interaction of G-proteins but Gai/o-linked receptors give best signal:noise ratio

cAMP measurement - direct (radioimmunoassay)

Radiometric assays not preferred for high-throughput screening (HTS)

cAMP measurement - direct (nonradiometric)a

High-affinity enzyme complementation (HitHunter™)

Bioluminescence resonance energy transfer (BRET) (e.g., ALPHAscreen)

Fluorescence resonance energy transfer (FRET) (e.g., LANCE™, PerkinElmer; HTRF®, CisBio)

Fluorescence polarization (FP)

cAMP measurement - reporter gene assay

Reporter gene (e.g., b-galactosidase, b-lactamase, GFP, luciferase) linked to a cAMP-response element (CRE)

Ca2+ - direct measurement

Organic fluorophores (fura-2, fluo-3, fluo-4, etc.)

Fluorescent proteins incorporating Ca2+ sensor (e.g., calmodulin)

Bioluminescence - emission upon irreversible binding of Ca2+ to aequorin

Ca2+ - reporter gene assay

Reporter gene (e.g., b-galactosidase, b-lactamase, GFP, luciferase) linked to a calcium-sensitive element such as activator protein 1 (AP1) or nuclear factor of activated T-cells (NFAT)


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