Pharmacological Assays

Radioligand binding assays - Saturation

Saturation binding and displacement assays use radiolabelled ligands to measure the binding of a drug to a receptor and are used to determine tissue expression of a given receptor (Bmax) and/or a drugs dissociation constant KD or Ki.

Assays are carried out using membrane fragments prepared from either whole cell cultures or primary tissue. The mass of membrane protein used will varied in a manner dependent on the receptor under investigation and the receptor expression of the tissue source.

Radiolabelled ligands can be either tritiated [3H] or iodinated [125I]. Which label is used will depend on which are available for a given ligand, the sensitivity required - [125I] is more sensitive and can be used to detect lower levels of protein expression, and cost - iodinated ligands are typically more expensive.

A good source of radiolabelled liagnds is Perkin Elmer:

Saturation binding basic methodology

Membrane fragments are incubated in 0.5ml volume of a binding buffer, typically a Tris-buffer the specific composition of which varies depending on the receptor under investigation. To this varying concentrations of radiolabel are added and non-specific binding (NSB) determined in the presence of saturating concentration of a non-radiolabelled drug. Reactions are typically incubated between between 1hr and 4hr and terminated via vacuum filtration through polyethyleneimine (PEI) soaked Whatman GF/B filters using a Brandel-harvester. Data are presented as a pKd, which is the negative log10 of the concentration required to occupy 50% of the receptors.

Radiolabelled binding assays - Displacement

Competition or displacement binding studies allow determination of binding affinities for non-labelled ligands. These binding studies utilise a fixed concentration of a radiolabel and the affinity of non-labelled ligands are determined by a drugs ability to compete for the same binding site. Increasing concentrations of non-labelled ligand are used to displace or out-compete the fixed concentration of a radiolabel, generating an IC50 for the competing ligand.

Displacement binding basic methodology

Methodolgy is essentially the same as that described for saturation binding except that a fixed mass of radioligand is used in the presence of varying concentrations of a test drug. The affinity of the non-labelled ligand is determined using the experimentally measured IC50 value in the Cheng and Prusoff equation (Cheng et al., 1973). The determined value is designated pKi and is the log concentration of the competing ligand required to displace 50% of the radiolabel through 50% occupancy of the total receptor population. This value is therefore equivalent to a pKd.

Radioligands we have experience with include:
Opioid Ligands Other GPCR Ligands
Diprenorphine [3H] Urotensin II (human) [125I]
Nociceptin [3H] and Nociceptin [125I]
Naltrindole [3H]

Functional second messenger assays

Functional assays measure the activation and response elements of a drug/receptor interaction that is intrinsic activity (efficacy) and potency (the concentration of ligand to cause a 50% response EC50).

  1. GTPγ[35S] binding assay

    The GTPγ[35S] assay measures the coupling of the receptor to a G-protein. Ligand binding at a G-protein coupled receptor alters the conformation of the receptor in a way that allows interaction with G-protein(s). This interaction leads to the exchange of GDP for GTP. Using a radiolabelled, stable analog of GTP, GTPγ[35S] it is possible to measure the exchange of GDP for GTPγ[35S]. The stimulated binding of GTPγ[35S] by a ligand can be used to determine a measure of its potency (EC50) and intrinsic activity (Emax). Experimental ligands are typically compared with a control ligand.

    GTPγ[35S] binding assay methodology

    Experiments are carried out on membrane fragments, typically between 20-50ug, prepared from either cultured cells or from primary tissue. Membranes are incubated in 0.5ml volumes of buffer containing bacitracin, , BSA and GTPγ[35S]. GDP is necessary to prevent radiolabel binding under basal conditions and optimal GDP concentrations need to be defined for a given receptor. NSB is determined in the presence of GTPγ in all experiments. Reactions are typically incubated at 30οC with gentle shaking for between 30-60mins.

    Figure 1. N/OFQ stimulated binding of GTPγ[35S] in membrane fragments from CHOhNOP cells. Data are presented as stimulation (stimulated GTPγ[35S] binding relative to basal GTPY[35S] binding).
  2. GTPγ[35S] antagonist assays

    GTPγ[35S] functional assays are ideally suited for measuring antagonist potency either by way of a full Schild Plot analysis or using a single antagonist concentration and analysis using the Gaddum equation (pkB = log(conc.ratio - 1) - log(antagonist conc.). In these experiments an agonist is incubated in the presence of a single or varying concentrations of an antagonist.

    Figure 1. Antagonism of N/OFQ stimulated GTPγ[35S] binding by UFP-101. Inserts show respective Schild plots.
  3. Adenylate cyclase

    Intracellular concentrations of cAMP can be measured using a competitive binding assay. Receptors which couple to second messenger pathways affecting adenylyl cyclase, such as G-protein couple receptors coupling to inhibitory Gi/o alpha subunits or stimulatory G-proteins coupling to Gs alpha subunits, change cyclic adenosine monophosphate (cAMP) production. cAMP measurements are made using a competitive binding assay which enables the pharmacology of drugs affecting this second pathway to be investigated.
    The effect on cAMP levels by a ligand can be used to determine a measures of both potency (EC50) and intrinsic activity (Emax).

    cAMP assay methodology

    Whole cell suspensions are incubated in the presence of isobutylmethylxanthine (IBMX) and forskolin, for inhinitory pathways, for 15 minutes. Drugs are included in various combinations and at different concentrations and reactions terminated using 10M HCl, the concentration of cAMP in these samples is measured using the protein binding method set out by (Brown et al., 1971). Our competitive binding protocol uses a binding protein for which labelled [3H]cAMP and unlabelled cAMP, either samples or standard controls, compete. The concentration of binding protein and [3H]cAMP are kept constant and hence the concentration of unlabelled cAMP present in a given sample can be determined from the relative displacement of [3H]cAMP from the binding protein.

    Figure 2. N/OFQ(1-13)NH2 inhibition of forskolin stimulated cAMP in CHOhNOP cells.

    Transfected Cell Lines Available

    • CHOhMOP - Chinese hamster ovary cells transfected human mu-opioid receptor
    • CHOhDOP - Chinese hamster ovary cells transfected human delta-opioid receptor
    • CHOhKOP - Chinese hamster ovary cells transfected human Kappa-opioid receptor
    • CHOhNOP - Chinese hamster ovary cells transfected human nociception orphanin FQ receptor
    • CHOhUT - Chinese hamster ovary cells transfected human Urotensin II receptor

    Control ligands we have experience with include:
    Cell Type Control Ligand
    CHOhMOP Endomorphin-1
    CHOhKOP Dynorphin A