Development and Evaluation in Rat and Monkey of a Candidate Homochiral Radioligand for PET Studies of Brain Receptor Interacting Protein Kinase 1: [18F](S)-1-(5-(3-Fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2,2-dimethylpropan-1-one
Susovan JanaMudasir MaqboolXuefeng YanJimmy E. JakobssonAdrian LeeJeih‐San LiowSami S. ZoghbiShawn WuPriscilla LongRobert B. InnisSanjay TeluVictor W. Pike
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Receptor interacting protein kinase 1 (RIPK1) crucially upregulates necroptosis and is a key driver of inflammation. An effective PET radioligand for imaging brain RIPK1 would be useful for further exploring the role of this enzyme in neuroinflammation and for assisting drug discovery. Here, we report our progress on developing a PET radioligand for RIPK1 based on the phenyl-1Keywords:
Radioligand
Radioligand Assay
Radioligand
Radioligand Assay
Ligand binding assay
Receptor–ligand kinetics
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Radioligand
Radioligand Assay
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Specific activity
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Radioligand
Radioligand Assay
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Abstract The sections in this article are: Identification of Receptors Based on Functional Properties Schild Analysis Identification of Receptors with Radioligand Binding Choosing the Biological Preparation Choosing a Radioligand Separating Bound From Free Radioligand Irrelevant Binding Establishing That the Radioligand Binding Detected Reflects Interaction With the Physiologically Significant Receptor Analysis of Radioligand Binding Data Based on the Law of Mass Action Fractional Occupancy Assumptions Inherent in the Law of Mass Action Saturation Binding Studies Defining Nonspecific Binding in a Radioligand Binding Assay Defining The Specificity of Radioligand Binding Using Competitive Binding Analysis Analyzing Competitive Binding Data Calculating the K i From the IC 50 Homologous Competitive Binding Curves Permit an Assessment of Both K d and B max Kinetic Analysis of Radioligand Binding Experiments Studies of Radioligand Dissociation Studies of Radioligand Association Complex Binding Phenomena Competitive Binding Experiments With Two (or More) Receptor Sites Saturation Binding Experiments With Two Receptor Sites Comparing One‐ and Two‐Site Models The Slope Factor of a Competitive Binding Curve Using Dissociation Experiments to Investigate Complex Binding Distinguishing Between Independent Receptor Subtypes and Negative Cooperativity Evaluating Allosteric Phenomena Obtaining Independent Data to Clarify the Biological Origin of Complex Binding Phenomena Agonist Binding and the Ternary Complex Summary
Radioligand
Radioligand Assay
Cooperative binding
Receptor–ligand kinetics
Competitive binding
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Abstract This unit presents two convenient radioligand binding assay methods for opioid receptors. These are applicable to all three of the opioid receptors that have been cloned to date (the mu, kappa and delta receptors), and can be used with numerous commercially available radiolabeled ligands. Moreover, they can serve as reasonable starting points in the development of new assays. A method for determining the binding of radioligand to cloned opioid receptors expressed on the surface of cultured cells is detailed along with a similar method to study radioligand binding to receptors from tissue homogenates. Also included is a procedure for titrating inhibitors of opioid receptor‐ligand binding, along with guidelines for analysis of the resulting data.
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κ-opioid receptor
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Subclasses of receptors exist for most neurotransmitters. Frequently, two subtypes of receptors coexist in the same tissue and, in some cases, they mediate the same physiological response. In tissues with two classes of binding sites for a given hormone, an estimate of the proportion of each class of binding sites is obtained by inhibiting the binding of a single concentration of a radioligand with a selective unlabeled ligand. Accurate estimates of the density of each class of receptors will only be obtained, however, if the radioligand is entirely nonselective. Selectivity of just 2- to 3-fold can markedly influence the results of subtype analysis. The conclusion that a radioligand is nonselective is usually based on the results of a saturation binding curve. If Scatchard analysis of such data results in a linear plot, then it is concluded that the radioligand is nonselective. However, Scatchard analysis cannot distinguish between a radioligand that is nonselective and one that is slightly selective. The use of a slightly selective radioligand can lead to errors of 50% or more, depending on the concentration of the radioligand relative to the Kd values of the two classes of sites. A new analytical method has been developed that can be used to quantitate 2- to 3-fold differences in the affinity of two distinct classes of binding sites for a radioligand. This new approach requires that a series of inhibition experiments with a selective unlabeled ligand be performed in the presence of increasing concentrations of the radioligand. Analysis of the resulting inhibition curves, utilizing the mathematical modeling program MLAB on the PROPHET system, yields accurate estimates of the density of each class of receptor as well as the affinity of each receptor for the labeled and unlabeled ligands. This approach was used to determine whether 125I-iodopindolol shows selectivity for beta 1- or beta 2-adrenergic receptors. A series of inhibition curves was generated with the unlabeled ligands ICI 89,406 (beta 1-selective) and ICI 118,551 (beta 2-selective), using membranes prepared from C6 glioma cells. These cells contain both beta 1- and beta 2-adrenergic receptors. 125I-Iodopindolol was determined to be 3-fold selective for beta 2-adrenergic receptors. Since the sensitivity of this approach is superior to that of Scatchard analysis, it is likely that other radioligands, previously thought to be nonselective, will be shown to be selective when analyzed by this method.
Radioligand
Radioligand Assay
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Radioligand binding has been used for many years to identify new binding sites, characterize receptors, and identify novel ligands. Although various techniques have been developed to improve the efficiency of preparing the biological source of the receptors and for detecting bound radioligand, the principles of the assays remain the same. This unit reviews theory and provides examples of the parameters that can be calculated from radioligand binding data to characterize ligand-receptor interactions. The important aspects of assay development and validation that allow meaningful interpretation are discussed. The selection of a radioligand, buffer and other assay components is critical to developing a useful binding assay. The nature of the binding interaction can also be probed by varying assay conditions.
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Radioligand Assay
Ligand binding assay
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The radioligand-binding assay is a relatively simple but powerful tool for studying G-protein-coupled receptors. There are three basic types of radioligand-binding experiments: (1) saturation experiments from which the affinity of the radioligand for the receptor and the binding site density can be determined; (2) inhibition experiments from which the affinity of a competing, unlabeled compound for the receptor can be determined; and (3) kinetic experiments from which the forward and reverse rate constants for radioligand binding can be determined. Detailed methods for typical radioligand-binding assays for G-protein-coupled receptors in membranes and intact cells are presented for these types of experiments. Detailed procedures for analysis of the data obtained from these experiments are also given.
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We describe a novel method of kinetic analysis of radioligand binding to neuroreceptors in brain in vivo, here applied to noradrenaline receptors in rat brain. The method uses positron emission tomography (PET) of [11C]yohimbine binding in brain to quantify the density and affinity of α 2 adrenoceptors under condition of changing radioligand binding to plasma proteins. We obtained dynamic PET recordings from brain of Spraque Dawley rats at baseline, followed by pharmacological challenge with unlabeled yohimbine (0.3 mg/kg). The challenge with unlabeled ligand failed to diminish radioligand accumulation in brain tissue, due to the blocking of radioligand binding to plasma proteins that elevated the free fractions of the radioligand in plasma. We devised a method that graphically resolved the masking of unlabeled ligand binding by the increase of radioligand free fractions in plasma. The Extended Inhibition Plot introduced here yielded an estimate of the volume of distribution of non-displaceable ligand in brain tissue that increased with the increase of the free fraction of the radioligand in plasma. The resulting binding potentials of the radioligand declined by 50-60% in the presence of unlabeled ligand. The kinetic unmasking of inhibited binding reflected in the increase of the reference volume of distribution yielded estimates of receptor saturation consistent with the binding of unlabeled ligand.
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