Development and validation of [3H]Cimbi-36 binding in mouse frontal cortex including affinity constant determination for classical psychedelics: Comparison to [3H]MDL100,907 binding

Receptor binding assays utilize ligands labeled with radioisotopes such as tritium ([³H]) to determine the pharmacology of non-radiolabeled compounds at a receptor of interest. We have determined the binding affinity of 10 compounds, including the agonist psychedelics psilocin, LSD and 5-MeO-DMT, at the 5-HT_2A receptor using the antagonist ligand [³H]MDL 100,907.

However, antagonists indiscriminately label receptors in the active and inactive state, whereas agonists only bind to the receptor in the active state. In this study, we developed a receptor binding assay in mouse brain using an agonist radioligand [³H]cimbi-36 and compared inhibition constants for 10 compounds at the 5-HT_2A receptor determined with an agonist and antagonist radioligand.

Antagonist (ketanserin, WAY-100635 and RS-102221) and the three agonists (psilocin, lisuride and LSD) had similar inhibition constants when receptors were labeled with an agonist and antagonist radioligand. The inhibition constants for the agonists 5-HT, DOI, 25CN-NBOH and 5-MeO-DMT were considerably lower when determined by an agonist radioligand compared to an antagonist (12, 9.7, 5.0 and 4.5-fold, respectively). It is theorized that the agonist-antagonist binding ratio could be used to predict functional activity. When establishing the binding affinity of compounds at a receptor, it is crucial to understand the pharmacology and design experiments accordingly.

Introduction

The pharmacological mode of action of classical psychedelics, such as psilocin, LSD and DMT is, largely, mediated by 5-HT_2A receptor agonism. Agonists bind with high affinity to the active form of the receptor, whereas antagonists do not discriminate between the active and inactive states. Competition binding assays determine the affinity of ligand for a receptor by displacing a known concentration of a radioligand. It was theorized that affinity defined by an agonist radioligand may differ from affinity defined by an antagonist radioligand. Therefore, the aim of the study was two-fold:

• Develop and validate an assay using an agonist radioligand [³H]cimbi-36 to label 5-HT_2A receptors in the mouse frontal cortex.
• Compare the binding affinity of 10 compounds at mouse 5-HT_2A receptors using an agonist [³H]cimbi-36 and antagonist [³H]MDL-100,907 to label receptors.

Methods

Membranes from mouse frontal cortex (400 μl) were incubated with 50 μl [³H]cimbi-36 and either 50 μl assay buffer (total binding) or 50 μl 1 μM 25CN-NBOH (non-specific binding) at 25 ^oC. Membrane-bound radioactivity was recovered by filtration under vacuum through 11731 filters, pre-soaked in 0.5% polyethylenimine (PEI) using a Skatron cell harvester. Filters were rapidly washed with ice-cold buffer and membrane-bound radioactivity determined by liquid scintillation counting (LSC).

Method development investigated:

• Buffer Constituents
• Buffer 1 (50 mM Tris, pH 7.4 containing 4 mM CaCl₂) versus buffer 2 (50 mM Tris, pH 7.4 containing 120 mM NaCl, 5 mM KCl, 1 mM MgCl₂ and 2 mM CaCl₂)
• Tissue Concentration
• Incubation Time
• Determination of K_d and B_max

Results assay development

• Specific binding increased linearly with tissue concentration and was greater with buffer 1 (Figure 1) than with buffer 2 (Figure 2). Percentage [³H]cimbi-36 bound with buffer 1 was 8.2 %, 16 % and 23 % at 0.5, 1 and 1.5 mg wet weight tissue/tube, respectively.
• The full association was reached within 60 minutes and was maintained until 120 minutes (Figure 3).
• The mean (n=6) Kd of [³H]cimbi-36 at 5-HT_2A receptors was 0.06 nM and Bmax was 11.5 fmoles/mg wet weight tissue (Figure 4).
• Competition assays were performed with 1 mg wet weight tissue/tube (400 μl) incubated with 0.06 nM [³H]cimbi-36 and either 50 μl buffer 1 (total binding), 50 μl 1 μM 25CN-NBOH (non-specific binding) or test compound (10 concentrations) at 25 ^oC for 90 minutes.

Figure 1. Effect of tissue concentration on 0.075 nM [³H]cimbi-36 binding to 5-HT_2A receptors in mouse frontal cortex.

Figure 2. Effect of tissue concentration on 0.075 nM [³H]cimbi-36 binding to 5-HT_2A receptors in mouse frontal cortex.

Figure 3. Incubation time course for 0.06 nM [³H]cimbi-36 binding to 5-HT_2A receptors in mouse frontal cortex.

Figure 4. Typical saturation curve and Scatchard plot for [³H]cimbi-36 binding to 5-HT_2A receptors.

Inhibition constants

• Ketanserin, lisuride, DOI, 25CN-NBOH and LSD had high affinity for 5-HT_2A receptors (Ki < 10 nM).
• Psilocin, 5-HT and 5-MeO-DMT had a moderate affinity for 5-HT_2A receptors (Ki 10 – 100 nM).
• WAY-100635 and RS-102221 had low affinity for 5-HT_2A receptors (Ki > 100 nM).
• The inhibition constants for 5-HT, DOI, 25CN-NBOH and 5-MeO-DMT determined by [³H]cimbi-36 were considerably less than those determined by [³H]MDL-100,907 (12, 9.7, 5.0 and 4.5-fold, respectively; Table 1).

Table 1. Inhibition constants (Ki) determined by [³H]cimbi-36 and [³H]MDL-100,907 binding. 

Figure 5. Comparison of inhibition constants (Ki) determined by [³H]cimbi-36 versus [³H]MDL-100,907 binding. Trendline excludes 25CN-NBOH, DOI, 5-HT & 5-MeO-DMT.

Summary

• We successfully developed a method for binding the agonist ligand [³H]cimbi-36 to 5-HT_2A receptors in mouse frontal cortex homogenates.
• Antagonists (ketanserin, RS-102221 and WAY-10063) exhibited similar binding affinities when receptors were labeled with either an agonist or antagonist radioligand.
• Agonists psilocin, lisuride and LSD exhibited similar binding affinities when receptors were labeled with either an agonist or antagonist radioligand.
• The agonists DOI and 25CN-NBOH were defined as having high affinity when receptors were labeled with an agonist radioligand but moderate affinity when labeled with an antagonist radioligand.
• The agonists 5-HT and 5-MeO-DMT were defined as having moderate affinity when receptors were labeled with an agonist radioligand but low affinity when labeled with an antagonist radioligand.
• To obtain reliable receptor binding data, it is crucial to understand the pharmacology of the compound and design assays accordingly.
• We theorize that agonist versus antagonist binding affinity could be used to predict functional activity. We will determine the functional activity of these 10 compounds at 5-HT_2A receptors and correlate with binding data.