Application note

Analyzing In Vitro Results for the 5-HT Ligands

Reaching a deeper understanding of ligand–receptor interactions

Effective drug discovery depends on a deep understanding of the function and therapeutic potential of a compound as well as a clear picture of the chemical structure and druggability of the compound. Using the example of serotonin receptors, this application note looks at how in vitro data from Reaxys® Medicinal Chemistry facilitate such studies on the interface of biology and chemistry

The study of serotonin (5-HT) receptor pharmacology has been and remains an area of major interest for pharmaceutical research. Indeed, in the last 50 years, drugs directly or indirectly targeting these receptors have emerged as important therapeutic agents, providing treatments for a broad range of clinical conditions.

Many different serotonin receptor subtypes have been described in the literature (1, 2) 1.Barnes, N.M. and Sharp, T. (1999) A review of central 5-HT receptors and their function. Neuropharmacology 38: 1083–1152.

2. Gershon, M.D. (2004) Serotonin receptors and transporters – roles in normal and abnormal gastrointestinal motility. Aliment. Pharmacol. Ther. 20: 3–14.
. The 5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6 and 5-HT7 receptor families all belong to the G-protein-coupled receptor (GPCR) class A superfamily (3) 3. IUPHAR receptor database: . The 5-HT1receptor family is one of the most complex in terms of molecular diversity and pharmacological interactions. Selective 5-HT1B/1D receptor agonists, such as sumatriptan and rizatriptan, have established a new standard in the acute treatment of migraine headaches.

Unlike other serotonin receptors, the 5-HT3 receptor is not a GPCR but a ligand-gated ion channel. Its ligands have been extensively studied. Selective antagonists at 5-HT3 receptors have transformed cancer therapy by preventing chemotherapy- and radiation-induced emesis. The most popular compounds in this category are ondansetron and tropisetron.

A systematic examination of the in vitro results and pharmacological properties for agonists and ligands for the 5-HT receptor families could reveal even broader possibilities for their therapeutic application. This study was performed to illustrate a potential methodology for such an examination.

Analytical method

All of the bioactivity data were retrieved using Reaxys Medicinal Chemistry. Data were initially viewed using the Heatmap and Analysis View in the web-based user interface. Then, all details on the bioactivity data were exported into Microsoft® Excel® for further manipulation. Queries focused on three members of the 5-HT receptor family: 5-HT1B, 5-HT1Dand 5-HT3. Table 1 shows the overall Reaxys Medicinal Chemistry statistics on these sub-families.

Table 1. Statistics on the 5-HT receptor data in Reaxys Medicinal Chemistry. 5-HT variants, isoforms and mutants are included. Valid as of August 2015.
Target Number of bioactivities Number of substances Number of bioactivities with pX > 7 Number of substances with pX > 7
5-HT all 531,564 153,560 126,550 57,447
5-HT1B 23,231 13,006 4,154 2,280
5-HT1D 26,790 13,526 5,645 3,060
5-HT3 27,416 15,457 5,678 2,890

To facilitate comparisons of bioactivity data from different publications and assay types, all in vitro data points in Reaxys Medicinal Chemistry have pX values. pX values are calculated by transforming parameters such as EC50, IC50 and Ki into the –Log equivalent (pEC50, pIC50, pKi). These are normalized values assigned to the data that enable easy quantification of compound–target affinity and comparison of information from all around the world.

Initially, the most active molecules against the targets had to be identified. Then, all of the biological responses could be analyzed in detail, focusing particularly on species and the organ, tissue or cell lines used as biological material. The affinities or activities reported in second messenger, isolated organ or electrophysiological protocols were also analyzed and correlated.

By setting the pX values at > 7 (affinity < 100 nM for IC50 or Ki values) and focusing on the most popular in vitro protocols (i.e., binding and second messenger for the GPCRs 5-HT1B and 5-HT1D; and binding and electrophysiology for the ion channel 5-HT3), the most active molecules could be retrieved with the help of the Reaxys Medicinal Chemistry Heatmap (Figure 1). The Heatmap visualizes the relationships between compounds and their targets in terms of key parameters, allowing rapid identification of relevant compound–target interactions. The highest pX values were selected for display in the Heatmap.

Analytical method Figure 1 | Elsevier
Figure 1. The Heatmap for inhibitors of all of the 5-HT receptors with pX > 7.0 (affinity < 100 nM)

In the Heatmap, biological affinities or activities are quantified as a pX value and displayed from 1 (low activity) in blue to 15 (high activity) in red. The color of the Heatmap cells represents the maximal pX retrieved for a given compound (row) against a given target (column). The thumbnail provides an overview of the entire Heatmap with a panel highlighting the section of the map currently displayed on the screen. The data density display enables analyses of the dataset, such as an examination of the number of compounds retrieved per target.