Discovery of α7-Nicotinic Receptor Ligands by Virtual Screening of the Chemical Universe Database GDB-13

The chemical universe database GDB-13 enumerates 977 million organic molecules up to 13 atoms of C, N, O, Cl, and S that are virtually possible following simple rules for chemical stability and synthetic feasibility. Analogs of nicotine were identified in GDB-13 using the city-block distance in MQN-space (CBD_MQN) as a similarity measure, combined with a restriction eliminating problematic structural elements. The search was carried out with a Web browser available at www.gdb.unibe.ch. This virtual screening procedure selected 31 504 analogs of nicotine from GDB-13, from which 48 were known nicotinic ligands reported in Chembl. An additional 60 virtual screening hits were purchased and tested for modulation of the acetylcholine signal at the human α7 nAChR expressed in Xenopus oocytes, which led to the identification of three previously unknown inhibitors. These experiments demonstrate for the first time the use of GDB-13 for ligand discovery

Discovery of Potent Positive Allosteric Modulators of the α3β2 Nicotinic Acetylcholine Receptor by a Chemical Space Walk in ChEMBL

While a plethora of ligands are known for the well studied α7 and α4β2 nicotinic acetylcholine receptor (nAChR), only very few ligands address the related α3β2 nAChR expressed in the central nervous system and at the neuromuscular junction. Starting with the public database ChEMBL organized in the chemical space of Molecular Quantum Numbers (MQN, a series of 42 integer value descriptors of molecular structure), a visual survey of nearest neighbors of the α7 nAChR partial agonist <i>N</i>-(3<i>R</i>)-1-azabicyclo­[2.2.2]­oct-3-yl-4-chlorobenzamide (PNU-282,987) pointed to <i>N</i>-(2-halobenzyl)-3-aminoquinuclidines as possible nAChR modulators. This simple “chemical space walk” was performed using a web-browser available at www.gdb.unibe.ch. Electrophysiological recordings revealed that these ligands represent a new and to date most potent class of positive allosteric modulators (PAMs) of the α3β2 nAChR, which also exert significant effects in vivo. The present discovery highlights the value of surveying chemical space neighbors of known drugs within public databases to uncover new pharmacology

Sequence analysis of <i>Alpo</i> CLR homologues.

<p>Anion-selective channels are indicated in yellow, (putative) cation-selective channels in blue. (A) Cladogram displaying the relationship between <i>Alpo</i>, known human CLR subunits and GluCl. (B) Pairwise sequence identity diagram of <i>Alpo</i> and the most closely related known human CLR subunits. The degree of sequence identity is displayed in shades of blue for putative cation-selective channels and in shades of yellow for anion-selective channels. (C) Multiple sequence alignment including sequences of <i>Alpo</i> (blue and yellow), CLRs with known structures (pink) and human CLR subunits with high sequence identity to the identified homologues. The degree of amino acid conservation is displayed in shades of blue, β-strands are indicated in green and the M2 helix in red. Secondary structure elements are retrieved from the m5-HT₃A R crystal structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151183#pone.0151183.ref017" target="_blank">17</a>]. Conserved aromatic residues and loops B, C and D involved in ligand binding are colored in orange, the vicinal disulphide is indicated in fuchsia, the ion selectivity filter is shown in purple and the eponymous Cys-loop in brown.</p

Subunit topology and construct design.

<p>β-strands are indicated in dark green, α-helices in red and eGFP in mint green. The signal sequence is colored blue, the His₈-tag yellow. EC: extracellular, IC: intracellular. (A) Cartoon representation of a single pLGIC subunit, seen parallel to the membrane plane. The arrow indicates the position of eGFP in the <i>Alpo</i>X-eGFP constructs. (B) Construct design of <i>Alpo</i>X-wt and <i>Alpo</i>X-eGFP.</p

List of 33 chemical compounds and their concentration used for ligand screening by TEVC.

<p>List of 33 chemical compounds and their concentration used for ligand screening by TEVC.</p

Melting curves of purified <i>Alpo</i>1-eGFP, <i>Alpo</i>4-eGFP and <i>Alpo</i>6-eGFP.

<p>Melting curves of <i>Alpo</i>1-eGFP, <i>Alpo</i>4-eGFP and <i>Alpo</i>6-eGFP purified in DDM, LMNG-CHAPS and LMNG-glycine respectively, generated by FSEC-TS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151183#pone.0151183.ref035" target="_blank">35</a>]. The deduced melting temperatures are indicated (n = 3).</p

Functional characterization of <i>Alpo</i>5-wt and <i>Alpo</i>6-wt by TEVC.

<p>Top: Currents derived from the activation of <i>Alpo</i>5-wt and <i>Alpo</i>6-wt by increasing concentrations of glycine, GABA or taurine. Bottom: Concentration-activation curves of glycine (blue), GABA (green) and taurine (purple) for <i>Alpo</i>5-wt and <i>Alpo</i>6-wt.</p

Ion selectivity of <i>Alpo</i>5-wt and <i>Alpo</i>6-wt.

<p>Current-voltage curves of <i>Alpo</i>5-wt and <i>Alpo</i>6-wt activated by 10 mM GABA recorded in the presence of extracellular chloride (green) or gluconate ions (red).</p

FSEC-based detergent screen [34].

<p>FSEC profiles of <i>Alpo</i>X-eGFP solubilized by the indicated detergents. The fluorescent signals were approximately equated with each other. * indicates a suitable detergent for the solubilization of the particular protein. A detergent was considered suitable if FSEC resulted in a chromatogram characterized by a high, symmetric peak around 13.5 mL and a lower peak around the void volume (7 mL).</p

Large-scale purification of <i>Alpo</i>1, <i>Alpo</i>4 and <i>Alpo</i>6.

<p>SEC-profiles of purified <i>Alpo</i> CLR homologues and SDS-PAGE analyses of the corresponding oligomeric peak fractions. Left: Purification of Alpo1-eGFP and <i>Alpo</i>1-wt in DDM. Middle: Purification of <i>Alpo</i>4-eGFP and <i>Alpo</i>4-wt in LMNG-CHAPS. Right: Purification of <i>Alpo</i>6-eGFP and <i>Alpo</i>6-wt in the presence of glycine and LMNG.</p