Mark H.P. Verheij

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines

GABAA receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABAA receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABAA receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.

Fragment library screening reveals remarkable similarities between the G protein-coupled receptor histamine H4 and the ion channel serotonin 5-HT3A

Graphical abstract

Identification of novel allosteric nonpeptidergic inhibitors of the human cytomegalovirus-encoded chemokine receptor US28.

Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28.

Small and colorful stones make beautiful mosaics: Fragment-Based Chemogenomics

Smaller stones with a wide variety of colors make a higher resolution mosaic. In much the same way, smaller chemical entities that are structurally diverse are better able to interrogate protein binding sites. This feature article describes the construction of a diverse fragment library and an analysis of the screening of six representative protein targets belonging to three diverse target classes (G protein-coupled receptors ADRB2, H1R, H3R, and H4R, the ligand-gated ion channel 5-HT3R, and the kinase PKA) using chemogenomics approaches. The integration of experimentally determined bioaffinity profiles across related and unrelated protein targets and chemogenomics analysis of fragment binding and protein structure allow the identification of: (i) unexpected similarities and differences in ligand binding properties, and (ii) subtle ligand affinity and selectivity cliffs. With a wealth of fragment screening data being generated in industry and academia, such approaches will contribute to a more detailed structural understanding of ligand-protein interactions.

VUF10166, a novel compound with differing activities at 5-HT₃A and 5-HT₃AB receptors.

The actions of a novel, potent 5-HT3 receptor ligand, [2-chloro-(4-methylpiperazine-1-yl)quinoxaline (VUF10166)], were examined at heterologously expressed human 5-HT3A and 5-HT3AB receptors. VUF10166 displaced [3H]granisetron binding to 5-HT3A receptors expressed in human embryonic kidney cells with high affinity (Ki = 0.04 nM) but was less potent at 5-HT3AB receptors (Ki = 22 nM). Dissociation of [3H]granisetron in the presence of VUF10166 was best fit with a single time constant (t1/2 = 53 min) at 5-HT3A receptors, but with two time constants (t1/2 = 55 and 2.4 min) at 5-HT3AB receptors. Electrophysiological studies in oocytes revealed that VUF10166 inhibited 5-HT-induced responses at 5-HT3A receptors at nanomolar concentrations, but inhibition and recovery were too slow to determine an IC50. At 5-HT3AB receptors, inhibition and recovery were faster, yielding an IC50 of 40 nM. Cysteine substitutions in the complementary (−), but not the principal (+), face of the 5-HT3B subunit produced heteromeric receptors in which the actions of VUF10166 resembled those at homomeric receptors. At 5-HT3A receptors, VUF10166 at higher concentrations also behaved as a partial agonist (EC50 = 5.2 μM; Rmax = 0.24) but did not elicit significant responses at 5-HT3AB receptors at ≤100 μM. Thus, we propose that VUF10166 binds to the common A+A− site of both receptor types and to a second A+B− modulatory site in the heteromeric receptor. The ability of VUF10166 to distinguish between 5-HT3A and 5-HT3AB receptors could help evaluate differences between these receptor types and has potential therapeutic value.

Design, Synthesis, and Structure−Activity Relationships of Highly Potent 5‑HT 3 Receptor Ligands

The 5-HT3 receptor, a pentameric ligand-gated ion channel (pLGIC), is an important therapeutic target. During a recent fragment screen, 6-chloro-N-methyl-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine (1) was identified as a 5-HT3R hit fragment. Here we describe the synthesis and structure–activity relationships (SAR) of a series of (iso)quinoline and quinazoline compounds that were synthesized and screened for 5-HT3R affinity using a [3H]granisetron displacement assay. These studies resulted in the discovery of several high affinity ligands of which compound 22 showed the highest affinity (pKi > 10) for the 5-HT3 receptor. The observed SAR is in agreement with established pharmacophore models for 5-HT3 ligands and is used for ligand–receptor binding mode prediction using homology modeling and in silico docking approaches.

An efficient and information-rich biochemical method design for fragment library screening on ion channels.

Drug discovery requires a simple, rapid, and cost-effective method for the early identification of novel leads and elimination of poor candidates. Here we present an experimental design that fulfils these criteria, using a ligand-gated ion channel expressed in a mammalian cell line, whose function can be probed using a voltage-sensitive dye. The experimental design is novel, as it uses the same screen to identify hit fragments and to characterize them as agonists or antagonists. The results were independently validated using radioligand binding, although the new technique has several advantages over radioligand methods. A number of novel high-affinity ligands were found. The method is broadly applicable to a wide range of receptor types including ligand-gated ion channels (LGICs), voltage-gated ion channels (VGICs), and G protein-coupled receptors (GPCRs), all of which are important drug targets.

4,6-Diphenylpyridines as Promising Novel Anti-Influenza Agents Targeting the PA–PB1 Protein–Protein Interaction: Structure–Activity Relationships Exploration with the Aid of Molecular Modeling

Influenza is an infectious disease that represents an important public health burden, with high impact on the global morbidity, mortality, and economy. The poor protection and the need of annual updating of the anti-influenza vaccine, added to the rapid emergence of viral strains resistant to current therapy make the need for antiviral drugs with novel mechanisms of action compelling. In this regard, the viral RNA polymerase is an attractive target that allows the design of selective compounds with reduced risk of resistance. In previous studies we showed that the inhibition of the polymerase acidic protein-basic protein 1 (PA-PB1) interaction is a promising strategy for the development of anti-influenza agents. Starting from the previously identified 3-cyano-4,6-diphenyl-pyridines, we chemically modified this scaffold and explored its structure-activity relationships. Noncytotoxic compounds with both the ability of disrupting the PA-PB1 interaction and antiviral activity were identified, and their mechanism of target binding was clarified with molecular modeling simulations.

Structure-activity relationships of quinoxaline-based 5-HT3A and 5-HT3AB receptor-selective ligands.

Until recently, discriminating between homomeric 5‐HT3A and heteromeric 5‐HT3AB receptors was only possible with ligands that bind in the receptor pore. This study describes the first series of ligands that can discriminate between these receptor types at the level of the orthosteric binding site. During a recent fragment screen, 2‐chloro‐3‐(4‐methylpiperazin‐1‐yl)quinoxaline (VUF10166) was identified as a ligand that displays an 83‐fold difference in [3H]granisetron binding affinity between 5‐HT3A and 5‐HT3AB receptors. Fragment hit exploration, initiated from VUF10166 and 3‐(4‐methylpiperazin‐1‐yl)quinoxalin‐2‐ol, resulted in a series of compounds with higher affinity at either 5‐HT3A or 5‐HT3AB receptors. These ligands reveal that a single atom is sufficient to change the selectivity profile of a compound. At the extremes of the new compounds were 2‐amino‐3‐(4‐methylpiperazin‐1‐yl)quinoxaline, which showed 11‐fold selectivity for the 5‐HT3A receptor, and 2‐(4‐methylpiperazin‐1‐yl)quinoxaline, which showed an 8.3‐fold selectivity for the 5‐HT3AB receptor. These compounds represent novel molecular tools for studying 5‐HT3 receptor subtypes and could help elucidate their physiological roles.

The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT3A and 5-HT3AB receptors.

VUF10166 (2-chloro-3-(4-methyl piperazin-1-yl)quinoxaline) is a ligand that binds with high affinity to 5-HT3 receptors. Here we synthesise [3H]VUF10166 and characterise its binding properties at 5-HT3A and 5-HT3AB receptors. At 5-HT3A receptors [3H]VUF10166 displayed saturable binding with a Kd of 0.18 nM. Kinetic measurements gave monophasic association (6.25 × 107 M−1 min−1) and dissociation (0.01 min−1) rates that yielded a similar Kd value (0.16 nM). At 5-HT3AB receptors two association (6.15 × 10−7, 7.23 M−1 min−1) and dissociation (0.024, 0.162 min−1) rates were seen, yielding Kd values (0.38 nM and 22 nM) that were consistent with values obtained in saturation (Kd = 0.74 nM) and competition (Ki = 37 nM) binding experiments respectively. At both receptor types, specific binding was inhibited by classical 5-HT3 receptor-selective orthosteric ligands (5-HT, allosetron, d-tubocurarine, granisetron, mCPBG, MDL72222, quipazine), but not by non-competitive antagonists (bilobalide, ginkgolide B, picrotoxin) or competitive ligands of other Cys-loop receptors (ACh, bicuculline, glycine, gabazine). To explore VUF10166 ligand–receptor interactions we used in silico modelling and docking, and tested the predictions using site directed mutagenesis. The data suggest that VUF10166 adopts a similar orientation to 5-HT3 receptor agonists bound in AChBP (varenicline) and 5HTBP (5-HT) crystal structures.