Jacqueline E. van Muijlwijk-Koezen

Surface Plasmon Resonance Biosensor Based Fragment Screening Using Acetylcholine Binding Protein Identifies Ligand Efficiency Hot Spots (LE Hot Spots) by Deconstruction of Nicotinic Acetylcholine Receptor α7 Ligands

The soluble acetylcholine binding protein (AChBP) is a homologue of the ligand-binding domain of the nicotinic acetylcholine receptors (nAChR). To guide future fragment-screening using surface plasmon resonance (SPR) biosensor technology as a label-free, direct binding, biophysical screening assay, a focused fragment library was generated based on deconstruction of a set of α7 nAChR selective quinuclidine containing ligands with nanomolar affinities. The interaction characteristics of the fragments and the parent compounds with AChBP were evaluated using an SPR biosensor assay. The data obtained from this direct binding assay correlated well with data from the reference radioligand displacement assay. Ligand efficiencies for different (structural) groups of fragments in the library were correlated to binding with distinct regions of the binding pocket, thereby identifying ligand efficiency hot spots (LE hot spots). These hot spots can be used to identity the most promising hit fragments in a large scale fragment library screen.

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

Graphical abstract

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.

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.

The Adenosine A3 Receptor and its Ligands

Publisher Summary The scientific history of adenosine and its receptors is described by the biological actions of adenine nucleosides and nucleotides in the cardiovascular system. Two types of receptors were proposed P 1 and P 2 ,, which were distinguished on basis of their preference for binding to either adenosine or adenine nucleotides. Subsequently, the P I receptor was divided into A l and A 2 adenosine receptors in accordance with their ability to inhibit or to stimulate C AMP production. A further subdivision of the adenosine A 2 receptor into A 2 A and A 2 B was proposed based on high and low affinity towards 2-(4-methoxyphenylamino) adenosine. The cloning of two different adenosine A 2 receptors confirmed the existence of these subtypes. The pharmacological classification, selective ligands, characterization of the subtypes by cloning, tissue distribution and therapeutic potential of the A 1 and A 2 A receptors has been also reviewed. It explores that the youngest member of the adenosine receptor family, the A 3 receptor, was discovered when a sequence from a rat testis cDNA library was characterized as an adenosine receptor subtype and called A 3. The receptor gene has now also been cloned and characterized in several species: Human, sheep, dog, and rabbit. Therefore, it also describes the cloned and pharmacologically characterized adenosine A 3 receptor remains the youngest member of the adenosine receptor family.

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.

Exploration of the molecular architecture of the orthosteric binding site in the α4β2 nicotinic acetylcholine receptor with analogs of 3-(dimethylamino)butyl dimethylcarbamate (DMABC) and 1-(pyridin-3-yl)-1,4-diazepane.

X-ray crystal structures of acetylcholine binding proteins (AChBPs) have revealed two different possible extensions to the classical ligand binding pocket known to accommodate various nicotinic agonists. One of the pockets is limited in size while the other is of considerable dimensions and protrudes along the interfacial cleft between subunits. To probe these putative extensions in functional nicotinic acetylcholine receptors (nAChRs), elongated analogs of 3-(dimethylamino)butyl dimethylcarbamate (DMABC) and 1-(pyridine-3-yl)-1,4-diazepane were prepared and characterized pharmacologically at neuronal heteromeric nAChRs. Although the new analogs, relative to parent compounds, displayed lower binding affinities, functional characterization of selected compounds revealed that they had retained partial α4β2 nAChR agonist activity. The structure-activity relationship data did not indicate an upper limit to the size of substituents as would have been expected if the ligand was bound in the smaller pocket. The data were better in agreement with a binding mode in which substituents protrude along the interfacial cleft of the receptor. This was further supported by docking into a homology model of the α4-β2 nAChR interface and by surface plasmon resonance biosensor analysis of binding of the compounds to acetylcholine-binding proteins, where they exhibit preference for Lymnaea stagnalis ACh binding protein (Ls-AChBP) over the Aplysia california ACh binding protein (Ac-AChBP). These results suggest new opportunities for expanding chemical space in the development of partial agonist and may be of interest in relation to development of novel smoking cessation aids.

Structure-based design, synthesis and structure-activity relationships of dibenzosuberyl- and benzoate-substituted tropines as ligands for acetylcholine-binding protein

Using structure-based optimization procedures on in silico hits, dibenzosuberyl- and benzoate substituted tropines were designed as ligands for acetylcholine-binding protein (AChBP). This protein is a homolog to the ligand binding domain of the nicotinic acetylcholine receptor (nAChR). Distinct SAR is observed between two AChBP species variants and between the α7 and α4β2 nAChR subtype. The AChBP species differences are indicative of a difference in accessibility of a ligand-inducible subpocket. Hereby, we have identified a region that can be scrutinized to achieve selectivity for nicotinic receptor subtypes.

Online parallel fragment screening and rapid hit exploration for nicotinic acetylcholine receptors

An online bioaffinity analysis system was used to screen our in-house fragment library on two related proteins, Ls- and Ac-AChBP, model proteins for nAChRs, in particular the α7 subtype. An efficient protocol for medium throughput fragment screening, hit validation and affinity ranking after single concentration injections was developed. The screening of the fragment library and the good correlation between online estimated pKi values (derived from a single injection) and pKi values measured with a radioligand binding assay (RBA, full range concentration curve) have proven the value of the online fluorescence enhancement assay in FBDD. The online bioaffinity system was also used for rapid hit exploration using single point injections of combinatorial libraries at 96-well format. This led to the discovery of an optimized hit with micromolar affinity towards the α7 nAChR.

Medicinal Chemistry of the Human Adenosine A3 Receptor.

Partial agonists and antagonists were synthesized and evaluated biologically for extended pharmacologic characterization of the human adenosine A3 receptor. The affinities of all compounds were determined at the human adenosine A3 receptor stably transfected in HEK 293 cells and in rat brain membranes for the adenosine A1 and A2A receptors. The partial agonists were also evaluated for their ability to stimulate [35S]GTPγ[S] binding in Chinese hamster ovary cells expressing the human adenosine A3 receptor to determine their intrinsic activities. 5′‐(Alkylthio)‐substituted analogs of N6‐(3‐iodobenzyl)adenosine were synthesized in 47–60% overall yields. The compounds proved to be potent and selective partial agonists for the A3 receptor, displaying affinities in the nanomolar range. N6‐(3‐iodobenzyl)adenosine (2), 5′‐deoxy‐N6‐(3‐iodobenzyl)‐5′‐methyl‐thioadenosine (4), and 5′‐deoxy‐N6‐(3‐iodobenzyl)‐5′‐ethylthioadenosine (5) had highest affinities for the A3 receptor with Ki values ranging from 9–28 nM. Compound 6 (5′‐deoxy‐N6‐(3‐iodobenzyl)‐5′‐n‐propyl‐thioadenosine) had the highest (over 200‐fold) A3 receptor selectivity. Of all partial agonists, 2 and 4 had the highest intrinsic activities. Subsequently, a series of 3‐(2‐pyridinyl)isoquinoline derivatives was synthesized as potential antagonists for the human adenosine A3 receptor. A structure‐activity relationship was performed at the 1‐position of this series. This analysis indicated that a phenyl group, when coupled by a spacer allowing conjugation on position 1 of the isoquinoline ring, increased the adenosine A3 receptor affinity. Of all spacers tested, a carboxamide proved to be optimal. N‐[2‐(2‐pyridinyl)isoquinolin‐4‐yl]‐benzamide (9) had an affinity of 200 nM at the adenosine A3 receptor. Furthermore, the effects of mono‐ and disubstitution of the benzamide ring of 9 were investigated. This led to the A3‐selective compound 4‐methoxy‐N‐[2‐(2‐pyridinyl)quinazolin‐4‐yl]‐benzamide (18) with an affinity of 17 nM at the human adenosine A3 receptor. These partial agonists and antagonists may be useful tools in the pharmacologic characterization and the investigation of the physiologic function of this receptor. Drug Dev. Res. 45:182–189, 1998.