The Role of Orthosteric Building Blocks of Bitopic Ligands for Muscarinic M1 Receptors

Abstract

The muscarinic M 1 acetylcholine receptor is an important drug target for the treatment of various neurological disorders. Designing M 1 receptor-selective drugs has proven challenging, mainly due to the high conservation of the acetylcholine binding site among muscarinic receptor subtypes. Therefore, less conserved and topographically distinct allosteric binding sites have been explored to increase M 1 receptor selectivity. In this line, bitopic ligands, which target orthosteric and allosteric binding sites simultaneously, may provide a promising strategy. Here, we explore the allosteric, M 1 -selective BQCAd scaffold derived from BQCA as a starting point for the design, synthesis, and pharmacological evaluation of a series of novel bitopic ligands in which the orthosteric moieties and linker lengths are systematically varied. Since βarrestin recruitment seems to be favorable to therapeutic implication, all the compounds were investigated by G protein and β-arrestin assays. Some bitopic ligands are partial to full agonists for G protein activation, some activate β-arrestin recruitment, and the degree of β-arrestin recruitment varies according to the respective modification. The allosteric BQCAd scaffold controls the positioning of the orthosteric ammonium group of all ligands, suggesting that this interaction is essential for stimulating G protein activation. However, β-arrestin recruitment is not affected. The novel set of bitopic ligands may constitute a toolbox to study the requirements of β-arrestin recruitment during ligand design for therapeutic usage.

Mechanistic Understanding from Molecular Dynamics in Pharmaceutical Research 2: Lipid Membrane in Drug Design

“…Due to the highly conserved structure of orthosteric pockets, e.g., among GPCRs, it is difficult to find a highly selective ligand based on this criterion alone; an inclusion of the allosteric site into the computational drug design approach allows for the necessary targeting specificity. The use of the allosteric site in addition to the orthosteric pocket in drug design has been referred to as the "exosite model" and "the design of bitopic ligands/drugs" by two separate research communities [938,939]. Modeling methods, including MD simulations, are one of the tools used in the design of bitopic drugs [900,940,941]; however, in these studies lipids are only considered as a passive component and their complete role in substrate selection is not fully elucidated.…”

Design, Synthesis, and Biological Evaluation of 4,4’-Difluorobenzhydrol Carbamates as Selective M1 Antagonists

“…To prioritize among the docked compounds, a distance filter has been used, dropping all poses whose cationic amine did not come within a distance of 5.5 Å to the Asp105 3.32 carboxyl oxygens. Since the chargecharge interaction between Asp105 3.32 and a ligand's cationic head is not restricted to a distinct spatial point, the ammonium group's position has some leeway [10]. The distance of tiotropium's positively charged amine to the Asp105 3.32 carboxyl oxygens is slightly below 5 Å, and for other known ligands, the distance is predicted to be in a similar range [18,21], hence a distance constraint of 5.5 Å was assumed to be reasonable.…”

“…For this purpose, CHO-hM 1 cells were harvested from culture flasks by trypsinization and seeded into 96-well microculture plates (Corning ® , Corning, NY, USA) in densities of 4000 cells/well (100 µL/well). After a 24 h preincubation, cells were exposed in triplicates for each concentration level to dilutions of the test compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) in complete culture medium (100 µL/well) for 72 h. At the end of the exposure period, the compound solutions were replaced with 100 µL of non-supplemented RPMI 1640 medium and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT reagent in PBS, 5 mg/mL) mixed in a 6:1 ratio. After incubation for 4 h, the medium was removed, and the formazan product was solved in DMSO (100 µL/well).…”

“…Inhibition constants (K i ) were determined by means of a competitive radioligand binding assay using 50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM EDTA, pH 7.4 as assay buffer as described previously [42]. 5 µL of test compound (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12) in DMSO, 50 µL of [ 3 H]NMS in assay buffer and 445 µL of membrane suspension in assay buffer were incubated for 90 min at 23 • C in PP tubes. Maximum binding was measured by using 5 µL DMSO, and nonspecific binding was measured by using 5 µL of 1 µM scopolamine in DMSO.…”

“…Instead, a range of side effect-plagued pan-muscarinic antagonists and inverse agonists is used in clinical practice, such as the antiemetic agent scopolamine, the bronchodilator tiotropium, or benztropine which is used to treat symptoms of Parkinson's disease (Figure 1). Thus, current research is increasingly devoted towards the discovery of more selective ligands targeting an allosteric site exhibiting less sequence homology or so-called bitopic ligands, simultaneously targeting the orthosteric and an allosteric site [9,10]. [6,7], a difficult task to achieve, but non-selective compounds suffer from dose-limiting adverse effects [8].…”