Apo2ph4: A Versatile Workflow for the Generation of Receptor-based Pharmacophore Models for Virtual Screening.

Pharmacophore models are widely used as efficient virtual screening (VS) filters for the target-directed enrichment of large compound libraries. However, the generation of pharmacophore models that have the power to discriminate between active and inactive molecules traditionally requires structural information about ligand-target complexes or at the very least knowledge of one active ligand. The fact that the discovery of the first known active ligand of a newly investigated target represents a major hurdle at the beginning of every drug discovery project underscores the need for methods that are able to derive high-quality pharmacophore models even without the prior knowledge of any active ligand structures. In this work, we introduce a novel workflow, called apo2ph4, that enables the rapid derivation of pharmacophore models solely from the three-dimensional structure of the target receptor. The utility of this workflow is demonstrated retrospectively for the generation of a pharmacophore model for the M2 muscarinic acetylcholine receptor. Furthermore, in order to show the general applicability of apo2ph4, the workflow was employed for all 15 targets of the recently published LIT-PCBA dataset. Pharmacophore-based VS runs using the apo2ph4-derived models achieved a significant enrichment of actives for 13 targets. In the last presented example, a pharmacophore model derived from the etomidate site of the α1ß2γ2 GABAA receptor was used in VS campaigns. Subsequent in vitro testing of selected hits revealed that 19 out of 20 (95%) tested compounds were able to significantly enhance GABA currents, which impressively demonstrates the applicability of apo2ph4 for real-world drug design projects.

Synthesis, Biological Evaluation, and Docking Studies of Antagonistic Hydroxylated Arecaidine Esters Targeting mAChRs.

The muscarinic acetylcholine receptor family is a highly sought-after target in drug and molecular imaging discovery efforts aimed at neurological disorders. Hampered by the structural similarity of the five subtypes' orthosteric binding pockets, these efforts largely failed to deliver subtype-selective ligands. Building on our recent successes with arecaidine-derived ligands targeting M1, herein we report the synthesis of a related series of 11 hydroxylated arecaidine esters. Their physicochemical property profiles, expressed in terms of their computationally calculated CNS MPO scores and HPLC-logD values, point towards blood-brain barrier permeability. By means of a competitive radioligand binding assay, the binding affinity values towards each of the individual human mAChR subtypes hM1-hM5 were determined. The most promising compound of this series 17b was shown to have a binding constant towards hM1 in the single-digit nanomolar region (5.5 nM). Similar to our previously reported arecaidine-derived esters, the entire series was shown to act as hM1R antagonists in a calcium flux assay. Overall, this study greatly expanded our understanding of this recurring scaffolds' structure-activity relationship and will guide the development towards highly selective mAChRs ligands.

Design, synthesis and preclinical evaluation of muscarine receptor antagonists via a scaffold-hopping approach.

Our research group recently identified a rearrangement product of pirenzepine as starting point for a comprehensive rational drug design approach towards orthosteric muscarinic acetylcholine receptor ligands. Chemical reduction and bioscaffold hop lead to the development of sixteen promising compounds featuring either a benzimidazole or carbamate moiety, all exhibiting comparable pharmacophoric characteristics. The synthesized compounds were characterized by NMR, HR-MS, and RP-HPLC techniques. Subsequent evaluation encompassed binding affinity assessment on CHO-hM1-5 cells, mode of action determination, and analysis of physico-chemical parameters. The CNS MPO score indicated favorable drug-like attributes and potential CNS activity for the antagonistic ligands. The most promising compounds displayed Ki-values within a desirable low nanomolar range, and their structural features allow for potential carbon-11 radiolabeling. Our optimization efforts resulted in compounds with a remarkable 138-fold increase in binding affinity compared to the previously mentioned rearrangement product towards human M5, suggesting their prospective utility in positron emission tomography applications.

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

Due to their important role in mediating a broad range of physiological functions, muscarinic acetylcholine receptors (mAChRs) have been a promising target for therapeutic and diagnostic applications alike; however, the list of truly subtype-selective ligands is scarce. Within this work, we have identified a series of twelve 4,4'-difluorobenzhydrol carbamates through a rigorous docking campaign leveraging commercially available amine databases. After synthesis, these compounds have been evaluated for their physico-chemical property profiles, including characteristics such as HPLC-logD, tPSA, logBB, and logPS. For all the synthesized carbamates, these characteristics indicate the potential for BBB permeation. In competitive radioligand binding experiments using Chinese hamster ovary cell membranes expressing the individual human mAChR subtype hM1-hM5, the most promising compound 2 displayed a high binding affinitiy towards hM1R (1.2 nM) while exhibiting modest-to-excellent selectivity versus the hM2-5R (4-189-fold). All 12 compounds were shown to act in an antagonistic fashion towards hM1R using a dose-dependent calcium mobilization assay. The structural eligibility for radiolabeling and their pharmacological and physico-chemical property profiles render compounds 2, 5, and 7 promising candidates for future position emission tomography (PET) tracer development.