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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.

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The development of SAR around substituted N-piperidinyl indole-based nociceptin opioid receptor (NOP) ligands led to the discovery of a novel series of 2-substituted N-piperidinyl indoles that provide both selective NOP full agonists and bifunctional NOP full agonists-µ opioid (MOP) receptor partial agonists. 2-substituted N-piperidinyl indoles have improved potency at the NOP receptor and are NOP full agonists, compared to our previously reported 3-substituted N-piperidinyl indoles that are selective NOP partial agonists. SAR in this series of 2-substituted N-piperidinyl indoles shows that 2-substitution versus 3-substitution on the indole moiety affects their intrinsic activity and opioid receptor selectivity. Molecular docking of these 2-substituted N-piperidinyl indoles in an active-state NOP homology model and MOP receptor structures provides a rationale for the differences observed in the binding, functional profiles and selectivity of 2-substituted versus 3-substituted N-piperidinyl indoles.

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Ozanimod, a sphingosine 1-phosphate (S1P) receptor modulator, binds with high affinity selectively to S1P receptor subtypes 1 (S1P1) and 5 (S1P5), and is approved in multiple countries for treating adults with relapsing forms of multiple sclerosis (MS) or moderately to severely active ulcerative colitis (UC). Other S1P receptor modulators have been approved for the treatment of MS or are in clinical development for MS or UC, but it is unknown whether these compounds bind competitively with each other to S1P1 or S1P5. We developed a competitive radioligand binding assay using tritiated ozanimod and demonstrate full displacement of ozanimod by S1P (endogenous ligand), suggesting that ozanimod binds to the S1P1 and S1P5 orthosteric binding sites. S1P receptor modulators FTY720-p, siponimod, etrasimod, ponesimod, KRP-203-p, and amiselimod-p also completely displacing radiolabeled ozanimod; thus, on a macroscopic level, all bind to the same site. Molecular docking studies support these results and predict the binding of each molecule to the orthosteric site of the receptors, creating similar interactions within S1P1 and S1P5. The absolute free energy perturbation method further validated key proposed binding modes. Functional potency tightly aligned with binding affinities across S1P1 and S1P5 and all compounds elicited S1P1-mediated ß-arrestin recruitment. Since all the S1P modulators included in this study display similar receptor pharmacology and compete for binding at the same site, they can be considered interchangeable with one another. The choice of any one particular agent should therefore be made on the basis of overall therapeutic profile, and patients can be offered the opportunity to switch S1P medications without the potential concern of additive S1P pharmacology.

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The orthosteric binding site of GABA-gated ion channels has been widely explored. Many residues in the binding site of GABA were studied. The interactions due to the binding of GABA into the binding site drive channel activation and determine the potency and efficacy of GABA response. The combined effect of a competitive ligand and GABA on GABA-ρ1 receptors has been poorly studied. Here, we used point mutations, molecular modeling, and electrophysiological studies to explore the role of two hydrophilic residues (Serine 168 and Serine 243) of the GABA-ρ1 receptors in response to the binding of GABA and other studied ligands. Our results suggested that Ser168 residue stabilizes either closed state or open conformation depending on the other determinant interactions of each state. On the other hand, Ser243 residue is predicted to form different inter-subunit interactions with residues in the adjacent subunit at different states of the channel. Our current findings enlighten us to reasonably explain the additive/inhibitive effects of applying a competitive ligand with GABA simultaneously. Understanding the mixed effect of potentiation and inhibition would facilitate the discovery of new drugs to work as a direct GABA's activity modulators with more selectivity at various subunits forming GABA-gated ion channels.

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Schizophrenia was first described by Emil Krapelin in the 19th century as one of the major mental illnesses causing disability worldwide. Since the introduction of chlorpromazine in 1952, strategies aimed at modifying the activity of dopamine receptors have played a major role for the treatment of schizophrenia. The introduction of atypical antipsychotics with clozapine broadened the range of potential targets for the treatment of this psychiatric disease, as they also modify the activity of the serotoninergic receptors. Interestingly, all marketed drugs for schizophrenia bind to the orthosteric binding pocket of the receptor as competitive antagonists or partial agonists. In recent years, a strong effort to develop allosteric modulators as potential therapeutic agents for schizophrenia was made, mainly for the several advantages in their use. In particular, the allosteric binding sites are topographically distinct from the orthosteric pockets, and thus drugs targeting these sites have a higher degree of receptor subunit specificity. Moreover, "pure" allosteric modulators maintain the temporal and spatial fidelity of native orthosteric ligand. Furthermore, allosteric modulators have a "ceiling effect", and their modulatory effect is saturated above certain concentrations. In this review, we summarize the progresses made in the identification of allosteric drugs for dopamine and serotonin receptors, which could lead to a new generation of atypical antipsychotics with a better profile, especially in terms of reduced side effects.

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The γ-aminobutyric acid (GABA) type B receptor (GABAB-R) belongs to class C of the G-protein coupled receptors (GPCRs). Together with the GABAA receptor, the receptor mediates the neurotransmission of GABA, the main inhibitory neurotransmitter in the central nervous system (CNS). In recent decades, the receptor has been extensively studied with the intention being to understand pathophysiological roles, structural mechanisms and develop drugs. The dysfunction of the receptor is linked to a broad variety of disorders, including anxiety, depression, alcohol addiction, memory and cancer. Despite extensive efforts, few compounds are known to target the receptor, and only the agonist baclofen is approved for clinical use. The receptor is a mandatory heterodimer of the GABAB1 and GABAB2 subunits, and each subunit is composed of an extracellular Venus Flytrap domain (VFT) and a transmembrane domain of seven α-helices (7TM domain). In this review, we briefly present the existing knowledge about the receptor structure, activation and compounds targeting the receptor, emphasizing the role of the receptor in previous and future drug design and discovery efforts.

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GABAA receptors (GABAARs) play a crucial role in mediating inhibition in the adult brain. In spite of progress in describing (mainly) the static structures of this receptor, the molecular mechanisms underlying its activation remain unclear. It is known that in the α1ß2γ2L receptors, the mutation of the ß2E155 residue, at the orthosteric binding site, strongly impairs the receptor activation, but the molecular and kinetic mechanisms of this effect remain elusive. Herein, we investigated the impact of the ß2E155C mutation on binding and gating of the α1ß2γ2L receptor. To this end, we combined the macroscopic and single-channel analysis, the use of different agonists [GABA and muscimol (MSC)] and flurazepam (FLU) as a modulator. As expected, the ß2E155C mutation caused a vast right shift of the dose-response (for GABA and MSC) and, additionally, dramatic changes in the time course of current responses, indicative of alterations in gating. Mutated receptors showed reduced maximum open probability and enhanced receptor spontaneous activity. Model simulations for macroscopic currents revealed that the primary effect of the mutation was the downregulation of the preactivation (flipping) rate. Experiments with MSC and FLU further confirmed a reduction in the preactivation rate. Our single-channel analysis revealed the mutation impact mainly on the second component in the shut times distributions. Based on model simulations, this finding further confirms that this mutation affects mostly the preactivation transition, supporting thus the macroscopic data. Altogether, we provide new evidence that the ß2E155 residue is involved in both binding and gating (primarily preactivation).

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The GABAB receptor (GABAB-R) is a heterodimeric class C G protein-coupled receptor comprised of the GABAB1a/b and GABAB2 subunits. The endogenous orthosteric agonist γ-amino-butyric acid (GABA) binds within the extracellular Venus flytrap (VFT) domain of the GABAB1a/b subunit. The receptor is associated with numerous neurological and neuropsychiatric disorders including learning and memory deficits, depression and anxiety, addiction and epilepsy, and is an interesting target for new drug development. Ligand- and structure-based virtual screening (VS) are used to identify hits in preclinical drug discovery. In the present study, we have evaluated classical ligand-based in silico methods, fingerprinting and pharmacophore mapping and structure-based in silico methods, structure-based pharmacophores, docking and scoring, and linear interaction approximation (LIA) for their aptitude to identify orthosteric GABAB-R compounds. Our results show that the limited number of active compounds and their high structural similarity complicate the use of ligand-based methods. However, by combining ligand-based methods with different structure-based methods active compounds were identified in front of DUDE-E decoys and the number of false positives was reduced, indicating that novel orthosteric GABAB-R compounds may be identified by a combination of ligand-based and structure-based in silico methods.

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The diversity seen in the mode of pharmacology and the numerous glutamate receptor subtypes have previously complicated drug development efforts. Nonetheless, recent clinical trials of drug candidates that accommodate the glutamatergic intricate pharmacology have yielded encouraging results. Target engagement is an important requirement for the advancement of central nervous system drug candidates into clinical trial. Positron emission tomography (PET) tracer technology has the unique ability to give the direct insight into the relationship between the level of receptor occupancy and the administered dose, thus establishing a direct link between the level of target exposure and the drug efficacy in human. This review is focused on the advancement of mGlu5 , mGlu 1 , and mGlu 2 receptor-related PET tracer technology: PET tracer development strategies, PET tracer selection in receptor occupancy studies, the scopes and limitations to use PET to measure receptor occupancy for the drug candidates with different pharmacology, and how to use the measurements of receptor occupancy as a translational biomarker for decision-making in an innovative drug development program.

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The serotonin-gated 5-HT3 receptor is a ligand-gated ion channel. Its location at the synapse in the central and peripheral nervous system has rendered it a prime pharmacological target, for example, for antiemetic drugs that bind with high affinity to the neurotransmitter binding site and prevent the opening of the channel. Advances in structural biology techniques have led to a surge of disclosed three-dimensional receptor structures; however, solving ligand-bound high-resolution 5-HT3 receptor structures has not been achieved to date. Ligand binding poses in the orthosteric binding site have been largely predicted from mutagenesis and docking studies. We report the synthesis of a series of photo-cross-linking compounds whose structures are based on the clinically used antiemetic drug granisetron (Kytril). These displaced [3H]granisetron from the orthosteric binding site with low nanomolar affinities and showed specific photo-cross-linking with the human 5-HT3 receptor. Detailed analysis by protein-MS/MS identified a residue (Met-228) near the tip of binding loop C as the covalent modification site.

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Neonicotinoid insecticides interact with the orthosteric sites of nicotinic acetylcholine receptors (nAChRs) formed at the interfaces of (a) two adjacent α subunits and (b) α and non-α subunits. However, little is known of the detailed contributions of these two orthosteric sites to neonicotinoid actions. We therefore applied voltage-clamp electrophysiology to the Dα1/chicken ß2 hybrid nAChR expressed in Xenopus laevis oocytes to explore the agonist actions of imidacloprid and thiacloprid on wild type receptors and following binding site mutations. First, we studied the S221E mutation in loop C of the ACh binding site of the Dα1 subunit. Secondly, we explored the impact of combining this mutation in loop C with others in the loop D-E-G triangle (R57S; E78K; K140T; S221E). The S221E loop C mutation alone reduced the affinity of the neonicotinoids tested, while hardly affecting the concentration-response curve for acetylcholine. Addition of the three R57S; E78K; K140T mutations in the loop D-E-G triangle led to a further reduction in neonicotinoid sensitivity, suggesting that all four binding site loops (C, D, E, G) in the Dα1 subunit, which are located upstream of loop B in the N-terminal, extracellular domain, contribute to the selective actions of neonicotinoid insecticides.

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