Drug Design for CNS Diseases: Polypharmacological Profiling of Compounds Using Cheminformatic, 3D-QSAR and Virtual Screening Methodologies.

HIGHLIGHTS Many CNS targets are being explored for multi-target drug designNew databases and cheminformatic methods enable prediction of primary pharmaceutical target and off-targets of compoundsQSAR, virtual screening and docking methods increase the potential of rational drug design The diverse cerebral mechanisms implicated in Central Nervous System (CNS) diseases together with the heterogeneous and overlapping nature of phenotypes indicated that multitarget strategies may be appropriate for the improved treatment of complex brain diseases. Understanding how the neurotransmitter systems interact is also important in optimizing therapeutic strategies. Pharmacological intervention on one target will often influence another one, such as the well-established serotonin-dopamine interaction or the dopamine-glutamate interaction. It is now accepted that drug action can involve plural targets and that polypharmacological interaction with multiple targets, to address disease in more subtle and effective ways, is a key concept for development of novel drug candidates against complex CNS diseases. A multi-target therapeutic strategy for Alzheimer's disease resulted in the development of very effective Multi-Target Designed Ligands (MTDL) that act on both the cholinergic and monoaminergic systems, and also retard the progression of neurodegeneration by inhibiting amyloid aggregation. Many compounds already in databases have been investigated as ligands for multiple targets in drug-discovery programs. A probabilistic method, the Parzen-Rosenblatt Window approach, was used to build a "predictor" model using data collected from the ChEMBL database. The model can be used to predict both the primary pharmaceutical target and off-targets of a compound based on its structure. Several multi-target ligands were selected for further study, as compounds with possible additional beneficial pharmacological activities. Based on all these findings, it is concluded that multipotent ligands targeting AChE/MAO-A/MAO-B and also D1-R/D2-R/5-HT2A -R/H3-R are promising novel drug candidates with improved efficacy and beneficial neuroleptic and procognitive activities in treatment of Alzheimer's and related neurodegenerative diseases. Structural information for drug targets permits docking and virtual screening and exploration of the molecular determinants of binding, hence facilitating the design of multi-targeted drugs. The crystal structures and models of enzymes of the monoaminergic and cholinergic systems have been used to investigate the structural origins of target selectivity and to identify molecular determinants, in order to design MTDLs

Development of novel ligands influencing neurotransmission in the central nervous system

The development of novel drugs targeting GPCRs is of particular interest since modulation of subfamilies of this receptor class highly influences neurotransmission in the central nervous system. This study has focused on the development of ligands for the dopamine D3 receptor. The receptor belongs to the dopamine D2-like family among the biogenic amine binding GPCRs. The dopamine D3 receptor is involved in neurological and neuropsychiatric disorders such as Parkinson’s disease, schizophrenia and drug addiction. Due to its close structural similarity to the dopamine D2 receptor subtype, it is still a challenge to identify and further optimize new leads. Therefore an in vitro screening assay, which also allows elucidating comprehensive structure-affinity relationships, is required. In this investigation the implementation and evaluation of radioligand binding assays for human dopamine D2S and dopamine D3 receptors and for the related aminergic human histamine H1 receptor stably expressed in Chinese hamster ovary (CHO) cells has been performed. Saturation binding experiments with [³H]spiperone at dopamine D2S and D3 receptors and with [³H]mepyramine at histamine H1 receptors were carried out. The determined equilibrium dissociation constant of radioligands (Kd) and the total number of specific binding sites (Bmax) of the receptor membrane preparations were in good agreement with reference data. Inhibition constants (Ki) of reference ligands obtained in radioligand competition binding experiments at dopamine hD2S, hD3 and histamine H1 receptors validated the reliability and reproducibility of the assay. In order to discriminate agonists from antagonists, a GTP shift assay has been investigated for dopamine D2S and D3 receptors. In competition binding studies at dopamine D2S receptors the high- and low affinity state in the absence of the GTP analogue Gpp(NH)p has been recognized for the agonists pramipexole and the seleno analogue 54. In the presence of Gpp(NH)p a decrease in affinity, referred to as “GTP shift”, has been revealed for agonists at dopamine D2S and D3 receptors. An effect of Gpp(NH)p on dopamine D2S receptor binding has not been observed for the antagonists ST 198 and BP 897, while a reverse “GTP shift” has been noticed at the dopamine D3 receptor. For the development of novel ligands with high affinity and selectivity for dopamine D3 receptors, investigation in refined structure-affinity relationships (SAR) of analogues of the lead BP 897 has been performed. Replacement of the naphthalen-2-carboxamide of BP 897 by aryl amide residues (1 - 4) had a clear influence on affinity binding and selectivity for dopamine D3 receptors. Introduction of the benzo[b]thiophen-2-carboxamide (1) has markedly improved binding with subnanomolar affinity and enhanced selectivity for dopamine D3 receptors. Exchanging the aryl substituted basic alkanamine residue of 1 by a 1,2,3,4-tetrahydroisoquinoline moiety (6) emphasized the benefit of the 4-(2-methoxyphenyl) piperazine residue of BP 897 regarding dopamine D2 and D3 receptor affinities. The change of particular elements of BP 897 and the rearrangement of the amide functionality resulted in inverse amide compounds with new chemical properties. Moderate affinity binding data, as obtained for the isoindol-1-carbonyl compound 11, suggest that inverse amides provide a worthwhile new lead structure with a novel structural scaffold. A hybrid approach combining privileged scaffolds of histamine H1 receptor antagonists and fragments of dopamine D3 receptor-preferring ligands, related to BP 897and analogues has been investigated. Various benzhydrylpiperazine derivatives and related structures have shown moderate to high affinities for dopamine D3 receptors with the impressive enhancement of the cinnamide substituted bamipine-related hybrid 39, exhibiting the highest affinity and selectivity for dopamine D3 receptors. Improved affinity profiles of structural modified histamine H1 receptor antagonists for dopamine D2 and D3 receptors and a refined SAR has been achieved. A SAR of derivatives of the dopamine agonist pramipexole and the related etrabamine has been studied. The propargyl substituted etrabamine derivative 61 demonstrated highest affinity and selectivity. The ligand attracts attention since neuroprotective properties have been reported for the propargyl functionality. Further development resulted in the most promising compound 64, a cinnamide derivative with 4-fluoro substitution on the phenyl ring. Subnanomolar affinity and remarkable selectivity for dopamine D3 receptors has aroused particular interest in this ligand due to its development potential as a radioligand for PET studies. Radioligand binding studies in combination with virtual screening and different classification techniques of chemoinformatic methods resulted in further elucidation of SAR. New leads with novel chemical scaffolds have been found in the bicycle[2.2.1]heptane derivative 95 and the benzhydrylidene substituted pyrrolidindione 112 and can be further optimized by chemical modifications. The outcome of the studies provides the development of various novel high affine and dopamine D3 receptor selective ligands. Modifications of lead structures or application of chemoinformatic tools in combination with radioligand competition binding assays have resulted in new leads with different chemical scaffolds. Furthermore, a comprehensive insight into structure-affinity relationships of ligands at dopamine D3 receptors has been revealed. This refined SAR is valuable to develop more affine and selective drug candidates with a designed pharmacological receptor profile.Das Ziel der Arbeit war die Entwicklung von neuen Liganden zur Beeinflussung der Neurotransmission im zentralen Nervensystem. Der Fokus lag auf dem Dopamin-D3-Rezeptor, der eine wichtige Rolle bei Morbus Parkinson, Schizophrenie und Drogenmissbrauch spielt. Aufgrund seiner Strukturähnlichkeit zum Dopamin-D2-Rezeptor ist es eine Herausforderung, neue, selektive Leitstrukturen für den Dopamin-D3-Rezeptor zu identifizieren bzw. zu optimieren. Ein in vitro Testsystem ist hierfür erforderlich und ermöglicht das Aufstellen von Struktur-Wirkungsbeziehungen (SAR) und ein rationales Wirkstoffdesign. Die Arbeit umfasste die Etablierung von Radioliganden Bindungsassays an Dopamin-D2S- und -D3-Rezeptoren, sowie am verwandten aminergen Histamin-H1-Rezeptor, die stabil in Zelllinien von Ovarien des Chinesischen Hamsters exprimiert wurden. Sättigungsstudien wurden mit [³H]Spiperon am Dopamin-D2S- und D3-Rezeptor und mit [³H]Mepyramin am Histamin-H1-Rezeptor durchgeführt. Die ermittelten Dissoziationskonstanten (Kd) und maximale Zahl der Bindungsstellen (Bmax) stimmten mit den Literaturwerten überein. Die in Verdrängungsstudien bestimmten Inhibitionskonstanten (Ki) von Referenzsubstanzen am Dopamin-D2S- und -D3-Rezeptor sowie am Histamin-H1-Rezeptor bestätigten die Zuverlässigkeit und Reproduzierbarkeit der Bindungsassays. Zur Unterscheidung der Agonisten von Antagonisten wurden „GTP-Shift“ Assays am Dopamin-D2S- und -D3-Rezeptor angewandt. Für Pramipexol und das Selenanaloga 54 wurden zwei Bindungszustände mit unterschiedlichen Affinitäten (ein so genannter „high- und low affinity state“) am Dopamin-D2S-Rezeptor in Abwesenheit von Gpp(NH)p beobachtet. Eine Affinitätsabnahme („GTP-Shift“) in Anwesenheit von Gpp(NH)p zeigte sich für die Agonisten am Dopamin-D2S- und -D3-Rezeptor. Dieser Einfluss des Gpp(NH)p konnte nicht für den Antagonisten ST 198 und den partiellen Agonisten BP 897 gezeigt werden. Für diese Verbindung wurde ein inverser „GTP-Shift“, also eine Affinitätsverbesserung am Dopamin-D3-Rezeptor beobachtet. Um neue Liganden mit hoher Affinität und Selektivität für den Dopamin-D3-Rezeptor zu entwickeln, wurden ausführliche SAR verschiedener Derivate der Leitstruktur BP 897 und ST 198 erstellt. Der Austausch des Naphthalen-2-carboxamid-Rests von BP 897 durch verschiedene Arylamid-Strukturen (1 – 4) zeigte deren deutlichen Einfluss auf die Dopamin-D3-Rezeptorbindungsaffinität und -selektivität. Die Einführung eines Benzo[b]thiophen-2-carboxamid-Rests führte in Verbindung 1 zu herausragender subnanomolarer Affinität am Dopamin-D3-Rezeptor sowie zu deutlich erhöhter Selektivität im Vergleich zu BP 897. Die Variation des lipophilen basischen Amin-Restes von 1 ergab das 1,2,3,4-Tetrahydroisochinolin-Derivat 6. Verdrängungsstudien konnten den Vorteil des 4-(2-Methoxyphenyl)piperazine-Substituenten von BP 897 bezüglich der Affinitäten am Dopamin-D2S- und -D3-Rezeptor deutlich zeigen. Modifikationen einzelner Elemente von BP 897 und ST 198 und die veränderte Integration der Amid-Funktion in dem lipophilen Aryl-Rest führten zur Substanzklasse der inversen Amide mit neuen chemischen Eigenschaften. Moderate Bindungsaffinitäten, wie für das Isoindol-1-carbonyl-Derivat 11 gezeigt, legen nahe, dass inverse Amide eine lohnenswerte neue Leitstruktur mit andersartigem strukturellem Gerüst darstellen. In einer Hybrid-Strategie wurden Strukturelemente von Histamin-H1-Rezeptorantagonisten mit Substrukturen von Liganden mit ausgeprägter Dopamin-D3-Rezeptorpräferenz kombiniert. Daraus resultierten Benzhydrylpiperazin-Derivative und verwandte Substanzen mit moderater bis hoher Affinität am Dopamin-D3-Rezeptor. Besonders hervorzuheben ist das Zimtsäureamid substituierte und zum Bamipin verwandte Hybrid 39, welches die besten Ergebnisse in dieser Serie hinsichtlich Affinität und Selektivität am Dopamin-D3-Rezeptor erbrachte. Verbesserte pharmakologische Profile der strukturell modifizierten Histamine-H1-Rezeptorantagonisten am Dopamin-D2S- und -D3-Rezeptor und eine differenzierte SAR wurden erreicht. Für Derivate des Dopaminrezeptoragonisten Pramipexol und des strukturähnlichen Etrabamin wurden SAR ausgearbeitet. Das Propargyl substituierte Etrabamin-Derivat 61 zeigte herausragende Dopamin-D3-Rezeptoraffinität und -selektivität. Der Ligand ist von Interesse, da für den Propargyl-Rest neuroprotektive Eigenschaften berichtet wurden. Die Weiterentwicklung führte zur Verbindung 64, einem Zimtsäureamid-Derivat mit 4-Fluor-Substitution am Phenylring. Subnanomolare Affinität und hohe Selektivität am Dopamin-D3-Rezeptor prädestinieren 64 zur Anwendung als potentiellen PET-Radioliganden. Radioliganden Bindungsstudien wurden auf die Ergebnisse von virtuellen Screeningstudien angewandt. Sie führten zur Identifizierung neuer Leitstrukturen und zum weiteren Verständnis der SAR. Als neue Leitstrukturen mit verschiedenartigen chemischen Gerüsten wurden unter anderem das Bicyclo[2.2.1]heptan-Derivat 95 und der Benzhydryliden substituierte Pyrrolidindion Ligand 112 gefunden. Diese können nun zur weiteren Optimierung chemisch modifiziert werden. Die in dieser Arbeit durchgeführten Radioliganden Bindungsstudien führten zur Identifizierung, Entwicklung und Optimierung von hoch affinen und selektiven Dopamin-D3-Rezeptor Liganden. Des Weiteren ermöglichten die Ergebnisse eine ausführliche Vertiefung der SAR. Die kombinierte Strategie von chemoinformatischen Methoden und Radioliganden Bindungsstudien hat das Finden neuer Leitstrukturen als potentielle Arzneistoffe erlaubt. Die Resultate ermöglichen in der Zukunft ein gezieltes Liganden-Design mit einem gerichteten pharmakologischen Rezeptorprofil

The significance of halogen bonding in ligand-receptor interactions : the lesson learned from molecular dynamic simulations of the D4D_{4} receptor

Recently, a computational approach combining a structure–activity relationship library containing pairs of halogenated ligands and their corresponding unsubstituted ligands (called XSAR) with QM-based molecular docking and binding free energy calculations was developed and used to search for amino acids frequently targeted by halogen bonding, also known as XB hot spots. However, the analysis of ligand–receptor complexes with halogen bonds obtained by molecular docking provides a limited ability to study the role and significance of halogen bonding in biological systems. Thus, a set of molecular dynamics simulations for the dopamine D4 receptor, recently crystallized with the antipsychotic drug nemonapride (5WIU), and the five XSAR sets were performed to verify the identified hot spots for halogen bonding, in other words, primary (V5x40), and secondary (S5x43, S5x461 and H6x55). The simulations confirmed the key role of halogen bonding with V5x40 and H6x55 and supported S5x43 and S5x461. The results showed that steric restrictions and the topology of the molecular core have a crucial impact on the stabilization of the ligand–receptor complex by halogen bonding

Structure-based development of caged dopamine D2/D3 receptor antagonists

Dopamine is a neurotransmitter of great physiological relevance. Disorders in dopaminergic signal transduction are associated with psychiatric and neurological pathologies such as Parkinson's disease, schizophrenia and substance abuse. Therefore, a detailed understanding of dopaminergic neurotransmission may provide access to novel therapeutic strategies for the treatment of these diseases. Caged compounds with photoremovable groups represent molecular tools to investigate a biological target with high spatiotemporal resolution. Based on the crystal structure of the D-3 receptor in complex with eticlopride, we have developed caged D-2/D-3 receptor ligands by rational design. We initially found that eticlopride, a widely used D-2/D-3 receptor antagonist, was photolabile and therefore is not suitable for caging. Subtle structural modification of the pharmacophore led us to the photostable antagonist dechloroeticlopride, which was chemically transformed into caged ligands. Among those, the 2-nitrobenzyl derivative 4 (MG307) showed excellent photochemical stability, pharmacological behavior and decaging properties when interacting with dopamine receptor-expressing cells

Dibenzazecine compounds with a novel dopamine/5HT(2A )receptor profile and 3D-QSAR analysis

BACKGROUND: Antipsychotics are divided into typical and atypical compounds based on clinical efficacy and side effects. The purpose of this study was to characterize in vitro a series of novel azecine-type compounds at human dopamine D(1)-D(5 )and 5HT(2A )receptors and to assign them to different classes according to their dopamine/5HT(2A )receptor profile. RESULTS: Regardless of using affinity data (pK(i )values at D(1)-D(5 )and 5HT(2A)) or selectivity data (15 log (K(i )ratios)), principal component analysis with azecine-type compounds, haloperidol, and clozapine revealed three groups of dopamine/5HT(2A )ligands: 1) haloperidol; 2) clozapine plus four azecine-type compounds; 3) two hydroxylated dibenzazecines. Reducing the number of K(i )ratios used for principal component analysis from 15 to two (the D(1)/D(2 )and D(2)/5HT(2A )K(i )ratios) obtained the same three groups of compounds. The most potent dibenzazecine clustering in the same group as clozapine was the non-hydroxylated LE410 which shows a slightly different D(2)-like receptor profile (D(2L )> D(3 )> D(4.4)) than clozapine (D(4.4 )> D(2L )> D(3)). The monohydroxylated dibenzacezine LE404 clusters in a separate group from clozapine/LE410 and from haloperidol and shows increased D(1 )selectivity. CONCLUSION: In conclusion, two compounds with a novel dopamine/5HT(2A )receptor profile, LE404 and LE410, with some differences in their respective D(1)/D(2 )receptor affinities including a validated pharmacophore-based 3D-QSAR model for D(1 )antagonists are presented

Characterization of ligand binding to Dopamine D3 receptor using fluorescence anisotropy and radioligand binding

There are five subtypes of dopamine receptors that play a role in the dopaminergic system. Due to their limited distribution and involvement in cognitive and emotional functions, Dopamine D3 receptors are attractive pharmacological targets for treatment of drug addiction and neuropsychiatric disorders. D3 receptor ligands have been labelled with a fluorescent dye or a radioisotope for direct monitoring of ligand binding to the receptor. However, there are not many fluorescent ligands that are available for studying D3 receptor. A2-TAMRA is a novel fluorescent ligand with high affinity for D3 receptor. Binding of [3H]-methylspiperone to D3R was studied in parallel to validate results from A2-TAMRA binding to D3R. The D3 receptor ligands had similar affinities in inhibiting A2-TAMRA and [3H]-methyl spiperone binding to D3 receptor, since a very good correlation (R2 = 0.94) was obtained between both methods. The affinities for the known antagonists had a good correlation with previously published data. Selectivity of A2-TAMRA towards two different subtypes was also studied and we found that A2-TAMRA prefers D3 receptor over D1 receptor

Molecular Determinants of the Intrinsic Efficacy of the Antipsychotic Aripiprazole

Partial agonists of the dopamine D2 receptor (D2R) have been developed to treat the symptoms of schizophrenia without causing the side effects elicited by antagonists. The receptor-ligand interactions that determine the intrinsic efficacy of such drugs, however, are poorly understood. Aripiprazole has an extended structure comprising a phenylpiperazine primary pharmacophore and a 1,2,3,4-tetrahydroquinolin-2-one secondary pharmacophore. We combined site-directed mutagenesis, analytical pharmacology, ligand fragments and molecular dynamics simulations to identify the D2R-aripiprazole interactions that contribute to affinity and efficacy. We reveal that an interaction between the secondary pharmacophore of aripiprazole and a secondary binding pocket defined by residues at the extracellular portions of transmembrane segments 1, 2 and 7 determine the intrinsic efficacy of aripiprazole. Our findings reveal a hitherto unappreciated mechanism through which to fine-tune the intrinsic efficacy of D2R agonists

Dopamine D3 receptor ligands—Recent advances in the control of subtype selectivity and intrinsic activity

AbstractVarious pharmacological studies have implicated the dopamine D3 receptor as an interesting therapeutic target in the treatment of different neurological disorders. Because of these putative therapeutic applications, D3 receptor ligands with diverse intrinsic activities have been an active field of research in recent years. Separation of purely D3-mediated drug effects from effects produced by interactions with similar biogenic amine receptors allows to verify the therapeutic impact of D3 receptors and to reduce possible side-effects caused by “promiscuous” receptor interactions. The requirement to gain control of receptor selectivity and in particular subtype selectivity has been a challenging task in rational drug discovery for quite a few years. In this review, recently developed structural classes of D3 ligands are discussed, which cover a broad spectrum of intrinsic activities and show interesting selectivities

The current status of pharmacotherapy for the treatment of Parkinson's disease: transition from single-target to multitarget therapy

Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons. Motor features such as tremor, rigidity, bradykinesia and postural instability are common traits of PD. Current treatment options provide symptomatic relief to the condition but are unable to reverse disease progression. The conventional single-target therapeutic approach might not always induce the desired effect owing to the multifactorial nature of PD. Hence, multitarget strategies have been proposed to simultaneously target multiple proteins involved in the development of PD. Herein, we provide an overview of the pathogenesis of PD and the current pharmacotherapies. Furthermore, rationales and examples of multitarget approaches that have been tested in preclinical trials for the treatment of PD are also discussed

Electrophysiology-based investigations of G protein-coupled receptor pharmacology

G protein-coupled receptors (GPCRs) constitute targets for ~34% of approved drugs. The muscarinic acetylcholine M2 receptor (M2R) activates G protein-coupled receptor inward rectifying potassium (GIRK) channels in the central nervous system and heart. Membrane potential modulates agonist potency at several GPCRs. However, the mechanism underlying the voltage sensitivity remains debated. A highly conserved aspartate residue (D2.5069) has been proposed to mediate the voltage-sensitivity of the M2R, although the low expression of D69 mutants has complicated further functional investigations. Dopamine D2 and D3 receptors (D2R and D3R) are pre- and postsynaptic inhibitory receptors in the central nervous system, involved in locomotion, cognition and endocrine functions. D2R antagonists and weak partial agonists are used clinically as antipsychotics but are associated with several side effects. Various strategies have been suggested to reduce the side-effect profile of novel antipsychotic drugs. One such strategy includes the selective targeting of non-canonical signaling pathways, e.g., the β-arrestin pathway, while leaving the classical, G protein pathway, undisturbed. Additionally, binding affinity and kinetics at the D2R, as well as ligand lipophilicity, have been suggested to be of significance in determining the side-effect liability of antipsychotics. In the thesis, M2R, D2R and D3R were investigated using two-electrode voltage-clamp in Xenopus laevis oocytes co-expressing the respective receptor and GIRK channels. M2R carrying a charge-neutralizing D69N mutation demonstrated a voltage-dependent shift of agonist-potency, similar to the wild type M2R. This finding is in line with a recent alternative hypothesis, which implicates three tyrosine residues in the M2R voltage sensor. The proposed β-arrestin-selective partial D2R agonist, UNC9994, was found to be a weak partial- and almost full agonist at D2R and D3R mediated GIRK activation, respectively. These findings are incongruent with β-arrestin-selectivity and suggest that the promising effects of UNC9994 in animal models of psychosis may be related, at least in part, to involvement of the D3R. Finally, the partial D2R agonist positron emission tomography ligand, SV-III-130, demonstrated an insurmountable, yet competitive, binding mechanism at the D2R. Mutations of residues in a secondary binding pocket, engaging the secondary pharmacophore, abolished the insurmountable binding. Kinetic models incorporating an irreversible, SV-III-130-bound state captured the experimentally observed data. Molecular dynamics simulations suggested that D2R extracellular linkers participate in an induced-fit binding mechanism. In summary, the thesis addresses the mechanism of voltage-dependent agonist-potency at GPCRs and contradicts earlier reports of a β-arrestin-selective action of the experimental antipsychotic, UNC9994, at the D2R. Finally, a two-step induced-fit binding mechanism was demonstrated for the aripiprazole analogue, SV-III-130, at the D2R. The findings may guide further mechanistic investigations and provide insights for the development of novel diagnostic and therapeutic GPCR ligands