Quantitative analysis of fluorescent ligand binding to dopamine D3 receptors using live-cell microscopy.

Dopamine receptors are G protein-coupled receptors that have several essential functions in the central nervous system. A better understanding of the regulatory mechanisms of ligand binding to the receptor may open new possibilities to affect the downstream signal transduction pathways. The majority of the available ligand binding assays use either membrane preparations, cell suspensions, or genetically modified receptors, which may give at least partially incorrect understanding of ligand binding. In this study, we implemented an assay combining fluorescence and bright-field microscopy to measure ligand binding to dopamine D3 receptors in live mammalian cells. For membrane fluorescence intensity quantification from microscopy images, we developed a machine learning-based user-friendly software membrane tools and incorporated it into a data management software aparecium that has been previously developed in our workgroup. For the experiments, a fluorescent ligand NAPS-Cy3B was synthesized by conjugating a dopaminergic antagonist N-(p-aminophenethyl)spiperone with a fluorophore Cy3B. The subnanomolar affinity of NAPS-Cy3B makes it a suitable ligand for the characterization of D3 receptors in live HEK293 cells. Using a microplate compatible automated widefield fluorescence microscope, together with the membrane tools software, enables the detection and quantification of ligand binding with a high-throughput. The live cell assay is suitable for the characterization of fluorescent ligand binding and also in the competition experiments for the screening of novel unlabeled dopaminergic ligands. We propose that this simple yet more native-like approach is feasible in GPCR research, as it enables the detection of ligand binding in an environment containing more components involved in the signal transduction cascade.

Structure of the dopamine D2 receptor in complex with the antipsychotic drug spiperone.

In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone's phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy.

Spiperone: Tritium labelling at high specific activity.

An efficient method is described to tritiate spiperone at high specific activity.

Biotinylated-spiperone ligands for quantum dot labeling of the dopamine D2 receptor in live cell cultures.

We have synthesized 3 analogs of the dopamine D2 receptor (D2 DR) antagonist spiperone that can be conjugated to streptavidin-coated quantum dots via a pegylated biotin derivative. Using fluorescent imaging we demonstrate that substitution on the spiro position is tolerated, whilst the length and rigidity of a spacer arm attached to spiperone is important in controlling specific labeling as well as minimizing nonspecific labeling to cells and the surface of cell culture dishes. The ligand with the most rigid linker IDT772 (4) had the best binding profile and had high specific binding to D2 DR expressing HEK-293T cells with low nonspecific binding to plates and HEK-293T cells that lacked the D2 DR.

Targeting G†Protein-Coupled Receptors by Capture Compound Mass Spectrometry: A Case Study with Sertindole.

Unbiased chemoproteomic profiling of small-molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G protein-coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates. We report a capture compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK 293 cells. The structure-activity relationship of sertindole for DRD2 binding was reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and capture compound mass spectrometry (CCMS). The activity pattern was rationalized by molecular modelling. The most-active CC showed activities very similar to that of unmodified sertindole. A concentration of DRD2 in living cells well below 100 fmol used as an experimental input was sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions.

The topological pressure-temperature phase diagram and crystal structures of the dimorphic system spiperone.

The topological pressure-temperature phase diagram for the dimorphism of spiperone, a potent neuroleptic drug, has been constructed using literature data and improved crystal structures obtained with new crystallographic data from single-crystal X-ray diffraction at various temperatures. It is inferred that form II, which is the more dense form and exhibits the lower melting temperature, becomes the more stable phase under pressure. Under ambient conditions, form I is more stable.

Structure and dynamics of DRD4 bound to an agonist and an antagonist using in silico approaches.

Human dopamine receptor D4 (DRD4), a member of G-protein coupled receptor (GPCR) family, plays a central role in cell signaling and trafficking. Dysfunctional activity of DRD4 can lead to several psychiatric conditions and, therefore, represents target for many neurological disorders. However, lack of atomic structure impairs our understanding of the mechanism regulating its activity. Here, we report the modeled structure of DRD4 alone and in complex with dopamine and spiperone, its natural agonist and antagonist, respectively. To assess the conformational dynamics induced upon ligand binding, all-atom explicit solvent molecular dynamics simulations in membrane environment were performed. Comprehensive analyses of simulations reveal that agonist binding triggers a series of conformational changes in the transmembrane region, including rearrangement of residues, characteristic of transmission and tyrosine toggle molecular switches. Further, the trajectories indicate that a loop region in the intracellular region--ICL3, is significantly dynamic in nature, mainly due to the side-chain movements of conserved proline residues involved in SH3 binding domains. Interestingly, in dopamine-bound receptor simulation, ICL3 represents an open conformation ideal for G protein binding. The structural and dynamical information presented here suggest a mode of activation of DRD4, upon ligand binding. Our study will help in further understanding of receptor activation, as acquiring structural information is crucial for the design of highly selective DRD4 ligands.

Modification of agonist binding moiety in hybrid derivative 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-1-ol/-2-amino versions: impact on functional activity and selectivity for dopamine D2/D3 receptors.

The goal of the present study was to explore, in our previously developed hybrid template, the effect of introduction of additional heterocyclic rings (mimicking catechol hydroxyl groups as bioisosteric replacement) on selectivity and affinity for the D3 versus D2 receptor. In addition, we wanted to explore the effect of derivatization of functional groups of the agonist binding moiety in compounds developed by us earlier from the hybrid template. Binding affinity (K(i)) of the new compounds was measured with tritiated spiperone as the radioligand and HEK-293 cells expressing either D2 or D3 receptors. Functional activity of selected compounds was assessed in the GTPγS binding assay. In the imidazole series, compound 10a exhibited the highest D3 affinity whereas the indole derivative 13 exhibited similar high D3 affinity. Functionalization of the amino group in agonist (+)-9d with different sulfonamides derivatives improved the D3 affinity significantly with (+)-14f exhibiting the highest affinity. However, functionalization of the hydroxyl and amino groups of 15 and (+)-9d, known agonist and partial agonist, to sulfonate ester and amide in general modulated the affinity. In both cases loss of agonist potency resulted from such derivatization.

Antagonist-radioligand binding to D2L-receptors in intact cells.

D(2)-dopamine receptors mediate most of the physiological actions of dopamine and are important recognition sites for antipsychotic drugs. Earlier binding studies were predominantly done with broken cell preparations with the tritiated D(2)-receptor antagonists [(3)H]-raclopride, a hydrophilic benzamide, and [(3)H]-spiperone, a highly hydrophobic butyrophenone. Here we compared [(3)H]-raclopride and [(3)H]-spiperone binding properties in intact Chinese Hamster Ovary cells stably expressing recombinant human D(2L)-receptors. Specific binding of both radioligands occurred to a comparable number of sites. In contrast to the rapid dissociation of [(3)H]-raclopride in both medium only and in the presence of an excess of unlabelled ligand [(3)H]-spiperone dissociation was only observed in the latter condition, and it was still slower than in broken cell preparations. However, this could not explain the pronounced difference in the potency of some unlabelled ligands to compete with both radioligands. To integrate these new findings, a model is proposed in which raclopride approaches the receptor from the aqueous phase, while spiperone approaches the receptor by lateral diffusion within the membrane.

Screening of 5-HT1A receptor antagonists using molecularly imprinted polymers.

Molecular imprinting produces network polymers with recognition sites for imprint molecules. The high binding affinity and selectivity in conjunction with the polymers' physical robustness positions molecular imprinted polymers (MIPs) as candidates for use as preliminary screens in drug discovery. As such, MIPs can serve as crude mimics of native receptors. In an effort to evaluate the relationship between MIPs and native receptors, imprinted polymers for WAY-100635, an antagonist of the serotonin (5-HT) receptor subtype 5-HT1A were prepared. The resulting MIP P(WAY) was evaluated as an affinity matrix in the screening of serotonin receptor antagonists with known affinities for the native receptor. Rough correlations in affinity between the synthetic P(WAY) and native receptor 5-HT1A were found. These findings provide some support for the analogy between MIPs and native receptors and their possible use as surrogates.

Modeling the similarity and divergence of dopamine D2-like receptors and identification of validated ligand-receptor complexes.

Focusing on the similarity and divergence of GPCR subtypes and their ligand interactions, we generated dopamine D2, D3, and D4 receptor models based on the rhodopsin crystal structure and refined these with an extensive MM/MD protocol. After validation by diagnostic experimental data, subtype-specific relative positions of TM1, 2, 6, and 7 and bending angles of TM7 were found. To sample the conformational space of the complex, we performed simulated-annealing runs of the receptor protein with the sub-nanomolar antagonist spiperone. Docking a representative set of ligands, we were able to identify one superior model for each subtype when excellent correlations between predicted energies of binding and experimental affinities (r2 = 0.72 for D2, 0.91 for D3 and 0.77 for D4) could be observed. Further analysis revealed general ligand interactions with ASP3.32 and aromatic residues in TM6/7 and individual key interactions with TM1 and TM2 residues of the D3 and D4 receptor models, respectively.

8-(Heteroaryl)phenalkyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-ones as opioid receptor modulators.

A series of N-biarylalkyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-ones were prepared and evaluated for biological activity at opioid (mu, delta, kappa) and opioid receptor like-1 (ORL-1) G-protein coupled receptors. Substitution on the biaryl moiety produced enhanced affinity for the mu-opioid receptor.

Spiperone: influence of spiro ring substituents on 5-HT2A serotonin receptor binding.

Spiperone (1) is a widely used pharmacological tool that acts as a potent dopamine D2, serotonin 5-HT1A, and serotonin 5-HT2A antagonist. Although spiperone also binds at 5-HT2C receptors, it is one of the very few agents that display some (ca. 1000-fold) binding selectivity for 5-HT2A versus 5-HT2C receptors and, hence, might serve as a useful template for the development of novel 5-HT2A antagonists if the impact of its various substituent groups on binding was known. In the present investigation we focused on the 1, 3,8-triazaspiro[4.5]decanone portion of spiperone and found that replacement of the N1-phenyl group with a methyl group only slightly decreased affinity for cloned rat 5-HT2A receptors. However, N1-methyl derivatives displayed significantly reduced affinity for 5-HT1A, 5-HT2C, and dopamine D2 receptors. Several representative examples were shown to behave as 5-HT2 antagonists. As such, N1-alkyl analogues of spiperone may afford entry into a novel series of 5-HT2A-selective antagonists.

[18F]fluoromethylbenzylsulfonate ester: a rapid and efficient synthetic method for the N-[18F]fluoromethylbenzylation of amides and amines.

We have prepared 4-[18F]fluoromethylbenzylsulfonate esters as fluoromethylbenzylating agents. These agents are readily prepared by an [18F]fluoride ion displacement of the corresponding bissulfonate esters. The application of these 4-[18F]fluoromethylbenzylsulfonate esters to N-alkylation reaction of spiperone and 1-phenylpiperazine shows that the products 3-N-(4-[18F]fluoromethylbenzyl)spiperone and 1-N-(4-[18F]fluoromethylbenzyl)-4-phenylpiperazine are rapidly produced with high radiochemical yields under a no-carrier-added condition.

A crystallographic and molecular modeling study of butyrophenones.

The X-ray crystal structures of four butyrophenone analogues have been completed and are reported herein. These include spiperone hydrochloride (I), N-methylspiperone hydrochloride (II), pimozide (III), and fluspirilene (IV). These structures were compared to other structurally similar molecules with similar pharmacological activity. In addition, a molecular modeling study was done in order to determine the low energy conformations of these molecules. It was found that calculations of parameters that describe the molecular conformations showed that all four molecules were structurally similar. Crystallographic data: [see text]

The incorporation of butyrophenones and related compounds into a pharmacophore for dopamine D2 antagonists.

This study is an attempt to incorporate the butyrophenones, an important class of nontricyclic antipsychotic drugs, into a previously proposed pharmacophore model of tricyclic dopamine D2 receptor antagonist ligands. Conformational energy calculations were performed using the MM3-92 program on spiperone, as a representative butyrophenone, and milenperone and R48455, as related compounds with more limited conformational freedom. Twenty seven conformers were evaluated for spiperone with MM3-92 calculations and nine of these were within 1.1 kcal/mole of the global minima indicating the flexibility of the compound. A conformational analysis of twenty crystal structures of butyrophenones was also performed and six distinct conformers were represented. All of the energy minimized conformers of spiperone were superimposed in a least squares sense onto loxapine as a relatively rigid, typical D2 antagonist and a pair of mirror image conformers, which are observed in one crystal structure of spiperone, were found to be the best fit. However, it was not possible to discriminate between these two conformers since they fit the pharmacophore model equally well. The para-fluoro and carbonyl group of the butyrophenones were found to correspond best to the oxygen and chlorine atoms of loxapine, respectively. The conformations of milenperone and R48455 were also consistent with the two putative biologically active forms of spiperone and the pharmacophore model. Conformational energy calculations were also performed on molindone, an antipsychotic drug in clinical use, which can be related to the butyrophenones since both have a carbonyl group adjacent to an aromatic ring. A putative biologically active form was proposed for molindone and this was related to the structure of piquindone, a rigid analog of molindone. All of the compounds were found to be entirely consistent with the pharmacophore model. However, as previously found, there is great variability in the distance between the ammonium nitrogen and the center of the relevant aromatic ring with the most extreme case in the present study being R48455 where the distance is 7.2 A. The results of the present study should also be relevant to the structures of novel, atypical antipsychotic drugs such as risperidone which appear to be analogs of the butyrophenones.

Determination of timiperone in rat plasma by high-performance liquid chromatography with electrochemical detection.

We report a sensitive new method for the determination of timiperone in rat plasma by using high-performance liquid chromatography with electrochemical detection. The method involves extraction of plasma samples with heptane-isoamyl alcohol at pH > 8, followed by back-extraction into dilute acetic acid. Separation was accomplished by reversed-phase high-performance liquid chromatography on an ODS column with the mobile phase consisting of 0.1 M phosphate buffer (pH 3.5)-acetonitrile-methanol (65:20:15, v/v). Recovery was greater than 80%. Calibration curve was linear over the concentration range 0.5-50.0 ng/ml. The limit of quantitation of timiperone was 0.5 ng/ml plasma.

Synthesis, characterization and biodistribution of neutral and lipid-soluble 99mTc-bisaminoethanethiol spiperone derivatives: possible ligands for receptor imaging with SPECT.

Using parts of the molecular structure of spiperone, two new ligand systems for complexation with [99mTc]technetium were prepared in order to develop potential receptor imaging agents for single photon emission computer tomography (SPECT). The bis-aminoethanethiols (BAT): 1-benzyl-4-(2-mercapto-2-methyl-4-aza-pentyl)-4-(2-mercapto-2-methyl- propylamino)-piperidine (benzylpiperidyl-BAT, BP-BAT) and 1-[3-(4-fluorobenzoyl)-propyl]-4-(2-mercapto-2-methyl-4-aza-pentyl)-4- (2-mercapto-2-methyl-propyl-amino)-piperidine (butyrophenoylpiperidyl-BAT, BUP-BAT) form stable, neutral and lipid soluble complexes with [99mTc]technetium at pH > or = 11 using SnCl2 as reducing agent in nearly quantitative radiochemical yields. Biodistribution of 99mTc-BP-BAT and 99mTc-BUP-BAT in rats showed a moderate clearance from blood and low uptake and retention in the liver, whereas brain uptake was moderate, however with prolonged brain retention. On the other hand, significant accumulations and retentions were observed in heart, kidney and lung with increasing oxygen/blood ratios up to 24 h. Within 24 h p.i. 22 and 29% of the injected dose (i.d.) of 99mTc-BP-BAT and 99mTc-BUP-BAT were eliminated by hepatobiliary excretion whereas 22% i.d. of both 99mTc-BAT complexes were excreted into the urine. Although first biodistribution studies of 99mTc-BP-BAT and 99mTc-BUP-BAT in rats showed relatively low brain uptake, the high uptake in peripheral, receptor rich organs indicates that compounds of this type may be used as a basis for further structural modification to develop agents with optimal properties for cerebral or peripheral receptor imaging with SPECT.

Vinylstannylated alkylating agents as prosthetic groups for radioiodination of small molecules: design, synthesis, and application to iodoallyl analogues of spiperone and diprenorphine.

The preparation and synthetic utility of p-toluenesulfonate esters of (E)- and (Z)-3-(tri-n-butylstannyl)-prop-2-en-1-ol as bifunctional reagents for radioiodination are described. These vinylstannylated alkylating agents are prepared in two steps from propargyl alcohol, and readily couple with nucleophilic functionality (amide nitrogen, secondary amine, tertiary alcohol) in good yields (48-95%) to provide derivatives of the neuroreceptor ligands spiperone and diprenorphine. Regio- and stereospecific radioiododestannylation with retention of configuration occurs under mild, no-carrier-added conditions to give the corresponding radiolabeled N- or O-iodoallyl analogues in good radiochemical yields (55-95%) with high specific radioactivities. The methodology is versatile and well-suited to selective labeling of small molecules with radioisotopes of iodine such as 125I or 123I.

Evaluation of N-alkyl derivatives of radioiodinated spiperone as radioligands for in vivo dopamine D2 receptor studies: effects of lipophilicity and receptor affinity on the in vivo biodistribution.

A series of radioiodinated spiperone (2'-ISP) derivatives bearing amide N-alkyl substituents (N-methyl-2'-ISP, N-ethyl-2'-ISP, and N-propyl-2'-ISP) were synthesized and evaluated as potential singlet photon emission computed tomographic radiopharmaceuticals for visualizing dopaminergic receptors. The lipophilicity of these ligands (i.e., the partition coefficient for octanol-phosphate buffer) increased as the chain length increased. Investigation of blood-brain barrier permeability in rats showed a parabolic relationship between the brain uptake index and the partition coefficient. In vitro competitive binding studies showed that the relative affinity for the dopamine D2 receptor was in the order of N-propyl-2'-ISP greater than 2'-ISP greater than N-methyl-2'-ISP approximately N-ethyl-2'-ISP. In vivo biodistribution studies showed that the initial brain uptake correlated fairly well with the brain uptake index and that the kinetics of the radioactivity specifically bound to the striatum were strongly influenced by the dopamine receptor binding affinity of the compounds. Thus, the in vivo behavior of these N-alkylated 2'-ISP derivatives involved a complex interplay between receptor affinity, lipophilicity, and blood-brain barrier permeability.