Dual Piperidine-Based Histamine H3 and Sigma-1 Receptor Ligands in the Treatment of Nociceptive and Neuropathic Pain.

In search of new dual-acting histamine H3/sigma-1 receptor ligands, we designed a series of compounds structurally based on highly active in vivo ligands previously studied and described by our team. However, we kept in mind that within the previous series, a pair of closely related compounds, KSK67 and KSK68, differing only in the piperazine/piperidine moiety in the structural core showed a significantly different affinity at sigma-1 receptors (σ1Rs). Therefore, we first focused on an in-depth analysis of the protonation states of piperazine and piperidine derivatives in the studied compounds. In a series of 16 new ligands, mainly based on the piperidine core, we selected three lead structures (3, 7, and 12) for further biological evaluation. Compound 12 showed a broad spectrum of analgesic activity in both nociceptive and neuropathic pain models based on the novel molecular mechanism.

Discovery of nanomolar ligands with novel scaffolds for the histamine H4 receptor by virtual screening.

The involvement of histamine H4 receptor (H4R) in immune cells chemotaxis and mediator release makes it an attractive target for the treatment of inflammation disorders. A decade of medicinal chemistry efforts has led to several promising ligands, although the chemical structures described so far possesses a singular limited diversity. We report here the discovery of novel structures, belonging to completely different scaffolds. The virtual screening was planed as a two-steps process. First, using a "scout screening" methodology, we have experimentally probed the H4R ligand binding site using a small size chemical library with very diverse structures, and identified a hit that further assist us in refining a raw 3D homology model. Second, the refined 3D model was used to conduct a widened virtual screening. This two-steps strategy proved to be very successful, both in terms of structural diversity and hit rate (23%). Moreover, the hits have high affinity for the H4R, with most potent ligands in the nanomolar range.

Synthesis and evaluation of a 2-benzothiazolylphenylmethyl ether class of histamine H4 receptor antagonists.

Synthesis and biological evaluation of a new class of histamine H4 receptor ligands, distinct from the previously reported chemotypes, are described. A virtual screening of our corporate compound collection identified a hit with an undesired dual H3R/H4R activity. Chemical exploration led to the discovery of a more potent and selective 2-benzothiazolylphenylmethyl ether lead compound.

Improving selectivity of dopamine D3 receptor ligands.

The seminal human dopamine D3 receptor (hD3R) ligand BP 897 has shown interesting properties during clinical trials. However, its lack of selectivity towards human adrenergic receptor impedes further development. Two approaches were followed to increase hD3R selectivity. The lead optimisation succeeded, we disclose here ligands with subnanomolar potency for D3R, combined with a good selectivity for the closely related human dopamine D2 and human adrenergic alpha-1 receptors.

Determination of the binding mode and interacting amino-acids for dibasic H3 receptor antagonists.

Due to its involvement in major CNS functions, the histamine H3 receptor (H3R) is the subject of intensive medicinal chemistry investigation, supported by the range of modern drug discovery tools, such as receptor modeling and ligand docking. Although the receptor models described to date share a majority of common traits, they display discrete alternatives in amino-acid conformation, rendering ligand binding modes quite different. Such variations impede structure-based drug design in the H3R field. In the present study, we used a combination of medicinal chemistry, receptor-guided and ligand-based methods to elucidate the binding mode of antagonists. The approaches converged towards a ligand orientation perpendicular to the membrane plane, bridging Glu206 of the transmembrane helix 5 to acidic amino acids of the extracellular loops. This consensus will help future structure-based drug design for H3R ligands.

Novel and highly potent histamine H3 receptor ligands. Part 3: an alcohol function to improve the pharmacokinetic profile.

Synthesis and biological evaluation of potent histamine H3 receptor antagonists incorporating a hydroxyl function are described. Compounds in this series exhibited nanomolar binding affinities for human receptor, illustrating a new possible component for the H3 pharmacophore. As demonstrated with compound BP1.4160 (cyclohexanol 19), the introduction of an alcohol function counter-intuitively allowed to reach high in vivo efficiency and favorable pharmacokinetic profile with reduced half-life.

Novel and highly potent histamine H3 receptor ligands. Part 1: withdrawing of hERG activity.

Pre-clinical investigation of some aryl-piperidinyl ether histamine H3 receptor antagonists revealed a strong hERG binding. To overcome this issue, we have developed a QSAR model specially dedicated to H3 receptor ligands. This model was designed to be directly applicable in medicinal chemistry with no need of molecular modeling. The resulting recursive partitioning trees are robust (80-85% accuracy), but also simple and comprehensible. A novel promising lead emerged from our work and the structure-activity relationships are presented.

Novel and highly potent histamine H3 receptor ligands. Part 2: exploring the cyclohexylamine-based series.

Synthesis and biological evaluation of novel and potent cyclohexylamine-based histamine H3 receptor inverse agonists are described. Compounds in this newly identified series exhibited subnanomolar binding affinities for human receptor and no significant interaction with hERG channel. One derivative (10t) demonstrated enhanced in vivo efficiency and preferential brain distribution, both properties suitable for potential clinical evaluation.

Homology Model Versus X-ray Structure in Receptor-based Drug Design: A Retrospective Analysis with the Dopamine D3 Receptor.

Structure-based design methods commonly used in medicinal chemistry rely on a three-dimensional representation of the receptor. However, few crystal structures are solved in comparison with the huge number of pharmaceutical targets. This often renders homology models the only information available. It is particularly true for G protein-coupled receptors (GPCRs), one of the most important targets for approved medicines and current drug discovery projects. However, very few studies have tested their validity in comparison with corresponding crystal structures, especially in a lead optimization perspective. The recent solving of dopamine D3 receptor crystal structure allowed us to assess our historical homology model. We performed a statistical analysis, by docking our in-house lead optimization library of 1500 molecules. We demonstrate here that the refined homology model suits at least as well as the X-ray structure. It is concluded that when the crystal structure of a given GPCR is not available, homology modeling can be an excellent surrogate to support drug discovery efforts.

Synthesis and evaluation of amides surrogates of dopamine D3 receptor ligands.

Isosteric replacement of the amide function and modulation of the arylpiperazine moiety of known dopamine D3 receptor ligands led to potent and selective compounds. Enhanced bioavailability and preferential brain distribution make compound 6c a good candidate for pharmacological and clinical evaluation.

Connexin 30 is expressed in the mouse sino-atrial node and modulates heart rate.

AIMS: This study aimed at characterizing expression and the functional role of the Gjb6 gene, encoding for connexin 30 (Cx30) protein, in the adult mouse heart. METHODS AND RESULTS: The expression of the Gjb6 gene in the mouse heart was investigated by RT-PCR and sequencing of amplified cDNA fragments. The sites of Gjb6 expression were identified in the adult heart using transgenic mice with reporter genes (Cx30(LacZ/LacZ) and Cx30(LacZ/LacZ)/Cx40(EGFP/EGFP) mice), as well as anti-HCN4 (hyperpolarization activated cyclic nucleotide-gated potassium channel 4) or anti-connexin antibodies. Cine-magnetic resonance imaging and telemetric ECG recordings were used to evaluate the impact of Cx30 deficiency on cardiac physiology. Gjb6 was shown to be expressed in the sinoatrial (SA) node of the adult mouse heart. Eighty from 100 nuclei on average were LacZ-positive in the SA node of Cx30(LacZ/LacZ) mice. No significant LacZ expression was seen in other cardiac tissues. Cx30 protein was identified in low abundance in the SA node of wild-type mice, as indicated by immunofluorescence experiments. Telemetric ECG recordings indicated that Cx30-deficient mice displayed a mean daily heart rate (HR) that was 9% faster than that measured in control mice (572 +/- 38 b.p.m. vs. 524 +/- 23, P < 0.05). This moderate tachycardia was still observed after inhibition of the autonomic nervous system, demonstrating that Cx30 deficiency resulted in changes in the intrinsic electrical properties of the SA node. Consistent with this hypothesis, Cx30(LacZ/LacZ) displayed a significant reduction of SDNN (standard deviation of the interbeat interval) compared with control mice. Increase of both the cardiac index (20%) and the end-diastolic volume to body weight ratio (16%) with no deficiency in ejection fraction or stroke volume were observed in mutant mice. An increase in cardiac index was interpreted as being a direct consequence of high HR, whereas large end-diastolic volume may be an indirect consequence of prolonged high HR. CONCLUSION: Cx30 is functionally expressed, in low abundance, in the SA node of the adult mouse heart where it participates in HR regulation.

Refined docking as a valuable tool for lead optimization: application to histamine H3 receptor antagonists.

Drug-discovery projects frequently employ structure-based information through protein modeling and ligand docking, and there is a plethora of reports relating successful use of them in virtual screening. Hit/lead optimization, which represents the next step and the longest for the medicinal chemist, is very rarely considered. This is not surprising because lead optimization is a much more complex task. Here, a homology model of the histamine H(3) receptor was built and tested for its ability to discriminate ligands above a defined threshold of affinity. In addition, drug safety is also evaluated during lead optimization, and "antitargets" are studied. So, we have used the same benchmarking procedure with the HERG channel and CYP2D6 enzyme, for which a minimal affinity is strongly desired. For targets and antitargets, we report here an accuracy as high as at least 70%, for ligands being classified above or below the chosen threshold. Such a good result is beyond what could have been predicted, especially, since our test conditions were particularly stringent. First, we measured the accuracy by means of AUC of ROC plots, i. e. considering both false positive and false negatives. Second, we used as datasets extensive chemical libraries (nearly a thousand ligands for H(3)). All molecules considered were true H(3) receptor ligands with moderate to high affinity (from microM to nM range). Third, the database is issued from concrete SAR (Bioprojet H(3) BF2.649 library) and is not simply constituted by few active ligands buried in a chemical catalogue.

Hybrid approach for the design of highly affine and selective dopamine D(3) receptor ligands using privileged scaffolds of biogenic amine GPCR ligands.

A series of compounds containing privileged scaffolds of the known histamine H(1) receptor antagonists cetirizine, mianserin, ketotifen, loratadine, and bamipine were synthesized for further optimization as ligands for the related biogenic amine binding dopamine D(3) receptor. A pharmacological screening was carried out at dopamine D(2) and D(3) receptors. In the preliminary testing various ligands have shown moderate to high affinities for dopamine D(3)receptors, for example, N-(4-{4-[benzyl(phenyl)amino]piperidin-1-yl}butylnaphthalen-2-carboxamide (19a) (hD(3)K(i)=0.3 nM; hD(2)K(i)=703 nM), leading to a selectivity ratio of 2343.

In situ hybridization: a technique to study localization of cardiac gene expression.

In situ hybridization allows the detection of specific gene transcripts in tissues, cells or, chromosomes. In the cardiovascular field, this powerful and rapid methodology provides precious insights into the complex gene organization and expression within an heterogeneous cell population. This technique is particularly useful to elucidate the genes and pathways involved in cardiac cells processes (differentiation, proliferation, apoptosis) or in the development of cardiovascular pathologies. In situ hybridization allows the precise localization of gene transcripts to the different heart regions and to individual cell types such as working cardiomyocytes, cells from conductive tissues and blood vessels displaying specific functions. This chapter describes the different technical procedures that are of crucial importance to carry on sensitive and specific in situ hybridization experiments in heart samples. The detection of transcripts within paraformaldehyde-fixed, paraffin-embedded cardiac tissue samples is illustrated here with the detection of cardiac sphingosine-1-phosphate receptor expression.

Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2-5 null mutants.

In humans, mutations of the gene encoding the transcription factor Nkx2-5 result in the heart in electrical conduction defects and morphological abnormalities. In this organ Nkx2-5 is expressed in both the myocardium and the endocardium. Connexins (Cxs) are gap junction channel proteins that have been shown to be involved in both heart development and cardiac electrical conduction, suggesting a possible correlation between expression of Cxs and Nkx2-5. To evaluate this correlation, the expression of Cxs has been investigated in the cardiovascular system of wild-type and Nkx2-5-/- 9.2 days post-conception (dpc) mouse embryos. The disruption of the Nkx2-5 gene results in the loss of Cx43 in the heart, due in part to the poor development of the ventricular trabecular network, as well as specific downregulation of Cx45 gene expression. In addition, the nuclear translocation of NFATc1 in the endocardial endothelial cells is inhibited in the Nkx2-5-/- embryos. These results indicate for the first time that Nkx2-5 is involved in the transcriptional regulation of the Cx45 gene expression. In the mutant embryos the aorta is collapsed, and the vascular endothelial Cxs, Cx40 and Cx37, are no longer expressed in its posterior region. Poor development of the trabeculae and downregulation of Cx45 may contribute both to failure of the myocardial function and to hemodynamic insufficiency. The latter, in turn, may result in the dysregulation of Cx40 and -37 expressions along the whole length of the aorta. Direct or indirect effects of Nkx2-5 inactivation on the Cx45 gene expression could explain the absence of the endocardial cushions in the heart of Nkx2-5-/- embryos.

Parallel synthesis and dopamine D3/D2 receptor screening of novel {4-[4-(2-methoxyphenyl)piperazin-1-yl]butyl}carboxamides.

We have applied a fast and high-yielding method for the parallel amidation of 4-[4-(2-methoxyphenyl)piperazin-1-yl]-butylamine yielding analogs of the partial dopamine receptor agonist BP 897. Using this amino scaffold prepared in solution and polymer-bound carboxylic acid equivalents, we have synthesized a series of high affinity dopamine D(3) receptor ligands. The novel compounds were obtained in good to excellent yield and purity. Biological evaluation included determination of binding affinities at hD(2S) and hD(3) receptor subtypes. From the 22 novel structures presented here, compound 4v showed high affinity (K(i) (hD(3)) 1.6nM) and a 136-fold preference for the D(3) receptor versus that for the D(2) receptor subtype. Our results suggest that this derivatization technique is a useful method to speed up structure-activity relationships studies on dopamine receptor subtype modulators.

Genomic organization and alternative transcripts of the human Connexin40 gene.

The human Cx40 gene (NT_004434.5) was sorted out from the GenBank database and as a result of a BLAST homology search, two ESTs (BE784549 from a human lung database, and BE732411 from a human placenta database) overlapping with the coding exon 2 sequence and upstream regions of the gene were identified. These ESTs correspond to two transcripts 1A and 1B, which diverge from each other in their 5' regions. The transcript 1A corresponds to the only transcript previously identified for the mouse and rat Cx40 genes; whereas the transcript 1B is a new transcript. The human Cx40 gene therefore comprises three exons: exon 1A (100 bp), exon 1B (132 bp) and coding exon 2, with the exons 1A and 1B at 14 and 1.3 kb of the exon 2, respectively. The expression of these transcripts is cell-type specific. Transcript 1A is expressed in endothelial cells. Its expression was demonstrated in human umbilical vein endothelial cells (HUVEC). Transcript 1B is expressed in placental cytotrophoblasts. Its expression was demonstrated in malignant trophoblastic cells, BeWo, JAR and JEG-3, and purified cytotrophoblasts from human first trimester placental tissues. Interestingly, both transcripts 1A and 1B are expressed in the right atrial appendages (RAA), although the cell-type expression of the two transcripts in this particular tissue has not yet been determined. Both transcripts were found to be expressed in the various heart regions investigated, where transcript 1B was found to always occur rarely in comparison with transcript 1A. Transcripts 1A and 1B are both more abundant in the atria than in the ventricles. Luciferase reporter gene assays demonstrated that two genomic regions containing the exons 1A and 1B induced a cell-type specific expression. The 1.2 kb sequence, containing the exon 1A, induced an increase of the luciferase activity in HUVEC; whereas the 1.9 kb sequence, containing the exon 1B, induces an increase of expression of the luciferase activity in BeWo cells. The DNA sequence upstream of the exon 1A contains SP1 binding sites, but no TATA- or CAAT-box; whereas the region upstream of the exon 1B is preceded by three CAAT-boxes. Thus, in contrast to the mouse and rat Cx40 genes, the human Cx40 gene organized in three exons and generates two transcripts, which are cell-type specific.

Cell type-specific localization of human cardiac S1P receptors.

Sphingosine 1-phosphate (S1P), which derives from the metabolism of sphingomyelin, is mainly synthesized, stored, and released from platelets after activation by physiological and pathophysiological events. S1P acts in cardiovascular tissues through cell surface G-protein-coupled receptors of the endothelial differentiation gene (EDG) family, i.e., EDG1, EDG3 and EDG5. The aim of the present study was to assess the precise distribution of EDG1, EDG3, and EDG5 receptors expressed in human cardiovascular tissues to investigate their respective physiological implication. When assessed by Northern blots, EDG1, EDG3, and EDG5 displayed wide expression levels in decreasing order, respectively. In particular, EDG3 was mainly detected in the aorta. Detailed analysis by in situ hybridization (ISH) and immunohistochemistry (IHC) revealed strong EDG1 expression in cardiomyocytes and in endothelial cells of cardiac vessels. In cardiomyocytes, the EDG1 receptor is likely to be co-expressed with EDG3 and EDG5, although EDG1 exhibits the most prominent expression pattern. Unlike EDG3 and EDG5, which are expressed in the smooth muscle cell layer of the human aorta, no signal corresponding to EDG1 expression could be detected in the aorta. Moreover, only EDG3 expression was also found in smooth muscle cells of cardiac vessels. The present results provide new insight into the expression pattern of S1P receptors in human cardiovascular tissues, indicating a differential pattern of expression for these receptors in human vessels.

Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere.

The Rho small GTPases are crucial proteins involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. It has been reported that these GTPases are directly associated with cardiovascular disorders. In this context, we have searched for novel modulators of Rho GTPases, and here we describe p63RhoGEF a new Db1-like guanine nucleotide exchange factor (GEF). P63RhoGEF encodes a 63 kDa protein containing a Db1 homology domain in tandem with a pleckstrin homology domain and is most closely related to the second Rho GEF domain of Trio. Northern blot and in situ analysis have shown that p63RhoGEF is mainly expressed in heart and brain. In vitro guanine nucleotide exchange assays have shown that p63RhoGEF specifically acts on RhoA. Accordingly, p63RhoGEF expression induces RhoA-dependent stress fiber formation in fibroblasts and in H9C2 cardiac myoblasts. Moreover, we show that p63RhoGEF activation of RhoA in intact cells is dependent on the presence of the PH domain. Using a specific anti-p63RhoGEF antibody, we have detected the p63RhoGEF protein by immunocytochemistry in human heart and brain tissue sections. Confocal microscopy shows that p63RhoGEF is located in the sarcomeric I-band mainly constituted of cardiac sarcomeric actin. Together, these results show that p63RhoGEF is a RhoA-specific GEF that may play a key role in actin cytoskeleton reorganization in different tissues, especially in heart cellular morphology.