9-Methyl-ß-carboline inhibits monoamine oxidase activity and stimulates the expression of neurotrophic factors by astrocytes.

ß-Carbolines (BC) are pyridoindoles, which can be found in various exogenous and endogenous sources. Recent studies revealed neurostimulative, neuroprotective, neuroregenerative and anti-inflammatory effects of 9-methyl-BC (9-Me-BC). Additionally, 9-me-BC increased neurite outgrowth of dopaminergic neurons independent of dopamine uptake into these neurons. In this study, the role of astrocytes in neurostimulative, neuroregenerative and neuroprotective properties of 9-me-BC was further explored.9-Me-BC exerted anti-proliferative effects without toxic properties in dopaminergic midbrain and cortical astrocyte cultures. The organic cation transporter (OCT) but not the dopamine transporter seem to mediate at least part the effect of 9-me-BC on astrocytes. Remarkably, 9-me-BC stimulated the gene expression of several important neurotrophic factors for dopaminergic neurons like Artn, Bdnf, Egln1, Tgfb2 and Ncam1. These factors are well known to stimulate neurite outgrowth and to show neuroprotective and neuroregenerative properties to dopaminergic neurons against various toxins. Further, we show that effect of 9-me-BC is mediated through phosphatidylinositol 3-kinase (PI3K) pathway. Additionally, 9-me-BC showed inhibitory properties to monoamine oxidase (MAO) activity with an IC50 value of 1 µM for MAO-A and of 15.5 µM for MAO-B. The inhibition of MAO by 9-me-BC might contribute to the observed increased dopamine content and anti-apoptotic properties in cell culture after 9-me-BC treatment in recent studies. Thus, 9-me-BC have a plethora of beneficial effects on dopaminergic neurons warranting its exploration as a new multimodal anti-parkinsonian medication.

Small molecules intercept Notch signaling and the early secretory pathway.

Notch signaling has a pivotal role in numerous cell-fate decisions, and its aberrant activity leads to developmental disorders and cancer. To identify molecules that influence Notch signaling, we screened nearly 17,000 compounds using automated microscopy to monitor the trafficking and processing of a ligand-independent Notch-enhanced GFP (eGFP) reporter. Characterization of hits in vitro by biochemical and cellular assays and in vivo using zebrafish led to five validated compounds, four of which induced accumulation of the reporter at the plasma membrane by inhibiting γ-secretase. One compound, the dihydropyridine FLI-06, disrupted the Golgi apparatus in a manner distinct from that of brefeldin A and golgicide A. FLI-06 inhibited general secretion at a step before exit from the endoplasmic reticulum (ER), which was accompanied by a tubule-to-sheet morphological transition of the ER, rendering FLI-06 the first small molecule acting at such an early stage in secretory traffic. These data highlight the power of phenotypic screening to enable investigations of central cellular signaling pathways.

The cooperative roles of the dopamine receptors, D1R and D5R, on the regulation of renal sodium transport.

Determining the individual roles of the two dopamine D1-like receptors (D1R and D5R) on sodium transport in the human renal proximal tubule has been complicated by their structural and functional similarity. Here we used a novel D5R-selective antagonist (LE-PM436) and D1R- or D5R-specific gene silencing to determine second messenger coupling pathways and heterologous receptor interaction between the two receptors. D1R and D5R colocalize in renal proximal tubule cells and physically interact, as determined by co-immunoprecipitation and fluorescent resonance energy transfer microscopy. Stimulation of renal proximal tubule cells with fenoldopam (D1R/D5R agonist) led to both adenylyl cyclase and phospholipase C (PLC) activation using real-time fluorescent resonance energy transfer biosensors ICUE3 and CYPHR, respectively. Fenoldopam increased cAMP accumulation and PLC activity and inhibited both NHE3 and NaKATPase activities. LE-PM436 and D5R siRNA blocked the fenoldopam-stimulated PLC pathway but not cAMP accumulation, whereas D1R siRNA blocked both fenoldopam-stimulated cAMP accumulation and PLC signaling. Either D1R or D5R siRNA, or LE-PM436 blocked the fenoldopam-dependent inhibition of sodium transport. Further studies using the cAMP-selective D1R/D5R agonist SKF83822 and PLC-selective D1R/D5R agonist SKF83959 confirmed the cooperative influence of the two pathways on sodium transport. Thus, D1R and D5R interact in the inhibition of NHE3 and NaKATPase activity, the D1R primarily by cAMP, whereas the D1R/D5R heteromer modulates the D1R effect through a PLC pathway.

Fishing for accessory binding sites at GPCRs with 'loop-hooks' - an approach towards selectivity? Part I.

Receptor-subtype selectivity is an important issue in medicinal chemistry and can become very difficult to achieve if the actual binding pockets of the respective receptors are highly conserved. For such cases, known unselective ligands could be equipped with a spacer that sticks outside the actual orthosteric binding pocket towards the extracellular loops. The end of the spacer bears certain functional groups to enable specific or unspecific interactions with the receptor residues outside the binding cavity. Our experiments indicated that it is possible to achieve selectivity within the dopamine D1 family with such 'loop-hooks'.

Modification of Branched Poly(ethylene imine) with d-Fructose for Selective Delivery of siRNA into Human Breast Cancer Cells.

Branched poly(ethylene imine) (bPEI) is frequently used in RNA interference (RNAi) experiments as a cationic polymer for the delivery of small interfering RNA (siRNA) because of its ability to form stable polyplexes that facilitate siRNA uptake. However, the use of bPEI in gene delivery is limited by its cytotoxicity and a need for target specificity. In this work, bPEI is modified with d-fructose to improve biocompatibility and target breast cancer cells through the overexpressed GLUT5 transporter. Fructose-substituted bPEI (Fru-bPEI) is accessible in three steps starting from commercially available protected fructopyranosides and bPEI. Several polymers with varying molecular weights, degrees of substitution, and linker positions on d-fructose (C1 and C3) are synthesized and characterized with NMR spectroscopy, size exclusion chromatography, and elemental analysis. In vitro biological screenings show significantly reduced cytotoxicity of 10 kDa bPEI after fructose functionalization, specific uptake of siRNA polyplexes, and targeted knockdown of green fluorescent protein (GFP) in triple-negative breast cancer cells (MDA-MB-231) compared to noncancer cells (HEK293T).

9-Methyl-ß-carboline-induced cognitive enhancement is associated with elevated hippocampal dopamine levels and dendritic and synaptic proliferation.

ß-Carbolines (BCs) belong to the heterogenous family of carbolines, which have been found exogenously, that is, in various fruits, meats, tobacco smoke, alcohol and coffee, but also endogenously, that is, blood, brain and CSF. These exogenous and endogenous BCs and some of their metabolites can exert neurotoxic effects, however, an unexpected stimulatory effect of 9-methyl-ß-carboline (9-me-BC) on dopaminergic neurons in primary mesencephalic cultures was recently discovered. The aim of the present study was to extend our knowledge on the stimulatory effects of 9-me-BC and to test the hypothesis that 9-me-BC may act as a cognitive enhancer. We found that 10 days (but not 5 days) of pharmacological treatment with 9-me-BC (i) improves spatial learning in the radial maze, (ii) elevates dopamine levels in the hippocampal formation, and (iii) results after 10 days of treatment in elongated, more complex dendritic trees and higher spine numbers on granule neurons in the dentate gyrus of 9-me-BC-treated rats. Our results demonstrate that beyond its neuroprotective/neurorestorative and anti-inflammatory effects, 9-me-BC acts as a cognitive enhancer in a hippocampus-dependent task, and that the behavioral effects may be associated with a stimulatory impact on hippocampal dopamine levels and dendritic and synaptic proliferation.

A novel non-phenolic dibenzazecine derivative with nanomolar affinities for dopamine receptors.

Dibenzazecines are a novel class of dopamine receptor antagonists, characterized by their high affinities as well as their tendency for D(1) selectivity. Hitherto, the most active dibenzazecines were phenolic in nature; a 3-OH substituent was found to result in the highest affinities. However, the phenolic nature of these compounds mostly renders them unsuitable for in vivo application, due to the poor pharmacokinetic profile, imparted by the phenolic group. A novel dibenzazecine derivative was prepared, with methylenedioxy moiety, connecting C(2) amd C(3), instead of the 3-OH group. The newly synthesized derivative 3 showed high affinities similar to the lead LE404, displaying nanomolar affinities for all dopamine receptor subtypes. Its dibrominated derivative 4, though exhibiting almost a fivefold decrease in affinities, still displayed nanomolar ones for all dopamine receptors, except for D(4) . In a functional Ca(2+) assay, both compounds 3 and 4 were found to possess antagonistic properties towards the dopamine receptors.

Residues at the indole-NH of LE300 modulate affinities and selectivities for dopamine receptors.

To further investigate SAR in the class of azecine-type dopamine receptor antagonists, we synthesized a series of derivatives, substituted at the indole-NH of the lead compound LE300 by different alkyl chains in addition to phenylpropyl, allyl, propargyl, and acetyl residues. The affinities of the target compounds for all human dopamine receptors (D(1) -D(5) ) were investigated by radioligand binding assay and their functionality by a calcium assay. Both the affinities and selectivities for the dopamine receptors were found to be affected by the nature of the substituent. The N14-methylated derivative displayed the highest affinities for all D-receptors. In general, the affinities decreased with increasing chain length of the N-alkyl. Different substituents, partly led to altered affinity, and selectivity profile when compared with our lead LE300.

The exceptional properties of 9-methyl-beta-carboline: stimulation, protection and regeneration of dopaminergic neurons coupled with anti-inflammatory effects.

Beta-carbolines (BCs) are potential endogenous and exogenous neurotoxins that may contribute to the pathogenesis of Parkinson's disease. However, we recently demonstrated protective and stimulatory effects of 9-methyl-BC (9-me-BC) in primary dopaminergic culture. In the present study, treatment with 9-me-BC unmasked a unique tetrad of effects. First, tyrosine hydroxylase (TH) expression was stimulated in pre-existing dopa decarboxylase immunoreactive neurons and several TH-relevant transcription factors (Gata2, Gata3, Creb1, Crebbp) were up-regulated. Neurite outgrowth of TH immunoreactive (THir) neurons was likewise stimulated. The interaction with tyrosine kinases (protein kinase A and C, epidermal growth factor-receptor, fibroblast growth factor-receptor and neural cell adhesion molecule) turned out to be decisive for these observed effects. Second, 9-me-BC protected in acute toxicity models THir neurons against lipopolysaccharide and 2,9-dime-BC(+) toxicity. Third, in a chronic toxicity model when cells were treated with 9-me-BC after chronic rotenone administration, a pronounced regeneration of THir neurons was observed. Fourth, 9-me-BC inhibited the proliferation of microglia induced by toxin treatment and installed an anti-inflammatory environment by decreasing the expression of inflammatory cytokines and receptors. Finally, 9-me-BC lowered the content of alpha-synuclein protein in the cultures. The presented results warrant the exploration of 9-me-BC as a novel potential anti-parkinsonian medication, as 9-me-BC interferes with several known pathogenic factors in Parkinson's disease as outlined above. Further investigations are currently under way.

Novel imidazoline compounds as partial or full agonists of D2-like dopamine receptors inspired by I2-imidazoline binding sites ligand 2-BFI.

Based on the well known biological versatility of the imidazoline nucleus, we prepared the novel derivatives 3a-k inspired by 2-BFI scaffold to assess imidazoline molecules as D(2)-like dopamine receptor ligands. Conservative chemical modifications of the lead structure, such as the introduction of an hydroxy group in the aromatic ring alone or associated with N-benzyl substitution, provided partial (3f) or nearly full (3e and 3h) agonists, all endowed with D(2)-like potency comparable to that of dopamine.

Ring expansions of tetrahydroprotoberberines and related dibenzo[c,g]azecines modulate the dopamine receptor subtype affinity and selectivity.

The affinities of tetrahydroprotoberberines for dopamine receptors dramatically decrease after cleaving the central C-N bond to the analogous ten-membered dibenzo[c,g]azecines [1]. In the present work, we also synthesized eleven-membered homologues of these heterocycles and measured the affinities of the resulting dibenzazaundecenes and their underlying homoberberines for human dopamine receptors as well as the cytotoxic effects of all target compounds on human glia cells. The tetracyclic iso-C-homoberberine-derivatives revealed to be D(4)-selective antagonists, while all other active compounds showed a significant D(1)/D(5) selectivity. Distances in energy-minimized conformations were measured in order to explain our findings.

Polymethine Dye-Functionalized Nanoparticles for Targeting CML Stem Cells.

In chronic myelogenous leukemia (CML), treatment with tyrosine kinase inhibitors (TKI) is unable to eradicate leukemic stem cells (LSC). Polymethine dye-functionalized nanoparticles can be internalized by specific cell types using transmembrane carrier proteins. In this study we investigated the uptake behavior of various polymethine dyes on leukemia cell lines and searched for carrier proteins that guide dye transport using RNA interference. The results show that the uptake of DY-635 is dependent on organic anion transport protein 1B3 (OATP1B3) in CML cells and immature myeloid precursor cells of CML patients. In contrast to nonspecific poly(lactide-co-glycolic acid) (PLGA) nanoparticle constructs, DY-635-functionalization of nanoparticles led to an uptake in CML cells. Investigation of these nanoparticles on bone marrow of CML patients showed a preferred uptake in LSC. The transcription of OATP1B3 is known to be induced under hypoxic conditions via the hypoxia-inducing factor 1 alpha (HIF1α), thus also in the stem cells niche. Since these cells have the potential to repopulate the bone marrow after CML treatment discontinuation, eliminating them by means of drug-loaded DY-635-functionalized PLGA nanoparticles deployed as a selective delivery system to LSC is highly relevant to the ongoing search for curative treatment options for CML patients.

Formulation of Liver-Specific PLGA-DY-635 Nanoparticles Loaded with the Protein Kinase C Inhibitor Bisindolylmaleimide I.

Bisindolylmaleimide I (BIM-I) is a competitive pan protein kinase C inhibitor with anti-inflammatory and anti-metastatic properties, suggested to treat inflammatory diseases and various cancer entities. However, despite its therapeutic potential, BIM-I has two major drawbacks, i.e., it has a poor water solubility, and it binds the human ether-à-go-go-related gene (hERG) ion channels, potentially causing deadly arrhythmias. In this case, a targeted delivery of BIM-I is imperative to minimize peripheral side effects. To circumvent these drawbacks BIM-I was encapsulated into nanoparticles prepared from poly(lactic-co-glycolic acid) (PLGA) functionalized by the near-infrared dye DY-635. DY-635 served as an active targeting moiety since it selectively binds the OATP1B1 and OATP1B3 transporters that are highly expressed in liver and cancer cells. PLGA-DY-635 (BIM-I) nanoparticles were produced by nanoprecipitation and characterized using dynamic light scattering, analytical ultracentrifugation, and cryogenic transmission electron microscopy. Particle sizes were found to be in the range of 20 to 70 nm, while a difference in sizes between the drug-loaded and unloaded particles was observed by all analytical techniques. In vitro studies demonstrated that PLGA-DY-635 (BIM-I) NPs prevent the PKC activation efficiently, proving the efficacy of the inhibitor after its encapsulation, and suggesting that BIM-I is released from the PLGA-NPs. Ultimately, our results present a feasible formulation strategy that improved the cytotoxicity profile of BIM-I and showed a high cellular uptake in the liver as demonstrated in vivo by intravital microscopy investigations.

Dibenzazecine scaffold rebuilding--is the flexibility always essential for high dopamine receptor affinities?

The moderately flexible 7-methyl-5,6,7,8,9,14-hexahydrodibenz[d,g]azecines are known to be potent dopamine receptor antagonists, whereas the corresponding rigid dibenzo[d,g]quinolizines are inactive. We built the scaffolds of dibenzo[c,g], [c,f] and [d,f]azecines and together with their ring closed, more rigid precursors, evaluated the affinities for the human D(1)-D(5) receptors (radioligand binding) as well as the functionalities (calcium assay) and thus investigated the influence of annelation and conformative flexibility of these compounds on their affinity for human cloned dopamine receptors.

Dopamine/serotonin receptor ligands. Part 17: a cross-target SAR approach: affinities of azecine-styled ligands for 5-HT(2A) versus D1 and D2 receptors.

Dibenzo- and benzindolo-azecines represent a novel class of high-affinity dopamine receptor antagonists. To further characterize these drugs as potential neuroleptics, we selected a set of azecine derivatives and ring expanded homologues and we measured their antagonist activity at the 5-HT(2A) receptor in the porcine coronary artery. SARs found for the 5-HT(2A) receptor resemble those for the D1 but not the D2 receptor. The protein-ligand interactions were discussed with respect to the different binding pockets.

Dopamine/serotonin receptor ligands. 16.(1) Expanding dibenz[d,g]azecines to 11- and 12-membered homologues. Interaction with dopamine D(1)-D(5) receptors.

Oxygenated 7-methyl-5,6,7,8,9,14-hexahydrodibenz[d,g]azecines are potent dopamine receptor antagonists, preferentially at D1/D5. We synthesized the hydroxylated, methoxylated, and chlorinated 11-membered and 12-membered homologues of these 10-membered heterocycles. Their affinities for the human cloned D1-D5 receptors (radioligand binding) and functionalities (calcium assay) were measured. Enlarging the dibenzazecines to the corresponding dibenzazacycloundecenes and dibenzazacyclododecenes generally maintains the high antagonistic affinity for D1/D5 but also leads to a compound with a clozapine-like binding profile due to additional affinity for D4.

Cytotoxicity of beta-carbolines in dopamine transporter expressing cells: structure-activity relationships.

Some beta-carbolines (BC) are natural constituents in the human brain deriving from tryptophan, tryptamine, and serotonin. In vitro and animal experiments suggest that BC-cations may cause neurodegeneration with a higher vulnerability of dopaminergic than of other neurons. Despite the possible implication of the BC-cations in the pathogenesis of Parkinson's disease (PD), the underlying mechanisms are poorly understood. The present study further explores the structural requirements for the cytotoxic effects of BCs and searches for additional compounds involved in the pathogenesis of PD. Previous studies were now extended to serotonin-derived BCs, tetrahydro-BCs, a BC-dimer, and a BC-enantiomer to reveal possible stereoselectivity. Neutral, rather lipophilic BCs may pass the plasma membrane and the outer and inner mitochondrial membranes by diffusion whereas the cationic, more polar compounds, can be transported by the dopamine transporter (DAT). In the present study, 4 out of 17 BC-cations caused DAT-independent toxicity. This number is unexpected in view of previous findings that all BC-cations are transported by DAT. 3-Carboxylated and 6-methoxylated BCs were poor substrates. The size alone does not seem to be a limiting factor. A dimeric BC-cation was readily transported by the DAT despite its much larger structure compared to dopamine. Furthermore, (R)-enantiomers were preferentially transported. The neutral BCs were approximately one order of magnitude less toxic than the cationic BCs. There are considerable differences of the transport efficiency between the BCs. Potent cytotoxic tetrahydro-BCs were detected. Because precursor tetrahydro-BCs are present in the brain, the search for the occurrence of these compounds in human brain is warranted.

Synthesis and Characterization of new Azecine-Derivatives as Potential Neuroleptics.

Dibenzo- and benzindolo-azecines represent a class of potential neuroleptics. To characterize the effectiveness at the dopamine and 5-HT2A-receptor representative structures were synthesized and tested by radio ligand binding studies, in vivo and in vitro studies.Neuroleptic potency and the risk of side effects of the prodrug 7-methyl-5,6,7,8,9,14-hexahydrodibenzo[d,g]azecin-3-yl isobutyrate, an ester derivative of the most promising azecine 7-methyl-5,6,7,8,9,14-hexahydrodibenzo[d,g]azecin-3-ol (LE404), was tested in vivo concerning conditioned avoidance response inhibition, locomotor activity and triggering of catalepsy vs. haloperidol as a reference. Also ester hydrolysis was examined using porcine liver esterase to thereby obtain an indication of the stability of the prodrug in vivo. An HPLC method was developed for purity control and determination of octanol/water-distribution coefficients.It has been shown that the tested substances in their efficacy are comparable to haloperidol and risperidone, but the therapeutic index in most cases is larger. Esterification as a prodrug principle leads to significantly prolonged effectiveness.

Dopamine/serotonin receptor ligands. 13: Homologization of a benzindoloazecine-type dopamine receptor antagonist modulates the affinities for dopamine D(1)-D(5) receptors.

Enlarging the 10-membered ring of 7-methyl-6,7,8,9,14,15-hexahydro-5H-indolo[3,2-f][3]benzazecine (1, LE 300) yielded two homologue antagonists. Their affinities and inhibitory activities at D(1)-D(5) receptors were measured by radioligand binding experiments and a functional Ca(2+) assay. Compared to 1, phenylpropyl homologue 3 was superior in selectivity and affinity for the D(5) subtype (K(i) = 0.6 nM), whereas the affinity of the indolylpropyl homologue 2 for all subtypes decreased. Compounds 2, 3, 10, 11, 17, and 18 are derivatives of novel heterocyclic ring systems.