Transcriptomic Profiling Discloses Molecular and Cellular Events Related to Neuronal Differentiation in SH-SY5Y Neuroblastoma Cells.

Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.

SAR and biological evaluation of SEN12333/WAY-317538: Novel alpha 7 nicotinic acetylcholine receptor agonist.

Alpha 7 nicotinic acetylcholine receptor (alpha(7) nAChR) agonists are promising therapeutic candidates for the treatment of cognitive impairment associated with a variety of disorders including Alzheimer's disease and schizophrenia. Alpha 7 nAChRs are expressed in brain regions associated with cognitive function, regulate cholinergic neurotransmission and have been shown to be down regulated in both schizophrenia and Alzheimer's disease. Herein we report a novel, potent small molecule agonist of the alpha 7 nAChR, SEN12333/WAY-317538. This compound is a selective agonist of the alpha(7) nAChR with excellent in vitro and in vivo profiles, excellent brain penetration and oral bioavailability, and demonstrates in vivo efficacy in multiple behavioural cognition models. The SAR and biological evaluation of this series of compounds are discussed.

Arnica montana Stimulates Extracellular Matrix Gene Expression in a Macrophage Cell Line Differentiated to Wound-Healing Phenotype.

Arnica montana (Arnica m.) is used for its purported anti-inflammatory and tissue healing actions after trauma, bruises, or tissue injuries, but its cellular and molecular mechanisms are largely unknown. This work tested Arnica m. effects on gene expression using an in vitro model of macrophages polarized towards a "wound-healing" phenotype. The monocyte-macrophage human THP-1 cell line was cultured and differentiated with phorbol-myristate acetate and Interleukin-4, then exposed for 24h to Arnica m. centesimal (c) dilutions 2c, 3c, 5c, 9c, 15c or Control. Total RNA was isolated and cDNA libraries were sequenced with a NextSeq500 sequencer. Genes with significantly positive (up-regulated) or negative (down-regulated) fold changes were defined as differentially expressed genes (DEGs). A total of 20 DEGs were identified in Arnica m. 2c treated cells. Of these, 7 genes were up-regulated and 13 were down-regulated. The most significantly up-regulated function concerned 4 genes with a conserved site of epidermal growth factor-like region (p<0.001) and three genes of proteinaceous extracellular matrix, including heparin sulphate proteoglycan 2 (HSPG2), fibrillin 2 (FBN2), and fibronectin (FN1) (p<0.01). Protein assay confirmed a statistically significant increase of fibronectin production (p<0.05). The down-regulated transcripts derived from mitochondrial genes coding for some components of electron transport chain. The same groups of genes were also regulated by increasing dilutions of Arnica m. (3c, 5c, 9c, 15c), although with a lower effect size. We further tested the healing potential of Arnica m. 2c in a scratch model of wound closure based on the motility of bone marrow-derived macrophages and found evidence of an accelerating effect on cell migration in this system. The results of this work, taken together, provide new insights into the action of Arnica m. in tissue healing and repair, and identify extracellular matrix regulation by macrophages as a therapeutic target.

Novel Hits in the Correction of ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Protein: Synthesis, Pharmacological, and ADME Evaluation of Tetrahydropyrido[4,3-d]pyrimidines for the Potential Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is a lethal genetic disease caused by mutations of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) with a prevalence of the ΔF508 mutation. Whereas the detailed mechanisms underlying disease have yet to be fully elucidated, recent breakthroughs in clinical trials have demonstrated that CFTR dysfunction can be corrected by drug-like molecules. On the basis of this success, a screening campaign was carried out, seeking new drug-like compounds able to rescue ΔF508-CFTR that led to the discovery of a novel series of correctors based on a tetrahydropyrido[4,3-d]pyrimidine core. These molecules proved to be soluble, cell-permeable, and active in a disease relevant functional-assay. The series was then further optimized with emphasis on biological data from multiple cell systems while keeping physicochemical properties under strict control. The pharmacological and ADME profile of this corrector series hold promise for the development of more efficacious compounds to be explored for therapeutic use in CF.

Interactive SAR studies: rational discovery of super-potent and highly selective dopamine D3 receptor antagonists and partial agonists.

Starting from dopamine receptor ligand BP897, an interactive drug discovery process leading to heterocyclic bioisosteres is demonstrated. The four step strategy involved a careful optimization of geometric and electronic properties by systematic modification of the attachment points and heteroatoms, respectively. Efficacy tuning by modification of the phenyl substituents led to both D3 partial agonists and full antagonists. The benzothiophenes 3c (FAUC346) and 3d (FAUC365) revealed outstanding D3 affinity and subtype selectivity.

Attenuation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity by the novel selective dopamine D3-receptor partial agonist FAUC 329 predominantly in the nucleus accumbens of mice.

We previously synthesised a novel dopamine (DA) partial agonist FAUC 329 with high affinity and selectivity for the DA D(3) receptor. This is the first in vivo study to investigate the protective effects of FAUC 329 in a MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease. Adult male C57bl/6 mice were injected with FAUC 329 (0, 0.1, 0.5, 0.75, or 1 mg/kg) 30 min before MPTP (2 x 30 mg/kg, 4 hr apart). One week later, accumbal and striatal tissue was processed for DA and metabolite HPLC determination as well as immunohistochemical analysis of DA transporter positive neurons in the substantia nigra pars compacta and ventral tegmental area was carried out. FAUC 329 showed a significant attenuation of MPTP-induced DA reduction in the nucleus accumbens (0.5, 0.75 and 1 mg/kg) in a dose-dependent manner. FAUC 329 (0.75 mg/kg) partly protected against DA depletion in the dorsal striatum as well as protected against loss of DA transporter immunoreactivity in the substantia nigra pars compacta. The highest dose of FAUC 329 (1 mg/kg), however, showed a non-significant tendency to augment the MPTP-induced striatal DA reduction. The protective effect of FAUC 329 against MPTP-induced DA depletion was most pronounced in the nucleus accumbens and appears to be linked to the preferential abundance of D(3) receptors in this region. Targeting the mesolimbic DA system may have implications for improvement of impaired motor behaviour and particularly non-motor functions related to the nucleus accumbens.

Fused 3-Hydroxy-3-trifluoromethylpyrazoles Inhibit Mutant Huntingtin Toxicity.

Here, we describe the selection and optimization of a chemical series active in both a full-length and a fragment-based Huntington's disease (HD) assay. Twenty-four thousand small molecules were screened in a phenotypic HD assay, identifying a series of compounds bearing a 3-hydroxy-3-trifluoromethylpyrazole moiety as able to revert the toxicity induced by full-length mutant Htt by up to 50%. A chemical exploration around the series led to the identification of compound 4f, which demonstrated to be active in a Htt171-82Q rat primary striatal neuron assay and a PC12-Exon-1 based assay. This compound was selected for testing in R6/2 mice, in which it was well-tolerated and showed a positive effect on body weight and a positive trend in preventing ventricular volume enlargment. These studies provide strong rationale for further testing the potential benefits of 3-hydroxy-3-trifluoromethylpyrazoles in treating HD.

N-[5-(5-fluoropyridin-3-yl)-1H-pyrazol-3-yl]-4-piperidin-1-ylbutyramide (SEN78702, WYE-308775): a medicinal chemistry effort toward an α7 nicotinic acetylcholine receptor agonist preclinical candidate.

α7 Nicotinic acetylcholine receptors (α7 nAChR) represent promising therapeutic candidates for the treatment of cognitive impairment associated with Alzheimer's disease (AD) and schizophrenia. A medicinal chemistry effort around previously reported compound 1 (SEN15924, WAY-361789) led to the identification of 12 (SEN78702, WYE-308775) a potent and selective full agonist of the α7 nAChR that demonstrated improved plasma stability, brain levels, and efficacy in behavioral cognition models.

Discovery of a novel alpha-7 nicotinic acetylcholine receptor agonist series and characterization of the potent, selective, and orally efficacious agonist 5-(4-acetyl[1,4]diazepan-1-yl)pentanoic acid [5-(4-methoxyphenyl)-1H-pyrazol-3-yl] amide (SEN15924, WAY-361789).

Alpha-7 nicotinic acetylcholine receptors (α7 nAChR) are implicated in the modulation of many cognitive functions such as attention, working memory, and episodic memory. For this reason, α7 nAChR agonists represent promising therapeutic candidates for the treatment of cognitive impairment associated with Alzheimer's disease (AD) and schizophrenia. A medicinal chemistry effort, around our previously reported chemical series, permitted the discovery of a novel class of α7 nAChR agonists with improved selectivity, in particular against the α3 receptor subtype and better ADME profile. The exploration of this series led to the identification of 5-(4-acetyl[1,4]diazepan-1-yl)pentanoic acid [5-(4-methoxyphenyl)-1H-pyrazol-3-yl] amide (25, SEN15924, WAY-361789), a novel, full agonist of the α7 nAChR that was evaluated in vitro and in vivo. Compound 25 proved to be potent and selective, and it demonstrated a fair pharmacokinetic profile accompanied by efficacy in rodent behavioral cognition models (novel object recognition and auditory sensory gating).

Dopamine D2, D3, and D4 selective phenylpiperazines as molecular probes to explore the origins of subtype specific receptor binding.

Assembling phenylpiperazines with 7a-azaindole via different spacer elements, we developed subtype selective dopamine receptor ligands of types 1a,c, 2a, and 3a preferentially interacting with D4, D2, and D3, respectively. To complete this set, the methylthio analogues 2b and 3b exceeding the affinity of 2a and 3a by one order of magnitude and the structural intermediate 1b were synthesized. These chemically similar but biologically divergent target compounds served as molecular probes for radioligand displacement experiments, mutagenesis, and docking studies on homology models based on the recent crystal structure of the beta2-adrenergic receptor. Specific interactions with the highly conserved amino acids Asp3.32 and His6.55 and less conserved residues at positions 2.61, 2.64, 3.28, and 3.29 were identified. Inclusion of a carefully modeled extracellular loop 2 displayed two nonconserved residues in EL2 that differently contribute to ligand binding. Obviously, subtype selectivity is caused by nonconserved but frequently mediated by conserved amino acids.

Chirospecific and subtype selective dopamine receptor binding of heterocyclic methoxynaphthamide analogs.

Employing the D(3) and D(4) selective methoxynaphthalines nafadotride and FAUC 182, respectively, as lead compounds, the pyrazolo[1,5-a]pyridine-3-carboxamides of type 1a and 2a as well as their 2-substituted regioisomers 1b and 2b were synthesized when following an ex-chiral pool approach. Dopamine receptor binding studies involving the target compounds (1a,b, 2a,b) and the respective optical antipodes ent-1a,b and ent-2a,b revealed the heterocyclic carboxamide 2a as a strong and selective D(4) ligand (K(i) = 8.6 nM). According to a mitogenesis assay, 2a shows D(4) partial agonist effects (29%, EC(50) = 6.7 nM) and, thus, might be of interest for the treatment of sexual dysfunction.

Parallel synthesis and biological screening of dopamine receptor ligands taking advantage of a click chemistry based BAL linker.

The click-chemistry-derived formyl indolyl methyl triazole (FIMT) resin 1a was evaluated for the parallel solid-phase synthesis of a series of BP-897-type arylcarboxamides. By application of a five-step sequence (including loading by reductive amination, subsequent amide coupling, deprotection, palladium-catalyzed N-arylation, and acidic cleavage), a focused library of putative dopamine D3 receptor ligands was constructed. The final products revealed good to excellent purity and were screened for binding at monoaminergic G-protein-coupled receptors when selected library members proved to show excellent binding affinity, especially toward the dopamine D3 receptor subtype.