Nurr1 Is Not an Essential Regulator of BDNF in Mouse Cortical Neurons

Nurr1 and brain-derived neurotrophic factor (BDNF) play major roles in cognition. Nurr1 regulates BDNF in midbrain dopaminergic neurons and cerebellar granule cells. Nurr1 and BDNF are also highly expressed in the cerebral cortex, a brain area important in cognition. Due to Nurr1 and BDNF tissue specificity, the regulatory effect of Nurr1 on BDNF in different brain areas cannot be generalized. The relationship between Nurr1 and BDNF in the cortex has not been investigated previously. Therefore, we examined Nurr1-mediated BDNF regulation in cortical neurons in activity-dependent and activity-independent states. Mouse primary cortical neurons were treated with the Nurr1 agonist, amodiaquine (AQ). Membrane depolarization was induced by KCl or veratridine and reversed by nimodipine. AQ and membrane depolarization significantly increased Nurr1 (p < 0.001) and BDNF (p_AQ < 0.001, p_KCl < 0.01) as assessed by real-time qRT-PCR. However, Nurr1 knockdown did not affect BDNF gene expression in resting or depolarized neurons. Accordingly, the positive correlation between Nurr1 and BDNF expression in AQ and membrane depolarization experiments does not imply co-regulation because Nurr1 knockdown did not affect BDNF gene expression in resting or depolarized cortical neurons. Therefore, in contrast to midbrain dopaminergic neurons and cerebellar granule cells, Nurr1 does not regulate BDNF in cortical neurons.     

合成化学新概念——组合化学

简明地介绍 2 0世纪 90年代刚刚问世的组合化学新概念以及组合合成基本方法 ,着重叙述了“一珠一肽”混合均分合成法的基本原理及其在新药合成及筛选研究中的应用 ,并对组合合成中编码技术作了初步介绍     

Imaging of selective nuclear receptor modulator-induced conformational changes in the nuclear receptor to allow interaction with coactivator and corepressor proteins in living cells

Selective nuclear receptor modulators (SNRMs), which are used clinically for the treatment of NR-related diseases, display mixed agonistic/antagonistic activity in a tissue-selective manner depending on the cellular concentrations of coregulator proteins, that is, coactivators and corepressors. The molecular details of the SNRM function provided us with an idea for a rational method for the high-throughput screening of SNRMs in real time in intact living cells. We have developed genetically encoded fluorescent indicators based on the principle of ligand-induced coactivator and/or corepressor recruitment to NR ligand binding domain in single living cells. We demonstrated that an SNRM induces a distinct conformational change in the NR LBD, which is different from that induced by a full agonist or antagonist, but favorable for the recruitment of a coactivator or corepressor protein to the NR. The molecular details of an SNRM binding to a NR, and the subsequently induced conformational changes and recruitment of coregulator protein(s) are important features for the understanding of SNRM action in the living body. Our fluorescent indicators are capable of distinguishing among agonists, antagonists, and SNRMs, and can therefore serve as versatile molecular sensors that predict the pharmacological character of ligands, which is important for an accurate cure of a disease.     

The predicted 3D structures of the human M1 muscarinic acetylcholine receptor with agonist or antagonist bound

The muscarinic acetylcholine G-protein-coupled receptors are implicated in diseases ranging from cognitive dysfunctions to smooth-muscle disorders. To provide a structural basis for drug design, we used the MembStruk computational method to predict the 3D structure of the human M1 muscarinic receptor. We validated this structure by using the HierDock method to predict the binding sites for three agonists and four antagonists. The intermolecular ligand-receptor contacts at the predicted binding sites agree well with deductions from available mutagenesis experiments, and the calculated relative binding energies correlate with measured binding affinities. The predicted binding site of all four antagonists is located between transmembrane (TM) helices 3, 4, 5, 6, and 7, whereas the three agonists prefer a site involving residues from TM3, TM6, and TM7. We find that Trp 157(4) contributes directly to antagonist binding, whereas Pro 159(4) provides an indirect conformational switch to position Trp 157(4) in the binding site (the number in parentheses indicates the TM helix). This explains the large decrease in ligand binding affinity and signaling efficacy by mutations of Trp 157(4) and Pro 159(4) not previously explained by homology models. We also found that Asp 105(3) and aromatic residues Tyr 381(6), Tyr 404(7), and Tyr 408(7) are critical for binding the quaternary ammonium head group of the ligand through cation-pi interactions. For ligands with a charged tertiary amine head group, we suggest that proton transfer from the ligand to Asp 105(3) occurs upon binding. Furthermore, we found that an extensive aromatic network involving Tyr 106(3), Trp 157(4), Phe 197(5), Trp 378(6), and Tyr 381(6) is important in stabilizing antagonist binding. For antagonists with two terminal phenyl rings, this aromatic network extends to Trp 164(4), Tyr 179(extracellular loop 2), and Phe 390(6) located at the extracellular end of the TMs. We find that Asn 382(6) forms hydrogen bonds with selected antagonists. Tyr381(6) and Ser 109(3) form hydrogen bonds with the ester moiety of acetylcholine, which binds in the gauche conformation.     

ADP受体抑制剂研究进展

二磷酸腺苷（ADP）是血小板聚集重要活性因子之一,可以促进血小板聚集。ADP受体中的P2Y1和P2Y12受体是抗血小板聚集的重要靶点。简述ADP受体抑制剂作用机制,重点介绍ADP受体抑制剂的结构类型和构效关系。     

Variability in Behavioral Phenotypes after Forced Swimming-Induced Stress in Rats Is Associated with Expression of the Glucocorticoid Receptor,Nurr1, and IL-1β in the Hippocampus

Individual differences in coping with stress may determine either a vulnerable or resilient phenotype. Therefore, it is important to better understand the biology underlying the behavioral phenotype. We assessed whether individual behavioral phenotype to acute stress is related with the hippocampal expression of glucocorticoid receptor (GR), Nurr1, interleukin-1 beta (IL-1β) or brain-derived neurotrophic factor (BDNF). Wistar male rats were exposed to forced swimming for 15 min and sacrificed at different times. Behavioral response was analyzed, and it was compared with the gene and protein expression of GR, Nurr1, IL-1β and BDNF in the hippocampus for each time point. Behavioral phenotyping showed a group with high immobility (vulnerable) while another had low immobility (resilient). No significant differences were found in the Nurr1, IL-1β and BDNF mRNA levels between resilient and vulnerable rats at different recovery times except for Nr3c1 (gene for GR). However, exposure to stress caused significantly higher levels of GR, Nurr1 and IL-1β proteins of vulnerable compared to resilient rats. This variability of behavioral phenotypes is associated with a differential molecular response to stress that involves GR, Nurr1, and IL-1β as mediators in coping with stress. This contributes to identifying biomarkers of susceptibility to stress.     

Design, synthesis, and biological evaluation of aminoboronic acids as growth-factor receptor inhibitors of EGFR and VEGFR-1 tyrosine kinases

A series of aminoboronic acids was synthesized based on the structure of lavendustin pharmacophore 1. Their inhibitory activities against the epidermal growth-factor receptor (EGFR) and vascular endothelial growth-factor receptor-1 (VEGFR-1, Flt-1) protein tyrosine kinases, and various protein kinases, PKA, PKC, PTK, and eEF2K were evaluated. Selective inhibition activities were observed in a series of aminoboronic acids. 4-Methoxy-3-((2- methoxyphenylamino)methyl)phenylboronic acid 10 inhibited EGFR tyrosine kinase, whereas 4-(2,5-dihydroxybenzylamino)phenylboronic acid 12 inhibited Flt-1 protein kinase, although lavendustin pharmacophore 1 inhibited both EGFR and Flt-1 kinases at a compound concentration of 1.0 microg mL(-1). The selective inhibition of EGFR by 10 is considered to be due to the substitution of the dihydroxy groups on the benzyl moiety for a boronic acid group at the para position, whereas the selective inhibition of Flt-1 by 12 is due to the substitution of the carboxyl group on the aniline moiety in the lavendustin pharmacophore 1 for a boronic acid group.     

Malassezin, a novel agonist of the aryl hydrocarbon receptor from the yeast Malassezia furfur, induces apoptosis in primary human melanocytes

Pityriasis versicolor is the most common skin mycosis in humans worldwide. Yeasts of the genus Malassezia, particularly M. furfur, a saprophyte occurring widely on human skin, are generally regarded as the causative agents. Pityriasis versicolor is often accompanied by a long-lasting depigmentation that persists even after successful antimycotic therapy. M. furfur is able to convert tryptophan into a variety of indole alkaloids, some of them showing biological properties that correlate well with certain clinical features of pityriasis versicolor. This suggests a possible role for these compounds in the depigmentation process. We now report that human melanocytes undergo apoptosis when exposed to the crude mixture of tryptophan metabolites from M. furfur. The active compound was identified as malassezin, previously isolated by us from the same source and characterized as an agonist of the aryl hydrocarbon (Ah) receptor. The compound could, therefore, contribute to the marked depigmentation observed during the course of pityriasis versicolor.     

Generation and Screening of Oxime Libraries Addressing the Neuronal GABA Transporter GAT1

The objective of the present study was to transfer the concept of library screening by MS binding assays, so far applied to pseudostatic hydrazine libraries, to static oxime libraries to screen for new potent inhibitors of mGAT1, the most abundant GABA transporter in the central nervous system that represents a validated drug target for the treatment of epilepsy. Library generation was performed by reaction of guvacine derivatives possessing a hydroxylamine functionality with various sets of four aldehydes. After dilution, the libraries were screened by competitive MS binding assays. Deconvolution experiments allowed hits in the most active libraries to be identified, and they were resynthesized for biological evaluation. That way a series of compounds was identified that displayed binding affinities ≥8.00 (pK_i) at mGAT1, one of which was found to be the most potent mGAT1 inhibitor known to date in a functional GABA uptake assay with a pIC₅₀ value of 8.27±0.03.     

Proteomic Tools for the Quantitative Analysis of Artificial Peptide Libraries: Detection and Characterization of Target-Amplified PD-1 Inhibitors

We report a quantitative proteomics data analysis pipeline, which coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted, amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.     

Substituted pyrazolo[3,4-b]pyridines as potent A1 adenosine antagonists: synthesis, biological evaluation, and development of an A1 bovine receptor model

Sixty-eight new substituted pyrazolo[3,4-b]pyridine derivatives were synthesized and tested for enriching a library of active A(1) adenosine receptor (AR) antagonists belonging to the same class. These compounds were also used as an external test set to check the reliability of a 3D QSAR model recently reported by us. To investigate the binding mode of pyrazolopyridine derivatives, a model of the bovine A(1)AR (bA(1)AR) was developed by a novel homology modeling approach and used to evaluate the main interactions of the ligands with the receptor through docking studies. Results suggest important interactions of the ligands mainly with L3.33(88), T3.36(91), Q3.37(92) and H6.52(251), in agreement with mutagenesis data. The racemic mixture of the most active compound was separated into the corresponding enantiomers which showed a bA(1)AR affinity in the nanomolar range, with the R enantiomer sevenfold more active than the S enantiomer, according to results derived from calculations on the receptor model. Analysis of the bovine/human A(1)AR affinity profile of ligands supported the hypothesis that such receptors should be characterized by a different size of their binding site, responsible for the different affinity of the antagonists.     

Carbon-13 nuclear magnetic resonance study of ethylene-1-octene and ethylene-4-methyl-1-pentene copolymers

Detailed assignments of ¹³C n.m.r. signals of ethylene-1-octene and ethylene-4-methyl-1-pentene copolymers, members of the linear low-density polyethylene (LLDPE) family, are presented. The equations relating signal intensities to the monomer sequences are given. Using these equations, the characterization of these copolymers by the triad monomer sequences is possible. From the analysis by the triad sequence, it is suggested that these 1-olefins have a tendency to be present isolated in the copolymer chain.     

Identification of Cyclophilin A as a Potential Anticancer Target of Novel Nargenicin A1 Analog in AGS Gastric Cancer Cells

We recently discovered a novel nargenicin A1 analog, 23-demethyl 8,13-deoxynargenicin (compound 9), with potential anti-cancer and anti-angiogenic activities against human gastric adenocarcinoma (AGS) cells. To identify the key molecular targets of compound 9, that are responsible for its biological activities, the changes in proteome expression in AGS cells following compound 9 treatment were analyzed using two-dimensional gel electrophoresis (2-DE), followed by MALDI/TOF/MS. Analyses using chemical proteomics and western blotting revealed that compound 9 treatment significantly suppressed the expression of cyclophilin A (CypA), a member of the immunophilin family. Furthermore, compound 9 downregulated CD147-mediated mitogen-activated protein kinase (MAPK) signaling pathway, including c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) by inhibiting the expression of CD147, the cellular receptor of CypA. Notably, the responses of AGS cells to CypA knockdown were significantly correlated with the anticancer and antiangiogenic effects of compound 9. CypA siRNAs reduced the expression of CD147 and phosphorylation of JNK and ERK1/2. In addition, the suppressive effects of CypA siRNAs on proliferation, migration, invasion, and angiogenesis induction of AGS cells were associated with G2/M cell cycle arrest, caspase-mediated apoptosis, inhibition of MMP-9 and MMP-2 expression, inactivation of PI3K/AKT/mTOR pathway, and inhibition of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression. The specific interaction between compound 9 and CypA was also confirmed using the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA) approaches. Moreover, in silico docking analysis revealed that the structure of compound 9 was a good fit for the cyclosporin A binding cavity of CypA. Collectively, these findings provide a novel molecular basis for compound 9-mediated suppression of gastric cancer progression through the targeting of CypA.     

Investigations on the 4-quinolone-3-carboxylic acid motif part 5: modulation of the physicochemical profile of a set of potent and selective cannabinoid-2 receptor ligands through a bioisosteric approach

Three heterocyclic systems were selected as potential bioisosteres of the amide linker for a series of 1,6-disubstituted-4-quinolone-3-carboxamides, which are potent and selective CB2 ligands that exhibit poor water solubility, with the aim of improving their physicochemical profile and also of clarifying properties of importance for amide bond mimicry. Among the newly synthesized compounds, a 1,2,3-triazole derivative (1-(adamantan-1-yl)-4-[6-(furan-2-yl)-1,4-dihydro-4-oxo-1-pentylquinolin-3-yl]-1H-1,2,3-triazole) emerged as the most promising in terms of both physicochemical and pharmacodynamic properties. When assayed in vitro, this derivative exhibited inverse agonist activity, whereas, in the formalin test in mice, it produced analgesic effects antagonized by a well-established inverse agonist. Metabolic studies allowed the identification of a side chain hydroxylated derivative as its only metabolite, which, in its racemic form, still showed appreciable CB2 selectivity, but was 150-fold less potent than the parent compound.