In vivo staging of colitis, adenoma and carcinoma in CRC progression by combination of H4R/DRD4-targeted fluorescent probes.

Colorectal cancer (CRC) is the third most prevalent malignancy and the second leading cause of cancer-related mortality worldwide. Currently, CRC staging heavily relies on invasive surgical procedures for in vitro pathological analysis, which entails long detection cycles and increases the risk of metastasis. There is an urgent need for specific biomarkers to classify adenomas and cancers, while early in vivo staging detection could potentially reduce mortality and morbidity rates. This study focused on Type IV histamine receptor (H4R), which is highly expressed only in the inflammatory stage, and Dopamine receptor D4 (DRD4), which is highly expressed in colorectal adenoma and carcinoma stages. Fluorescent targeted molecular probes H4R-Cy5 and DRD4-M were constructed respectively. The in vitro cell level proves that H4R-Cy5 only has high specificity for RAW264.7 cells, and DRD4-M only has good affinity for HT29 cells. In inflammation-HT29 subcutaneous tumors, H4R-Cy5 and DRD4-M can target inflammation and tumor lesions respectively. In addition, this study is the first to combine the two probes to explore the feasibility of in vivo non-invasive staging on CRC mouse models. The results show that H4R-Cy5 can distinguish and identify the stages of inflammation in vivo, and the DRD4-M probe can accurately identify the stages of colorectal adenoma and carcinoma in vivo. The combination of these two probes can achieve precise non-invasive staging of colitis, adenoma and carcinoma, which is a major advance in the development of accurate diagnostic methods for colorectal precancerous lesions and has important implications for the selection of treatment strategies.

The Pivotal Distinction between Antagonists' and Agonists' Binding into Dopamine D4 Receptor-MD and FMO/PIEDA Studies.

The dopamine D4 receptor (D4R) is a promising therapeutic target in widespread diseases, and the search for novel agonists and antagonists appears to be clinically relevant. The mechanism of binding to the receptor (R) for antagonists and agonists varies. In the present study, we conducted an in-depth computational study, teasing out key similarities and differences in binding modes, complex dynamics, and binding energies for D4R agonists and antagonists. The dynamic network method was applied to investigate the communication paths between the ligand (L) and G-protein binding site (GBS) of human D4R. Finally, the fragment molecular orbitals with pair interaction energy decomposition analysis (FMO/PIEDA) scheme was used to estimate the binding energies of L-R complexes. We found that a strong salt bridge with D3.32 initiates the inhibition of the dopamine D4 receptor. This interaction also occurs in the binding of agonists, but the change in the receptor conformation to the active state starts with interaction with cysteine C3.36. Such a mechanism may arise in the case of agonists unable to form a hydrogen bond with the serine S5.46, considered, so far, to be crucial in the activation of GPCRs. The energy calculations using the FMO/PIEDA method indicate that antagonists show higher residue occupancy of the receptor binding site than agonists, suggesting they could form relatively more stable complexes. Additionally, antagonists were characterized by repulsive interactions with S5.46 distinguishing them from agonists.

Dopamine receptor D4 (DRD4) negatively regulates UCP1- and ATP-dependent thermogenesis in 3T3-L1 adipocytes and C2C12 muscle cells.

The activation of beige fat and muscle tissues is an interesting and encouraging target for therapeutic intervention in obesity owing to their remarkable lipolytic activity and energy-consuming futile cycles. This study examined the effect of dopamine receptor D4 (DRD4) on lipid metabolisms as well as UCP1- and ATP-dependent thermogenesis in Drd4-silenced 3T3-L1 adipocytes and C2C12 muscle cells. Silencing of Drd4, followed by quantitative real-time PCR, immunoblot analysis, immunofluorescence, and staining methods, were applied to evaluate the effects of DRD4 on diverse target genes and proteins of both cells. The findings showed that DRD4 was expressed in the adipose and muscle tissues of normal and obese mice. Furthermore, the knockdown of Drd4 upregulated the expression of brown adipocyte-specific genes and proteins while downregulating lipogenesis and the adipogenesis marker proteins. Drd4 silencing also upregulated the expression of key signaling molecules involved in ATP-dependent thermogenesis in both cells. This was further elucidated by mechanistic studies showing that a Drd4 knockdown mediates UCP1-dependent thermogenesis via the cAMP/PKA/p38MAPK pathway in 3T3-L1 adipocytes and UCP1-independent thermogenesis via the cAMP/SLN/SERCA2a pathway in C2C12 muscle cells. In addition, siDrd4 also mediates myogenesis via the cAMP/PKA/ERK1/2/Cyclin D3 pathway in C2C12 muscle cells. Silencing of Drd4 promotes ß3-AR-dependent browning in 3T3-L1 adipocytes and α1-AR/SERCA-based thermogenesis through an ATP-consuming futile process in C2C12 muscle cells. Understanding the novel functions of DRD4 on adipose and muscle tissues in terms of its ability to enhance energy expenditure and regulate whole-body energy metabolism will aid in developing novel obesity intervention techniques.

Highly Potent and Selective Dopamine D4 Receptor Antagonists Potentially Useful for the Treatment of Glioblastoma.

To better understand the role of dopamine D4 receptor (D4R) in glioblastoma (GBM), in the present paper, new ligands endowed with high affinity and selectivity for D4R were discovered starting from the brain penetrant and D4R selective lead compound 1-(3-(4-phenylpiperazin-1-yl)propyl)-3,4-dihydroquinolin-2(1H)-one (6). In particular, the D4R antagonist 24, showing the highest affinity and selectivity over D2R and D3R within the series (D2/D4 = 8318, D3/D4 = 3715), and the biased ligand 29, partially activating D4R Gi-/Go-protein and blocking ß-arrestin recruitment, emerged as the most interesting compounds. These compounds, evaluated for their GBM antitumor activity, induced a decreased viability of GBM cell lines and primary GBM stem cells (GSC#83), with the maximal efficacy being reached at a concentration of 10 µM. Interestingly, the treatment with both compounds 24 and 29 induced an increased effect in reducing the cell viability with respect to temozolomide, which is the first-choice chemotherapeutic drug in GBM.

Discovery of potent dual ligands for dopamine D4 and σ1 receptors.

During our work on exploration of molecules with some piperidine-triazole scaffolds, we realized that our compounds display chemical similarity with some σ, as well as dopaminergic receptor ligands. Here we show that this series of molecules has indeed strong affinity both for σ1 and dopamine D4 receptors. Moreover, they appear selective towards σ2, dopamine paralogues D1, D2, D3 and D5 receptors and hERG channel. Extensive molecular dynamics with our lead compound AVRM-13 were carried out on σ1, supporting agonist activity of the ligand. Unexpectedly, several observations suggested the existence of a cation binding domain, a probable regulatory site for calcium.

Circuits regulating pleasure and happiness - focus on potential biomarkers for circuitry including the habenuloid complex.

INTRODUCTION: The multiplicity and complexity of the neuronal connections in the central nervous system make it difficult to disentangle circuits that play an essential role in the development or treatment of (neuro)psychiatric disorders. By choosing the evolutionary development of the forebrain as a starting point, a certain order in the connections can be created. The dorsal diencephalic connection (DDC) system can be applied for the development of biomarkers that can predict treatment response. MATERIALS AND METHODS: After providing a brief introduction to the theory, we examined neuroanatomical publications on the connectivity of the DDC system. We then searched for neurochemical components that are specific for the habenula. RESULTS AND DISCUSSION: The best strategy to find biomarkers that reflect the function of the habenular connection is to use genetic variants of receptors, transporters or enzymes specific to this complex. By activating these with probes and measuring the response in people with different functional genotypes, the usefulness of biomarkers can be assessed. CONCLUSIONS: The most promising biomarkers in this respect are those linked to activation or inhibition of the nicotine receptor, dopamine D4 receptor, µ-opioid receptor and also those of the functioning of habenular glia cells (astrocytes and microglia).

Disordered eating in early childhood: DRD4 and DAT1 gene polymorphisms and quality of mother-child interaction.

PURPOSE: Eating disturbances are complex heritable conditions that can be influenced by both genetic and environmental factors but are poorly studied in early development. The aim of this research was to investigate the association of genetic polymorphisms within dopaminergic pathways with early feeding problems. METHODS: We analyzed the presence of VNTR polymorphisms of DRD4 (rs1805186) and DAT1 (rs28363170) in overeating (N = 45), undereating (N = 48) and control (N = 44) young children. We also assessed presence of externalizing, internalizing and dysregulation symptoms by the Child Behavior Checklist and quality of mother-child interactions during feeding by the Italian adaptation of the Scale for the Assessment of Feeding Interaction, respectively. RESULTS: Both polymorphisms were associated with children's eating behavior, psychological symptoms and quality of interaction with their mothers, suggesting that: (a) the DRD4 4-repeat allele behaves as a protective factor, the 2-repeats and 7-repeats alleles as risk factors, for undereating behavior, the general quality of mother-child interaction and internalizing, externalizing and dysregulated symptoms; and (b) the DAT1 9-repeats allele behaves as a protective factor, the 10-repeats allele as a risk factor, for overeating behavior, the general quality of mother-child interaction, internalizing, externalizing and dysregulated symptoms. Finally, a gene x gene interaction is suggested between the DAT1 9-repeat or 10-repeat allele and the DRD4 4-repeat allele. CONCLUSIONS: Our results suggest a role for DRD4 and DAT1 in an early susceptibility to eating disturbances. LEVEL OF EVIDENCE III: Evidence obtained from well-designed case-control analytic study.

The Dopamine D4 Receptor Regulates Gonadotropin-Releasing Hormone Neuron Excitability in Male Mice.

Gonadotropin-releasing hormone (GnRH)-secreting neurons control fertility. The release of GnRH peptide regulates the synthesis and release of both luteinizing hormone (LH) and Follicle stimulation hormone (FSH) from the anterior pituitary. While it is known that dopamine regulates GnRH neurons, the specific dopamine receptor subtype(s) involved remain unclear. Previous studies in adult rodents have reported juxtaposition of fibers containing tyrosine hydroxylase (TH), a marker of catecholaminergic cells, onto GnRH neurons and that exogenous dopamine inhibits GnRH neurons postsynaptically through dopamine D1-like and/or D2-like receptors. Our microarray data from GnRH neurons revealed a high level of Drd4 transcripts [i.e., dopamine D4 receptor (D4R)]. Single-cell RT-PCR and immunocytochemistry confirmed GnRH cells express the Drd4 transcript and protein, respectively. Calcium imaging identified changes in GnRH neuronal activity during application of subtype-specific dopamine receptor agonists and antagonists when GABAergic and glutamatergic transmission was blocked. Dopamine, dopamine with D1/5R-specific or D2/3R-specific antagonists or D4R-specific agonists decreased the frequency of calcium oscillations. In contrast, D1/5R-specific agonists increased the frequency of calcium oscillations. The D4R-mediated inhibition was dependent on Gαi/o protein coupling, while the D1/5R-mediated excitation required Gαs protein coupling. Together, these results indicate that D4R plays an important role in the dopaminergic inhibition of GnRH neurons.

Chronic pain and delinquency partially explain the effect of the DRD4 gene polymorphism on adult substance use.

Background: The dopamine receptor D4 [DRD4] has been reported to be associated with substance use. Yet, the roles that health conditions and behaviors may play in such association are understudied.Objective: This longitudinal study investigated the potential mediation effects of chronic pain and delinquency in adolescence on the association between the DRD4 2-repeat allele and substance use in adulthood. Sex, witnessing violence, and experiencing violence were also examined as potential moderators for the mediation pathways.Methods: We used the restricted and candidate gene data from the National Longitudinal Study of Adolescent to Adult Health (Waves I-IV) to conduct secondary analysis (N = 8,671; 47% male). A two-step approach was adopted to examine the mediation effects regarding four substance use outcomes in adulthood: number of lifetime alcohol use disorder symptoms, lifetime regular smoker status, past-month smoking, and lifetime "pain killer" misuse. The moderation effects were investigated using stratification and permutation.Results: The DRD4 2-repeat allele was associated with all adulthood substance use outcomes through adolescent chronic pain and delinquency (AORs/IRR range 1.08-3.78; all ps<0.01). The association between delinquency and smoking was higher among females. The association between delinquency and substance use was lower among the participants who witnessed violence in adolescence.Conclusions: This study identified modifiable mediators underlying the association between the DRD4 2-repeat allele and substance use behaviors, concluding that chronic pain and delinquency partially explain the effect of the DRD4 gene polymorphism on adult substance use.

Methylation quantitative locus rs3758653 in the DRD4 gene is associated with duration from first heroin exposure to addiction.

Opioid addiction is a chronic brain disease with a high heritability. However, the genetic underpinnings remain uncertain. DNA methylation is involved in the adaptive changes in neuroplasticity after prolonged drug use. The dopamine receptor D4 (DRD4) has an essential role in the reward processes associated with addictive drugs. To further elucidate the potential role and mechanism of the DRD4 gene variants in heroin addiction, we detected the methylation level of 46 CpG sites in the promoter region and the genotypes of three SNPs in the DRD4 gene. Correlations between the SNPs and methylation levels of the CpG sites, i.e., the analysis of methylation quantitative trait loci (mQTLs) was conducted. Following the identification of mQTLs that are unique in the heroin addiction group, we performed an association study between the mQTLs and traits of heroin addiction. Our results revealed that there were several correlations of SNPs rs3758653 and rs11246226 with the methylation levels of some CpG sites in the DRD4 gene. Among these SNP-CpG pairs, rs3758653-DRD4_04, rs3758653-DRD4_05, rs3758653-DRD4_13 and rs3758653-DRD4_03 were unique in the heroin addiction group. Moreover, we found that mQTL rs3758653 was associated with duration from first heroin exposure to addiction, and the expression level of the DRD4 gene in human brain regions of the frontal cortex and hippocampus. Our findings suggested that some mQTLs in the genome may be associated with traits of opioid addiction through implicating the processes of DNA methylation and gene expression.

Recent findings leading to the discovery of selective dopamine D4 receptor ligands for the treatment of widespread diseases.

Since its discovery, the dopamine D4 receptor (D4R) has been suggested to be an attractive target for the treatment of neuropsychiatric diseases. Novel findings have renewed the interest in such a receptor as an emerging target for the management of different diseases, including cancer, Parkinson's disease, alcohol or substance use disorders, eating disorders, erectile dysfunction and cognitive deficits. The recently resolved crystal structures of D4R in complexes with the potent ligands nemonapride and L-745870 strongly improved the knowledge on the molecular mechanisms involving the D4R functions and may help medicinal chemists in drug design. This review is focused on the recent development of the subtype selective D4R ligands belonging to classical or new chemotypes. Moreover, ligands showing functional selectivity toward G protein activation or ß-arrestin recruitment and the effects of selective D4R ligands on the above-mentioned diseases are discussed.

Dopamine improves chemotherapeutic efficacy for pancreatic cancer by regulating macrophage-derived inflammations.

Pancreatic cancer is an inflammatory malignancy, and tumor-associated macrophages (TAMs) are the predominant inflammatory cells in tumor tissue. TAMs have complicated interactions with pancreatic cancer cells, however, the details and mechanisms remain largely unknown. In this study, transcriptomics and proteomics analyses were performed to explore the interactions between murine pancreatic cancer cells and TAMs. Dopamine (DA) has been reported to suppress inflammations. However, its roles in TAMs of pancreatic cancer have not been reported. Herein, the roles and mechanisms of DA to affect the chemotherapeutic efficacy for pancreatic cancer were studied. Multi-omics results revealed that there was a tumor-promoting vicious cycle involving murine pancreatic cancer cells and TAMs. DA substantially improved the chemotherapeutic efficacy both in vitro study and in immunocompetent murine pancreatic cancer models by suppression of the M2 characters of TAMs. Further studies found that activation of DRD4 by DA led to the decrease of cAMP, and then inhibited the activation of PKA/p38 signal pathway, which suppressed the tumor-promoting inflammation of TAMs. This study uncovers the reciprocal interactions between TAMs and pancreatic cancer cells using multi-omics techniques and presents that DA has synergistic roles with chemotherapy for pancreatic cancer by suppressing of TAM-derived inflammations.

DRD4 genotyping may differentiate symptoms of attention-deficit/hyperactivity disorder and sluggish cognitive tempo

Objective: Studies to reduce the heterogeneity of attention-deficit/hyperactivity disorder (ADHD) have increased interest in the concept of sluggish cognitive tempo (SCT). The aim of this study was to investigate if the prevalence of two variable-number tandem repeats (VNTRs) located within the 3′-untranslated region of the DAT1 gene and in exon 3 of the dopamine D4 receptor (DRD4) gene differ among four groups (31 subjects with SCT but no ADHD, 146 individuals with ADHD but no SCT, 67 subjects with SCT + ADHD, and 92 healthy controls). Methods: We compared the sociodemographic profiles, neurocognitive domains, and prevalence of two VNTRs in SCT and ADHD subjects versus typically developing (TD) controls. Results: The SCT without ADHD group had a higher proportion of females and lower parental educational attainment. Subjects in this group performed worse on neuropsychological tests, except for psychomotor speed and commission errors, compared to controls. However, the ADHD without SCT group performed significantly worse on all neuropsychological domains than controls. We found that 4R homozygosity for the DRD4 gene was most prevalent in the ADHD without SCT group. The SCT without ADHD group had the highest 7R allele frequency, differing significantly from the ADHD without SCT group. Conclusion: The 7R allele of DRD4 gene was found to be significantly more prevalent in SCT cases than in ADHD cases. No substantial neuropsychological differences were found between SCT and ADHD subjects.

Genetically predicted gene expression of prefrontal DRD4 gene and the differential susceptibility to childhood emotional eating in response to positive environment.

Genetic differential susceptibility states that individuals may vary both by exhibiting poor responses when exposed to adverse environments, and disproportionally benefiting from positive settings. The dopamine D4 receptor gene (DRD4) may be particularly implicated in these effects, including disturbed eating behaviors that might lead to obesity. Here, we explore differential susceptibility to positive environments according to the predicted genetically regulated gene expression of prefrontal cortex DRD4 gene. Using MAVAN as the discovery cohort (Maternal Adversity, Vulnerability and Neurodevelopment) and GUSTO as the replication cohort (Growing Up in Singapore Towards Healthy Outcomes), we analyzed the interaction between a) a Positive postnatal environmental score, that accounts for positive outcomes in the postnatal period and b) the genetically regulated gene expression of prefrontal DRD4, computed using a machine learning prediction method (PrediXcan). The outcome measures were the pro-intake domains (Emotional over-eating, Food Responsiveness, Food Enjoyment and Desire to Drink) from the Child Eating Behavior Questionnaire at 48 months of age (MAVAN) and 60 months of age (GUSTO). The interaction between the positive environment and the predicted prefrontal DRD4 gene expression was significant for emotional over-eating in MAVAN (ß = -0.403, p < 0.02), in which the high gene expression group had more or less emotional eating according to the exposure to lower or higher positive environment respectively, showing evidence of differential susceptibility criteria. In the replication cohort, a similar result was found with the pro-intake domain Desire to drink (ß = -0.583, p < 0.05). These results provide further evidence for the genetic differential susceptibility, accounting for the benefit of positive environments.

Methylation-related metabolic effects of D4 dopamine receptor expression and activation.

D4 dopamine receptor (D4R) activation uniquely promotes methylation of plasma membrane phospholipids, utilizing folate-derived methyl groups provided by methionine synthase (MS). We evaluated the impact of D4R expression on folate-dependent phospholipid methylation (PLM) and MS activity, as well as cellular redox and methylation status, in transfected CHO cells expressing human D4R variants containing 2, 4, or 7 exon III repeats (D4.2R, D4.4R, D4.7R). Dopamine had no effect in non-transfected CHO cells, but increased PLM to a similar extent for both D4.2R- and D4.4R-expressing cells, while the maximal increase was for D4.7R was significantly lower. D4R expression in CHO cells decreased basal MS activity for all receptor subtypes and conferred dopamine-sensitive MS activity, which was greater with a higher number of repeats. Consistent with decreased MS activity, D4R expression decreased basal levels of methylation cycle intermediates methionine, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH), as well as cysteine and glutathione (GSH). Conversely, dopamine stimulation increased GSH, SAM, and the SAM/SAH ratio, which was associated with a more than 2-fold increase in global DNA methylation. Our findings illustrate a profound influence of D4R expression and activation on MS activity, coupled with the ability of dopamine to modulate cellular redox and methylation status. These previously unrecognized signaling activities of the D4R provide a unique link between neurotransmission and metabolism.

Eckol as a Potential Therapeutic against Neurodegenerative Diseases Targeting Dopamine D3/D4 Receptors.

The G protein-coupled receptor (GPCR) family of proteins comprises signaling proteins that mediate cellular responses to various hormones and neurotransmitters, and serves as a prime target for drug discovery. Towards our goal of discovering secondary metabolites from natural sources that can function as neuronal drugs, we evaluated the modulatory effect of eckol on various GPCRs via cell-based functional assays. In addition, we conducted in silico predictions to obtain molecular insights into the functional effects of eckol. Functional assays revealed that eckol had a concentration-dependent agonist effect on dopamine D3 and D4 receptors. The half maximal effective concentration (EC50) of eckol for the dopamine D3 and D4 receptors was 48.62 ± 3.21 and 42.55 ± 2.54 µM, respectively, while the EC50 values of dopamine as a reference agonist for these two receptors were 2.9 and 3.3 nM, respectively. In silico studies revealed that a low binding energy in addition to hydrophilic, hydrophobic, π⁻alkyl, and π⁻π T-shaped interactions are potential mechanisms by which eckol binds to the dopamine receptors to exert its agonist effects. Molecular dynamics (MD) simulation revealed that Phe346 of the dopamine receptors is important for binding of eckol, similar to eticlopride and dopamine. Our results collectively suggest that eckol is a potential D3/D4 agonist for the management of neurodegenerative diseases, such as Parkinson's disease.

ADHD risk genes involved in dopamine signaling and metabolism are associated with reduced estimated life expectancy at young adult follow-up in hyperactive and control children.

ADHD is associated with an elevated risk of mortality and reduced estimated life expectancy (ELE) by adulthood. Reduced life expectancy is substantially related to the trait of behavioral disinhibition; a correlate of both ADHD and of several dopamine genes related to dopamine signaling and metabolism. We therefore hypothesized that several ADHD risk genes related to dopamine might also be predictive of reduced ELE. Using a longitudinal study of 131 hyperactive children and 71 control cases followed to young adulthood, we examined whether several polymorphisms involving DRD4, DAT1, and DBH were related to ELE. The homozygous 9/9 allele of DAT1 and the heterozygous allele of DBH TaqI were associated with 5- and 2-year reductions, respectively, in total ELE. They did not operate on ELE through any relationships to ADHD specifically or behavioral disinhibition more generally. Instead, they showed links to alcohol use (DBH), reduced education, smoking, and reduced exercise (DAT1) employed in the computation of ELE. We conclude that polymorphisms of two dopamine genes are linked to reductions in ELE independently of their association with ADHD.

Significance of molecular classification of ependymomas: C11orf95-RELA fusion-negative supratentorial ependymomas are a heterogeneous group of tumors.

Extensive molecular analyses of ependymal tumors have revealed that supratentorial and posterior fossa ependymomas have distinct molecular profiles and are likely to be different diseases. The presence of C11orf95-RELA fusion genes in a subset of supratentorial ependymomas (ST-EPN) indicated the existence of molecular subgroups. However, the pathogenesis of RELA fusion-negative ependymomas remains elusive. To investigate the molecular pathogenesis of these tumors and validate the molecular classification of ependymal tumors, we conducted thorough molecular analyses of 113 locally diagnosed ependymal tumors from 107 patients in the Japan Pediatric Molecular Neuro-Oncology Group. All tumors were histopathologically reviewed and 12 tumors were re-classified as non-ependymomas. A combination of RT-PCR, FISH, and RNA sequencing identified RELA fusion in 19 of 29 histologically verified ST-EPN cases, whereas another case was diagnosed as ependymoma RELA fusion-positive via the methylation classifier (68.9%). Among the 9 RELA fusion-negative ST-EPN cases, either the YAP1 fusion, BCOR tandem duplication, EP300-BCORL1 fusion, or FOXO1-STK24 fusion was detected in single cases. Methylation classification did not identify a consistent molecular class within this group. Genome-wide methylation profiling successfully sub-classified posterior fossa ependymoma (PF-EPN) into PF-EPN-A (PFA) and PF-EPN-B (PFB). A multivariate analysis using Cox regression confirmed that PFA was the sole molecular marker which was independently associated with patient survival. A clinically applicable pyrosequencing assay was developed to determine the PFB subgroup with 100% specificity using the methylation status of 3 genes, CRIP1, DRD4 and LBX2. Our results emphasized the significance of molecular classification in the diagnosis of ependymomas. RELA fusion-negative ST-EPN appear to be a heterogeneous group of tumors that do not fall into any of the existing molecular subgroups and are unlikely to form a single category.

The novel piperazine-containing compound LQFM018: Necroptosis cell death mechanisms, dopamine D4 receptor binding and toxicological assessment.

Piperazine is a promising scaffold for drug development due to its broad spectrum of biological activities. Based on this, the new piperazine-containing compound LQFM018 (2) [ethyl 4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)piperazine-1-carboxylate] was synthetized and some biological activities investigated. In this work, we described its ability to bind aminergic receptors, antiproliferative effects as well as the LQFM018 (2)-triggered cell death mechanisms, in K562 leukemic cells, by flow cytometric analyses. Furthermore, acute oral systemic toxicity and potential myelotoxicity assessments of LQFM018 (2) were carried out. LQFM018 (2) was originally obtained by molecular simplification from LASSBio579 (1), an analogue compound of clozapine, with 33% of global yield. Binding profile assay to aminergic receptors showed that LQFM018 (2) has affinity for the dopamine D4 receptor (Ki = 0.26 µM). Moreover, it showed cytotoxicity in K562 cells, in a concentration and time-dependent manner; IC50 values obtained were 399, 242 and 119 µM for trypan blue assay and 427, 259 and 50 µM for MTT method at 24, 48 or 72 h, respectively. This compound (427 µM) also promoted increase in LDH release and cell cycle arrest in G2/M phase. Furthermore, it triggered necrotic morphologies in K562 cells associated with intense cell membrane rupture as confirmed by Annexin V/propidium iodide double-staining. LQFM018 (2) also triggered mitochondrial disturb through loss of ΔΨm associated with increase of ROS production. No significant accumulation of cytosolic cytochrome c was verified in treated cells. Furthermore, it was verified an increase of expression of TNF-R1 and mRNA levels of CYLD with no involviment in caspase-3 and -8 activation and NF-κB in K562 cells. LQFM018 (2) showed in vitro myelotoxicity potential, but it was orally well tolerated and classified as UN GHS category 5 (LD50 > 2000-5000 mg/Kg). Thus, LQFM018 (2) seems to have a non-selective action considering hematopoietic cells. In conclusion, it is suggested LQFM018 (2) promotes cell death in K562 cells via necroptotic signaling, probably with involvement of dopamine D4 receptor. These findings open new perspectives in cancer therapy by use of necroptosis inducing agents as a strategy of reverse cancer cell chemoresistance.

Neuregulin and Dopamine D4 Receptors Contribute Independently to Depotentiation of Schaffer Collateral LTP by Temperoammonic Path Stimulation.

Prior studies have found that dopamine (DA), acting at D4 receptors, and neuregulin (NRG), likely acting at ErbB4 receptors, are involved in a form of depotentiation of long-term potentiation (LTP) at Schaffer collateral (SC) synapses in the hippocampus. Furthermore, DA and NRG actions are intertwined in that NRG induces DA release. We previously found that low-frequency stimulation (LFS) of temperoammonic (TA) inputs to area CA1 also depotentiates previously established SC LTP through a complex signaling pathway involving endocannabinoids, GABA, adenosine, and mitogen-activated protein kinases (MAPKs), but not glutamate. In the present studies, we found that TA-induced SC depotentiation in hippocampal slices from Sprague-Dawley albino rats also involves activation of both D4 receptors and NRG-activated ErbB receptors, but that the roles of these two modulator systems are independent with D4 receptor antagonism failing to alter chemical depotentiation by NRG1ß. Furthermore, a selective D4 receptor agonist was unable to depotentiate SC LTP when administered alone, suggesting that D4 receptor activation is necessary but not sufficient for TA-induced SC depotentiation. Chemical depotentiation by NRG1ß was inhibited by a Pan-ErbB antagonist and by picrotoxin (PTX), an antagonist of GABA-A receptors (GABAARs), indicating that NRG likely promotes SC depotentiation via effects on GABA and interneurons. These findings have implications for understanding the role of DA and NRG in cognitive dysfunction associated with neuropsychiatric illnesses.