HECW1 restrains cervical cancer cell growth by promoting DVL1 ubiquitination and downregulating the activation of Wnt/ß-catenin signaling.

HECW1 belongs to ubiquitin ligase (E3) HECT family, and is found to be involved in tumorigenesis and tumor progression. However, the function of HECW1 in cervical cancer (CC) remains unknown. Clinical analysis showed that HECW1 is significantly decreased in CC tumor tissues. Ectopic expression of HECW1 suppressed cell growth, promoting cell cycle arrest and apoptosis in CC cells, while downregulation of HECW1 reversed these trends, impeded proliferation and accelerated cell cycle progression of CC cells. Overexpressing of HECW1 reduced mitochondrial membrane potential and the protein expression of voltage-dependent anion channel 1 (VDAC1). In addition, upregulation of HECW1 inhibited nuclear ß-catenin accumulation, downregulated ß-catenin/TCF/LEF-mediated transcriptional activity and the expression of downstream gene c-Myc, whereas inhibition of HECW1 received opposite results. Further results confirmed HECW1 affects the protein expression of dishevelled-1 (DVL1), a potent activator of Wnt/ß-catenin, and inhibition of HECW1 inhibited the ubiquitination of DVL1, upregulating its expression. Inhibition of DVL1 restrained the promotion effect of HECW1 suppression on cell proliferation. In vivo experiments also verified that HECW1 suppression promoted the tumor formation of CC cells. Summary, we demonstrated that HECW1 inhibits CC cell proliferation and tumor formation by downregulating DVL1 induced Wnt/ß-catenin signaling pathway activation.

MUC16 promotes EOC proliferation by regulating GLUT1 expression.

As a common malignancy in females with a higher incidence rate, epithelial ovarian cancer (EOC) is a heterogeneous disease with complexity and diversity in histology and therapeutic response. Although great progress has been made in diagnosis and therapeutic strategies, novel therapeutic strategies are required to improve survival. Although the promoting effect of mucin 16 (MUC16) on tumour progression has been reported, the potential mechanisms remain unclear. In our study, we reported that overexpression of MUC16 was significantly related to cell proliferation and disease progression in EOC. Results from clinical specimen analysis and cell experiment support this conclusion. Patients with a high MUC16 expression usually had a worse prognosis that those with a low expression. Cell proliferation ability was significantly decreased in EOC cell lines when the knockdown of MUC16. Further study shows that the function of MUC16 in cell proliferation is based on the regulation of glucose transporter 1 (GLUT1) expression. MUC16 can control glucose uptake by regulating GLUT1 in EOC cells, thereby promoting glycogen synthesis, so that tumour cells produce more energy for proliferation. This conclusion is based on two findings. First, the significant correlation between MUC16 and GLUT1 was verified by clinical specimen and TCGA data analysis. Then, alteration of MUC16 expression levels can affect the expression of GLUT1 and glucose uptake was also verified. Finally, this conclusion is further verified in vivo by tumour-bearing mice model. To summarize, our results suggest that MUC16 promotes EOC proliferation and disease progression by regulating GLUT1 expression.

Gene expression pattern of trophoblast-specific transcription factors in trophectoderm by analysis of single-cell RNA-seq data of human blastocyst.

The dysfunction of placenta development is correlated to the defects of pregnancy and fetal growth. The detailed molecular mechanism of placenta development is not identified in humans due to the lack of material in vivo. Trophoblast (TB) lineage derived from human embryonic stem cells (hESCs) induced by bone morphogenetic protein 4 (BMP4) has been applied as a model for studying TB lineage specification in vitro. With the development of single-cell sequencing technology, it became possible to detect the transcriptome of the post-implantation embryo at unprecedented precision. In this study, we reanalyzed single-cell RNA-seq of post-implantation embryos derived from two separate groups and identified different subtypes of trophoblast cells and their marker, respectively. At the same time, we focused on the gene expression patterns of trophoblast-specific transcription factors in different models. Our analysis sheds new light on the transcription regulation mechanism of trophoblast differentiation at the early stage of pregnancy establishment in human.

MicroRNA-545 suppresses progression of ovarian cancer through mediating PLK1 expression by a direct binding and an indirect regulation involving KDM4B-mediated demethylation.

BACKGROUND: Ovarian cancer (OC) is a life-threatening gynecological malignancy where dysregulation of microRNAs (miRNAs) is frequently implicated. This study focuses on the function of miR-545 on OC development and the molecules involved. METHODS: miR-545 expression in OC tissues and cell lines was determined, and its link to the survival of patients was analyzed. Altered expression of miR-545 was induced to determine its role in proliferation, apoptosis, migration and invasion of OC cells and the angiogenesis ability of human umbilical vein endothelial cells (HUVECs). The targeting mRNAs of miR-545 were predicted and validated through luciferase assays. Gain-of-function studies of KDM4B and PLK1 were performed to explore their involvements in OC development. In vivo experiments were conducted by inducing xenograft tumors in nude mice. RESULTS: Poor expression of miR-545 was found in OC tissues and cells compared to the normal ones and it indicated unfavorable prognosis in patients. Overexpression of miR-545 suppressed growth, migration, invasion and angiogenesis of OC cells as well as the angiogenesis ability of HUVECs. miR-545 was found to target mRNAs of KDM4B and PLK1, while KDM4B promoted the transcription of the PLK1 promoter through demethylation of H3K9me3. Either overexpression of KDM4B or PLK1 partially blocked the inhibitory effects of miR-545 mimic on OC cell growth, especially the former one. The in vitro results were reproduced in vivo. CONCLUSION: This study evidenced that miR-545 suppresses progression of OC through mediating PLK1 expression by a direct binding and an indirect regulation involving KDM4B-mediated demethylation.

[Diagnosis of glutaric acidemia type â ¡C by fetal whole exome sequencing].

OBJECTIVE: To explore the genetic basis of a fetus with enlargement and enhanced echo of the kidneys. METHODS: The imaging data of the fetus were collected, in addition with 20 mL amniotic fluid sample and 2 mL peripheral blood samples of both parents. Amniotic DNA was extracted for library construction and whole exome sequencing, and Sanger sequencing was carried out to verify candidate variant associated with the fetal phenotype. RESULTS: Prenatal ultrasound showed that the fetus had enlargement and enhanced echo of the kidneys, in addition with many small renal cysts. Whole exome sequencing showed that the fetus carried pathogenic compound heterozygous variants of the ETFDH gene, namely c.3G>C and c.1436dupA. Sanger sequencing of the family suggested that the variants were inherited from its mother and father, respectively. CONCLUSION: By combining its clinical manifestations and results of whole exome sequencing, the fetus was diagnosed as glutaric acidemia type ⅡC due to the compound heterozygous variants of the ETFDH gene. Above results have provided a basis for prenatal diagnosis and genetic counseling. Fetal exome sequencing has provided an important tool for prenatal diagnosis.

Melatonin enhances TNF-α-mediated cervical cancer HeLa cells death via suppressing CaMKII/Parkin/mitophagy axis.

BACKGROUND: Tumor necrosis factor-α (TNF-α) immunotherapy controls the progression of human cervical cancer. Here, we explored the detailed molecular mechanisms played by melatonin in human cervical cancer (HeLa cells) death in the presence of TNF-α injury, with a particular attention to the mitochondrial homeostasis. METHODS: HeLa cells were incubated with TNFα and then cell death was determined via MTT assay, TUNEL staining, caspase ELISA assay and western blotting. Mitochondrial function was detected via analyzing mitochondrial membrane potential using JC-1 staining, mitochondrial oxidative stress using flow cytometry and mitochondrial apoptosis using western blotting. RESULTS: Our data exhibited that treatment with HeLa cells using melatonin in the presence of TNF-α further triggered cancer cell cellular death. Molecular investigation demonstrated that melatonin enhanced the caspase-9 mitochondrion death, repressed mitochondrial potential, increased ROS production, augmented mPTP opening rate and elevated cyt-c expression in the nucleus. Moreover, melatonin application further suppressed mitochondrial ATP generation via reducing the expression of mitochondrial respiratory complex. Mechanistically, melatonin augmented the response of HeLa cells to TNF-α-mediated cancer death via repressing mitophagy. TNF-α treatment activated mitophagy via elevating Parkin expression and excessive mitophagy blocked mitochondrial apoptosis, ultimately alleviating the lethal action of TNF-α on HeLa cell. However, melatonin supplementation could prevent TNF-α-mediated mitophagy activation via inhibiting Parkin in a CaMKII-dependent manner. Interestingly, reactivation of CaMKII abolished the melatonin-mediated mitophagy arrest and HeLa cell death. CONCLUSIONS: Overall, our data highlight that melatonin enhances TNF-α-induced human cervical cancer HeLa cells mitochondrial apoptosis via inactivating the CaMKII/Parkin/mitophagy axis.

Gα12/13 signaling promotes cervical cancer invasion through the RhoA/ROCK-JNK signaling axis.

Several reports have indicated a role for the members of the G12 family of heterotrimeric G proteins (Gα12 and Gα13) in oncogenesis and tumor cell growth. The aims of the present study were to evaluate the role of G12 signaling in cervical cancer. We demonstrated that expression of the G12 proteins was highly upregulated in cervical cancer cells. Additionally, expression of the activated forms of Gα12/Gα13 but not expression of activated Gαq induced cell invasion through the activation of the RhoA family of G proteins, but had no effect on cell proliferation in the cervical cancer cells. Inhibition of G12 signaling by expression of the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (p115-RGS) blocked thrombin-stimulated cell invasion, but did not inhibit cell proliferation in cervical cells, whereas the inhibition of Gαq (RGS2) had no effect. Furthermore, G12 signaling was able to activate Rho proteins, and this stimulation was inhibited by p115-RGS, and Gα12-induced invasion was blocked by an inhibitor of RhoA/B/C (C3 toxin). Pharmacological inhibition of JNK remarkably decreased G12-induced JNK activation. Both a JNK inhibitor (SP600125) and a ROCK inhibitor (Y27632) reduced G12-induced JNK and c-Jun activation, and markedly inhibited G12-induced cellular invasion. Collectively, these findings demonstrate that stimulation of G12 proteins is capable of promoting invasion through RhoA/ROCK-JNK activation.

Suramin inhibits cell proliferation in ovarian and cervical cancer by downregulating heparanase expression.

BACKGROUND: Aberrant expression of heparanase (Hpa) is associated with apoor prognosis in ovarian and cervical cancer patients. Inhibitors of Hpa can prevent the growth and metastasis of malignant tumor cells, and suramin may be such a compound that has strong anti-proliferative effects on several kinds of cancer cells. We have therefore tested whether the growth inhibiting effect of suramin on ovarian and cervical cancer cells is due to downregulation of Hpa expression. RESULTS: Suramin at 300-600 µg/ml significantly inhibited HO-8910 PM and HeLa cell growth at 24 h, in both a time-dependent and dose-dependent manner, with an IC50 of 320 µg/ml and 475 µg/ml, respectively. Suramin at 300 µg/ml significantly decreased the expression of Hpa mRNA (P < 0.005) and protein (P < 0.005) in both HO-8910 PM and HeLa cells at 48 h. CONCLUSIONS: The inhibitory effect of suramin on Hpa enzyme may be due to downregulating of its expression in cancer cells. These findings confirm the importance of Hpa in tumor growth and the potential clinical application of Hpa inhibitors in the treatment of ovarian and cervical cancer.

[Effect of 1,3-O,N spiroheterocyclic inhibitors of heparanase on the growth of HeLa cells].

OBJECTIVE: To provide the theoretical supportting for targeted heparanase (HPA) inhibition of cervical cancer through observing the anti-proliferative effect of the HPA inhibitor on HeLa cell line of cervical cancer. METHODS: The two series of 13 kinds of novel HPA inhibitors were synthesized and optimized. Heparan degrading enzyme assay kit was used to test the effect of the inhibitors on the inhibition of HPA enzyme activity. Methyl thiazolyl tetrazolium (MTT) method and scratch test were used to observe the anti-proliferative and the migration effect of the inhibitors on HeLa cells. Flow cytometry was performed to determine the cell cycles and apoptosis. The expression of HPA was evaluated by reverse transcription (RT)-PCR, western blot and immunocytochemistry. RESULTS: All tested inhibitors could inhibit the activity of HPA enzyme [the range of 50% inhibiting concentration (IC50) values from 4.47 to 47.19 µmol/L] and the growth of HeLa cells (the range of IC50 values from 48.16 to 96.64 µmol/L). Among them, No.16 compound exhibits the strongest inhibition against the growth of HeLa, which could arrest the cell into G0/G1 and G2/M phases. The rate of cell apoptosis in the group treated with 50 µmol/L No.16 for 48 hours [(11.9 ± 1.2)%] was significantly higher than that [(6.6 ± 1.8)%] in untreated group (P = 0.013). Real time PCR and western blot showed that expression levels of HPA mRNA (1.23 ± 0.46) and protein (0.46 ± 0.31) significantly decreased in the treated group as compared with the levels of HPA mRNA (3.43 ± 0.45) and protein (1.30 ± 0.58) in the untreated group (both P < 0.05). Immunocytochemistry also showed that the treatment of No.16 significantly reduced the average optical density (0.39 ± 0.04) of HPA immuostaining signal compared with that in the control group (0.50 ± 0.09; P = 0.026). CONCLUSION: Novel 1,3-O,N spiroheterocyclic HPA inhibitors could inhibit the proliferation of HeLa cells, inhibit the HPA enzyme activity in different degree, and downregulate the expression of HPA protein.

Circ_0007445 inhibits trophoblast cell proliferation, migration and invasion by mediating the miR-4432/HTRA1 axis in preeclampsia.

BACKGROUND: : Circular RNAs (circRNAs) have been shown to be extensively involved in preeclampsia progression. At present, the role of circ_0007445 in preeclampsia progression is not clear. METHODS: A total of 30 preeclampsia patients and 30 normal pregnant women were recruited in our study. The function of trophoblast cells was explored to clarify the role and mechanism of circ_0007445 on the preeclampsia progression. The expression of circ_0007445, microRNA (miR)-4432 and high temperature requirement A1 (HTRA1) was analyzed by quantitative real-time PCR. The proliferation, migration and invasion of trophoblast cells were determined by cell counting kit 8 assay, EdU assay, colony formation assay, flow cytometry, and transwell assay. Protein expression was examined by western blot analysis. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay were used to assess RNA interaction relationships. RESULTS: Our data suggested that circ_0007445 had increased expression in preeclampsia patients. Knockdown of circ_0007445 enhanced trophoblast cell proliferation, migration and invasion. MiR-4432 was lowly expressed in preeclampsia patients, and it could be sponged by circ_0007445. MiR-4432 inhibitor overturned the promotion effects of circ_0007445 knockdown on trophoblast cell functions. HTRA1 was highly expressed in preeclampsia patients, and it could be targeted by miR-4432. HTRA1 overexpression could also reverse the proliferation, migration and invasion of trophoblast cells promoted by miR-4432 mimic. In addition, circ_0007445 positively regulated HTRA1 through targeting miR-4432. CONCLUSION: :Our results suggested that circ_0007445 facilitated the development of preeclampsia by suppressing trophoblast cell function through miR-4432/HTRA1 axis.

NR2F1 overexpression alleviates trophoblast cell dysfunction by inhibiting GDF15/MAPK axis in preeclampsia.

Abnormal functions of trophoblast cells are associated with the pathogenesis of preeclampsia (PE). Nuclear receptor subfamily 2 group F member 1 (NR2F1) acts as a transcriptionally regulator in many diseases, but its role in PE remains unknown. Hypoxia/reoxygenation (H/R)-stimulated HTR-8/SVneo cells were used to mimic PE injury in vitro. NR2F1 overexpression alleviated trophoblast apoptosis, as evidenced by the decreased number of TUNEL-positive cells and the downregulation of caspase 3 and caspase 9 expression in cells. NR2F1 overexpression increased the invasion and migration ability of HTR-8/SVneo cells, accompanied by increased protein levels of matrix metalloproteinase (MMP)-2 and MMP-9. mRNA-seq was applied to explore the underlying mechanism of NR2F1, identifying growth differentiation factor 15 (GDF15) as the possible downstream effector. Dual-luciferase reporter, ChIP-qPCR, and DNA pull-down assays confirmed that NR2F1 bound to the promoter of GDF15 and transcriptionally inhibited its expression. GDF15 overexpression increased apoptosis and decreased the ability of invasion and migration in HTR-8/SVneo cells expressing NR2F1. MAPK pathway was involved in the regulation of PE. Administration of p38 inhibitor, ERK inhibitor, and JNK inhibitor reversed the effect of simultaneous overexpression NR2F1 and GDF15 on trophoblast apoptosis, invasion, and migration. Our findings demonstrated that NR2F1 overexpression inhibited trophoblast apoptosis and promoted trophoblast invasion and migration. NR2F1 might negatively regulate GDF15 expression by binding to its promoter region, which further inhibited MAPK signaling pathway in PE. Our study highlights that NR2F1 might sever as a potential target in PE.

Positron emission tomography imaging of dopamine D2 receptors using a highly selective radiolabeled D2 receptor partial agonist.

A series of microPET imaging studies were conducted in anesthetized rhesus monkeys using the dopamine D2-selective partial agonist, [(11)C]SV-III-130. There was a high uptake in regions of brain known to express a high density of D2 receptors under baseline conditions. Rapid displacement in the caudate and putamen, but not in the cerebellum, was observed after injection of the dopamine D2/3 receptor nonselective ligand S(-)-eticlopride at a low dosage (0.025mg/kg/i.v.); no obvious displacement in the caudate, putamen and cerebellum was observed after the treatment with a dopamine D3 receptor selective ligand WC-34 (0.1mg/kg/i.v.). Pretreatment with lorazepam (1mg/kg, i.v. 30min) to reduce endogenous dopamine prior to tracer injection resulted in unchanged binding potential (BP) values, a measure of D2 receptor binding in vivo, in the caudate and putamen. d-Amphetamine challenge studies indicate that there is a significant displacement of [(11)C]SV-III-130 by d-Amphetamine-induced increases in synaptic dopamine levels.

Interference with XPO1 Suppresses the Stemness and Radioresistance of CD44 Positive Cervical Cancer Cells via Binding with Rad21.

OBJECTIVE: Cancer stem cells (CSCs) are responsible for cervical cancer progression and decreased radiosensitivity of tumor cells. The present work is meant to illuminate the impacts of exportin 1 (XPO1) on the aggressive behaviors and radiosensitivity of cervical cancer stemness cells and make a deeper inquiry into its regulatory mechanism despite that XPO1 has been supported to elicit significant activities on multiple malignancies. METHODS: XPO1 and Rad21 expression in HeLa (CD44+) cells was tested by RT-qPCR and western blot. Cell viability was estimated via CCK-8 assay. Cell stemness was examined via sphere formation assay and western blot. Following radiation treatment, cell proliferation was judged by CCK-8 assay, western blot as well as EdU staining whereas TUNEL assay, RT-qPCR and western blot analysis appraised cell apoptosis. Cell radiosensitivity was assessed through clonogenic survival assay. The levels of DNA damage markers were tested by western blot and related kits. STRING database and Co-IP assay respectively predicted and testified the binding of XPO1 with Rad21. RT-qPCR and western blot also examined the expression of XPO1 cargoes. RESULTS: The experimental data corroborated that XPO1 and Rad21 were overexpressed in cervical cancer tissues and cells. XPO1 inhibitor KPT-330 impeded the stemness while elevated the radiosensitivity of HeLa (CD44+) cells. XPO1 bond to Rad21 and positively modulated Rad21 expression. Moreover, Rad21 elevation reversed the impacts of KPT-330 on the behaviors of cervical cancer stemness cells. CONCLUSION: To sum up, XPO1 might impact the aggressive behavior and radioresistance of cervical cancer stemness cells through binding with Rad21.

Comprehensive analysis and experimental validation reveal elevated CLCN4 is a promising biomarker in endometrial cancer.

Several studies have reported the role of CLCN4 in tumor progression. However, its mechanism remains to be thoroughly studied. The objective of this study was to explore the potential pathogenic role of CLCN4 in endometrial carcinoma (UCEC) with a better understanding of the pathological mechanisms involved. The potential roles of CLCN4 in different tumors were explored based on The Cancer Genome Atlas (TCGA), the expression difference, mutation, survival, pathological stage, Immunity subtypes, Immune infiltration, tumor microenvironment (TME), tumor mutation burden (TMB), microsatellite instability (MSI), mismatch repair (MMR) related to CLCN4 were analyzed. Then, the expression, prognosis, mutation, and functional enrichment of CLCN4 in UCEC were analyzed. Immunohistochemical experiment was used to verify the expression of CLCN4 in endometrial cancer tissues and normal tissues. In vitro, we knocked down of CLCN4 in HEC-1-A cells and performed CCK8, WB, RT-PCR, wound-healing, transwell assays to further validation of the molecular function. Results revealed that high expression of CLCN4 was observed in 20 cancer types of TCGA. CLCN4 expression correlates with poor survival in MESO, BLCA, THCA, especially UCEC tumors. CLCN4 expression was significantly associated with CD4+ T-cell infiltration, especially CD4+ Th1-cell. Immunohistochemical experiment reveals that CLCN4 is high expressed in endometrial tumors, in vitro experiment reveals that knockdown of CLCN4 inhibits the cells proliferation, migration and invasion. Our study is the first to offer a comprehensive understanding of the oncogenic roles of CLCN4 on different tumors. CLCN4 may become a potential biomarker in UCEC.

Exosome-delivered circRNA circSYT15 contributes to cisplatin resistance in cervical cancer cells through the miR-503-5p/RSF1 axis.

The development of chemotherapy resistance is a major obstacle for cervical cancer (CC) patients. Exosome-mediated transfer of circular RNAs (circRNAs) was found to have relevance to the CC. This study is designed to explore the role and mechanism of exosomal circRNA synaptotagmin 15 (circSYT15) on cisplatin (DDP) resistance in CC. Cell proliferation ability and apoptosis rate were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), colony formation, and flow cytometry assays. CircSYT15, microRNA-503-5p (miR-503-5p), Remodeling spacing factor 1 (RSF1) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Exosomes were analyzed by a transmission electron microscope and nanoparticle tracking analysis. CD63, CD81, TSC101, Bcl-2, Bax, C-caspase 3, and RSF1 protein levels were examined by western blot assay. The binding between miR-503-5p and circSYT15 or RSF1 was predicted by circBank or Starbase and then verified by a dual-luciferase reporter and RNA Immunoprecipitation (RIP). The biological role of exosomal circSYT15 in DDP resistance of CC in vivo. CircSYT15 was upregulated in the DDP-resistant CC cells and exosomes isolated from DDP-resistant CC cells. CircSYT15 knockdown repressed the proliferation and drug resistance of CC and induced apoptosis in CC cells. Exosomes shuttled circSYT15 act as a sponge to affect RSF1 expression, thereby promoting proliferation and drug resistance and repressing apoptosis of sensitive CC cells. Exosomal circSYT15 boost DDP resistance of cervical cancer in vivo. Exosome-mediated transfer of circSYT15 enhanced DDP resistance in CC partly by targeting the miR-503-5p/RSF1 axis, providing a foundation for future clinical applications of CC drug resistance.

Hybrids of polyphenolic/quinone acids, the potential preventive and therapeutic drugs for PD: Disaggregate α-Syn fibrils, inhibit inclusions, and repair damaged neurons in mice.

Neurotoxic α-Syn fibers, the main components of Lewy bodies, play a key role in the development of PD characterized by a progressive loss of dopaminergic neurons. Here, we designed and synthesized the hybrids of polyphenolic/quinone acids. The candidate compounds showed high α-Syn aggregation inhibitory activities in vitro with IC50 down to 1.6 µM. The inhibition went through the aggregation process by stabilizing the conformation of α-Syn proteostasis and preventing ß-sheets aggregation, especially in the lag phase. Furthermore, the candidate drugs could disintegrate the preformed varisized aggregates into pony-size aggregates and functional monomers and continually inhibit the re-aggregation. The activities of anti-aggregation and aggregates depolymerization result in the reduction of inclusions in neuron cells. The candidate drugs also show high anti-oxidation and low cytotoxicity. They finally repair the damaged neurons in 6-OHDA-lesioned C57 mice and significantly improve PD-like symptoms of the PD model mice. The hybrids are promising molecules for PD prevention and therapy.© 2022 Elsevier Masson SAS. All rights reserved.

Exploration of ring size in a series of cyclic vicinal diamines with σ1 receptor affinity.

Imidazolidine and 1,4-diazepane analogs of N-(2-benzofuranyl)methyl-N'-(4-alkoxybenzyl)piperazines were prepared to explore the effect of ring contraction and expansion on σ receptor affinity and subtype selectivity within a series of cyclic diamines. In vitro receptor binding assays revealed that all cyclic vicinal diamines possessed affinity and selectivity for σ(1) receptors. The imidazolidines possessed nanomolar σ(1) affinities (K(i)=6.45-53.5 nM), and relatively low levels of subtype selectivity (σ(2)/σ(1)=58-237). However, the piperazines and diazepanes achieved picomolar σ(1) interactions, with K(i) ranges of 0.05-10.28 and 0.10-0.194 nM, respectively. Moreover, the piperazines and diazepanes showed excellent discrimination over the σ(2) receptor, with σ(1) selectivities of 143-16140 and 220-11542, respectively.

Synthesis and evaluation of in vitro bioactivity for vesicular acetylcholine transporter inhibitors containing two carbonyl groups.

To identify selective high-affinity ligands for the vesicular acetylcholine transporter (VAChT), we have incorporated a carbonyl group into the structures of trozamicol and prezamicol scaffolds, and also converted the secondary amines of the piperidines of trozamicols and prezamicols into amides. Of 18 new racemic compounds, 4 compounds displayed high affinity for VAChT (K(i)=10-20 nM) and greater than 300-fold selectivity for VAChT over σ(1) and σ(2) receptors, namely (4-(4-fluorobenzoyl)-4'-hydroxy-[1,3'-bipiperidin]-1'-yl)(3-methylthiophen-2-yl)methanone oxalate (9g) (K(i-VAChT)=11.4 nM, VAChT/σ(1)=1063, VAChT/σ(2)=370), (1'-benzoyl-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10c) (K(i-VAChT)=15.4 nM, VAChT/σ(1)=374, VAChT/σ(2)=315), (4'-hydroxy-1'-(thiophene-2-carbonyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10e) (K(i-VAChT)=19.0 nM, VAChT/σ(1)=1787, VAChT/σ(2)=335), and (4'-hydroxy-1'-(3-methylthiophene-2-carbonyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10g) (K(i-VAChT)=10.2 nM, VAChT/σ(1)=1500, VAChT/σ(2)=2030). These four compounds can be radiosynthesized with C-11 or F-18 to validate their possibilities of serving as PET probes for quantifying the levels of VAChT in vivo.

Synthesis and in vitro evaluation of α-synuclein ligands.

Accumulation of misfolded α-synuclein in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson's disease (PD). To identify ligands having high binding potency toward aggregated α-synuclein, we synthesized a series of phenothiazine derivatives and assessed their binding affinity to recombinant α-synuclein fibrils using a fluorescent thioflavin T competition assay. Among 16 new analogues, the in vitro data suggest that compound 11b has high affinity to α-synuclein fibrils (K(i)=32.10 ± 1.25 nM) and compounds 11d, 16a and16b have moderate affinity to α-synuclein fibrils (K(i)≈50-100 nM). Further optimization of the structure of these analogues may yield compounds with high affinity and selectivity for aggregated α-synuclein.