Synthesis and Biological Evaluation of Nipecotic Acid and Guvacine Derived 1,3-Disubstituted Allenes as Inhibitors of Murine GABA Transporter mGAT1

A new class of nipecotic acid and guvacine derivatives has been synthesized and characterized for their inhibitory potency at mGAT1–4 and binding affinity for mGAT1. Compounds of the described class are defined by a four-carbon-atom allenyl spacer connecting the nitrogen atom of the nipecotic acid or guvacine head with an aromatic residue. Among the compounds investigated, the mixture of nipecotic acid derivatives rac-{(R_a)-1-[4-([1,1′:2′,1′′-terphenyl]-2-yl)buta-2,3-dien-1-yl](3R)-piperidine-3-carboxylic acid} and rac-{(S_a)-1-[4-([1,1′:2′,1′′-terphenyl]-2-yl)buta-2,3-dien-1-yl](3R)-piperidine-3-carboxylic acid} ( 21 p ), possessing an o-terphenyl residue, was identified as highly selective and the most potent mGAT1 inhibitor in this study. For the (R)-nipecotic acid derived form of 21 p , the inhibitory potency in [³H]GABA uptake assays was determined as pIC₅₀=6.78±0.08, and the binding affinity in MS Binding Assays as pK_i=7.10±0.12. The synthesis of the designed compounds was carried out by a two-step procedure, generating the allene moiety via allenylation of terminal alkynes which allows broad variation of the terminal phenyl and biphenyl subunit.     

三甲铵乙内酯(甜菜碱)的合成

介绍了以氯乙酸钠和三甲胺为原料合成三甲铵乙内酯（甜菜碱）的方法,以室温为起始反应温度反应０．５ｈ,然后分别于５０℃～５５℃和８０℃～８５℃反应１ｈ,以强酸性阳离子交换树脂为反应液的分离提纯物质。其收率超过９７％,产品纯度达到９８．５％。该法具有操作简便、反应时间短、反应条件温和、产品纯度高等优点。     

Selective Fluorescent Probes for High-Throughput Functional Diagnostics of the Human Multidrug Transporter P-Glycoprotein (ABCB1)

The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dyes to examine the functional expression and properties of the ABCB1 multidrug transporter, especially in microplate-based high-throughput flow cytometry assays. In addition, we demonstrate the applicability of the TP dyes to efficiently select and separate even a very low number of Pgp-expressing intact cells.     

Antigen Peptide Transporter 1 (TAP1) Promotes Resistance to MEK Inhibitors in Pancreatic Cancers

Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors show limited benefit in Kirsten rat sarcoma (KRAS) mutant pancreatic cancer due to drug resistance. To identify mechanisms of resistance to MEK inhibitor (MEKi), we employed a differential expression analysis of MEKi-sensitive versus MEKi-resistant KRAS-mutant pancreatic cancer cell lines. Here, we report that the antigen peptide transporter 1 (TAP1) expression levels of MEKi-resistant cell lines were notably higher than those of MEKi-sensitive cell lines. Suppression of TAP1 significantly sensitized the MEKi-resistant pancreatic ductal adenocarcinoma (PDAC) cells to MEKi and induced higher apoptotic rate in vitro. Moreover, knockdown of TAP1 in MEKi-resistant tumor significantly decreased tumor growth in vivo. Consistently, overexpression of TAP1 in sensitive PDAC cells resulted in increased resistance to MEKi, both in vitro and in vivo. Mechanistic studies demonstrated that TAP1 promoted chemoresistance by enhancing the transport of MEKi out of PDAC cells, leading to reduced intracellular MEKi concentration and attenuated inhibition of KRAS signaling pathways. Moreover, TAP1 expression increased spheroid formation abilities of PDAC cells. These findings suggest that TAP1 could serve as a potential marker for predicting the response of patients to MEKi. Combination of TAP1 suppression and MEKi may provide a novel therapeutic strategy for PDAC treatment.     

Free Cholesterol Affects the Function and Localization of Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) and Organic Cation Transporter 1 (OCT1)

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are associated with obesity. They are accompanied by increased levels of free cholesterol in the liver. Most free cholesterol resides within the plasma membrane. We assessed the impact of adding or removing free cholesterol on the function and localization of two hepatocellular uptake transporters: the Na⁺/taurocholate cotransporting polypeptide (NTCP) and the organic cation transporter 1 (OCT1). We used a cholesterol–MCD complex (cholesterol) to add cholesterol and methyl-β-cyclodextrin (MCD) to remove cholesterol. Our results demonstrate that adding cholesterol decreases NTCP capacity from 132 ± 20 to 69 ± 37 µL/mg/min and OCT1 capacity from 209 ± 66 to 125 ± 26 µL/mg/min. Removing cholesterol increased NTCP and OCT1 capacity to 224 ± 65 and 279 ± 20 µL/mg/min, respectively. In addition, adding cholesterol increased the localization of NTCP within lipid rafts, while adding or removing cholesterol increased OCT1 localization in lipid rafts. These results demonstrate that increased cholesterol levels can impair NTCP and OCT1 function, suggesting that the free cholesterol content of the liver can alter bile acid and drug uptake into the liver. This could explain the increased plasma bile acid levels in NAFLD and NASH patients and potentially lead to altered drug disposition.     

Effects of Germination Process and Drying Temperature on Gamma-Aminobutyric Acid (GABA) and Starch Digestibility of Germinated Brown Rice

In this work, germination method, germination time, and drying temperature were investigated for their effects on gamma-aminobutyric acid (GABA) and starch digestibility. The germination method and germination time influenced the GABA and dietary fiber contents as well as starch and glucose, but both factors did not provide a faster hydrolysis of starch for germinated brown rice because of the dietary fiber. When the germinated samples were dried by a hot-air fluidized bed dryer at 130 or 150°C, the GABA content was not decreased and the amylose-lipid complexes occurred. Dissociation temperature of the complexes was given in a range of 100–117°C, which was lower than that of complexes in the non-germinated paddy. Thus, the amylose-lipid complexes in the germinated samples obtained at high temperature lost some crystalline structure when cooked by the boiling method. The corresponding rate of starch hydrolysis or glycemic index of the germinated samples changed insignificantly from that of the shade-dried germinated sample or non-germinated brown rice which was dried in shade.     

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient’s macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.     

Neuroprotective Effects of Growth Hormone (GH) and Insulin-Like Growth Factor Type 1 (IGF-1) after Hypoxic-Ischemic Injury in Chicken Cerebellar Cell Cultures

It has been reported that growth hormone (GH) and insulin-like growth factor 1 (IGF-1) exert protective and regenerative actions in response to neural damage. It is also known that these peptides are expressed locally in nervous tissues. When the central nervous system (CNS) is exposed to hypoxia-ischemia (HI), both GH and IGF-1 are upregulated in several brain areas. In this study, we explored the neuroprotective effects of GH and IGF-1 administration as well as the involvement of these endogenously expressed hormones in embryonic chicken cerebellar cell cultures exposed to an acute HI injury. To induce neural damage, primary cultures were first incubated under hypoxic-ischemic (<5% O₂, 1g/L glucose) conditions for 12 h (HI), and then incubated under normal oxygenation and glucose conditions (HI + Ox) for another 24 h. GH and IGF-1 were added either during or after HI, and their effect upon cell viability, apoptosis, or necrosis was evaluated. In comparison with normal controls (Nx, 100%), a significant decrease of cell viability (54.1 ± 2.1%) and substantial increases in caspase-3 activity (178.6 ± 8.7%) and LDH release (538.7 ± 87.8%) were observed in the HI + Ox group. On the other hand, both GH and IGF-1 treatments after injury (HI + Ox) significantly increased cell viability (77.2 ± 4.3% and 72.3 ± 3.9%, respectively) and decreased both caspase-3 activity (118.2 ± 3.8% and 127.5 ± 6.6%, respectively) and LDH release (180.3 ± 21.8% and 261.6 ± 33.9%, respectively). Incubation under HI + Ox conditions provoked an important increase in the local expression of GH (3.2-fold) and IGF-1 (2.5-fold) mRNAs. However, GH gene silencing with a specific small-interfering RNAs (siRNAs) decreased both GH and IGF-1 mRNA expression (1.7-fold and 0.9-fold, respectively) in the HI + Ox group, indicating that GH regulates IGF-1 expression under these incubation conditions. In addition, GH knockdown significantly reduced cell viability (35.9 ± 2.1%) and substantially increased necrosis, as determined by LDH release (1011 ± 276.6%). In contrast, treatments with GH and IGF-1 stimulated a partial recovery of cell viability (45.2 ± 3.7% and 53.7 ± 3.2%) and significantly diminished the release of LDH (320.1 ± 25.4% and 421.7 ± 62.2%), respectively. Our results show that GH, either exogenously administered and/or locally expressed, can act as a neuroprotective factor in response to hypoxic-ischemic injury, and that this effect may be mediated, at least partially, through IGF-1 expression.     

The First Cytoplasmic Loop in the Core Structure of the ABCC1 (Multidrug Resistance Protein 1; MRP1) Transporter Contains Multiple Amino Acids Essential for Its Expression

ABCC1 (human multidrug resistance protein 1 (hMRP1)) is an ATP-binding cassette transporter which effluxes xeno- and endobiotic organic anions and confers multidrug resistance through active drug efflux. The 17 transmembrane α-helices of hMRP1 are distributed among three membrane spanning domains (MSD0, 1, 2) with MSD1,2 each followed by a nucleotide binding domain to form the 4-domain core structure. Eight conserved residues in the first cytoplasmic loop (CL4) of MSD1 in the descending α-helix (Gly³⁹², Tyr⁴⁰⁴, Arg⁴⁰⁵), the perpendicular coupling helix (Asn⁴¹², Arg⁴¹⁵, Lys⁴¹⁶), and the ascending α-helix (Glu⁴²², Phe⁴³⁴) were targeted for mutagenesis. Mutants with both alanine and same charge substitutions of the coupling helix residues were expressed in HEK cells at wild-type hMRP1 levels and their transport activity was only moderately compromised. In contrast, mutants of the flanking amino acids (G392I, Y404A, R405A/K, E422A/D, and F434Y) were very poorly expressed although Y404F, E422D, and F434A were readily expressed and transport competent. Modeling analyses indicated that Glu⁴²² and Arg⁶¹⁵ could form an ion pair that might stabilize transporter expression. However, this was not supported by exchange mutations E422R/R615E which failed to improve hMRP1 levels. Additional structures accompanied by rigorous biochemical validations are needed to better understand the bonding interactions crucial for stable hMRP1 expression.     

KDRn3蛋白在人脐静脉血管内皮细胞中的表达及对其增殖的抑制作用

目的观察KDRn3蛋白在人脐静脉血管内皮细胞中的表达及对其增殖的抑制作用。方法将质粒pEGFP-N1/KDRn3扩增并鉴定后,以脂质体介导转染人脐静脉血管内皮细胞,培养一定时间后,在荧光显微镜下观察绿色荧光蛋白的表达,ELISA检测细胞培养上清中KDRn3的含量,MTT法检测KDRn3蛋白对人脐静脉血管内皮细胞增殖的影响。结果转染后48h,在荧光显微镜下可见pEGFP-N1/KDRn3转染组人脐静脉血管内皮细胞发出绿色荧光,72h发出绿色荧光的细胞增多,强度增强;转染后24、48和72h,细胞培养上清中KDRn3含量与转染前比较均提高,且差异有显著意义;转染后48和72h,pEGFP-N1/KDRn3转染组与对照组比较,细胞增殖有明显的抑制,且差异有显著意义。结论KDRn3蛋白可在人脐静脉血管内皮细胞中表达,并能抑制其增殖。     

Amino Acid Transporter LAT1 (SLC7A5) Mediates MeHg-Induced Oxidative Stress Defense in the Human Placental Cell Line HTR-8/SVneo

The placental barrier can protect the fetus from contact with harmful substances. The potent neurotoxin methylmercury (MeHg), however, is very efficiently transported across the placenta. Our previous data suggested that L-type amino acid transporter (LAT)1 is involved in placental MeHg uptake, accepting MeHg-L-cysteine conjugates as substrate due to structural similarity to methionine. The aim of the present study was to investigate the antioxidant defense of placental cells to MeHg exposure and the role of LAT1 in this response. When trophoblast-derived HTR-8/SVneo cells were LAT1 depleted by siRNA-mediated knockdown, they accumulated less MeHg. However, they were more susceptible to MeHg-induced toxicity. This was evidenced in decreased cell viability at a usually noncytotoxic concentration of 0.03 µM MeHg (~6 µg/L). Treatment with ≥0.3 µM MeHg increased cytotoxicity, apoptosis rate, and oxidative stress of HTR-8/SVneo cells. These effects were enhanced under LAT1 knockdown. Reduced cell number was seen when MeHg-exposed cells were cultured in medium low in cysteine, a constituent of the tripeptide glutathione (GSH). Because LAT1-deficient HTR-8/SVneo cells have lower GSH levels than control cells (independent of MeHg treatment), we conclude that LAT1 is essential for de novo synthesis of GSH, required to counteract oxidative stress. Genetic predisposition to decreased LAT1 function combined with MeHg exposure could increase the risk of placental damage.     

Maternal Separation Alters Ethanol Drinking and Reversal Learning Processes in Adolescent Rats: The Impact of Sex and Glycine Transporter Type 1 (GlyT1) Inhibitor

Adverse early life experiences are associated with an enhanced risk for mental and physical health problems, including substance abuse. Despite clinical evidence, the mechanisms underlying these relationships are not fully understood. Maternal separation (MS) is a commonly used animal model of early neglect. The aim of the current study is to determine whether the N-methyl-D-aspartate receptor (NMDAR)/glycine sites are involved in vulnerability to alcohol consumption (two-bottle choice paradigm) and reversal learning deficits (Barnes maze task) in adolescent rats subjected to the MS procedure and whether these effects are sex dependent. By using ELISA, we evaluated MS-induced changes in the NMDAR subunits (GluN1, GluN2A, GluN2B) expression, especially in the glycine-binding subunit, GluN1, in the prefrontal cortex (PFC) and ventral striatum (vSTR) of male/female rats. Next, we investigated whether Org 24598, a glycine transporter 1 (GlyT1) inhibitor, was able to modify ethanol drinking in adolescent and adult male/female rats with prior MS experience and reversal learning in the Barnes maze task. Our findings revealed that adolescent MS female rats consumed more alcohol which may be associated with a substantial increase in GluN1 subunit of NMDAR in the PFC and vSTR. Org 24598 decreased ethanol intake in both sexes with a more pronounced decrease in ethanol consumption in adolescent female rats. Furthermore, MS showed deficits in reversal learning in both sexes. Org 24598 ameliorated reversal learning deficits, and this effect was reversed by the NMDAR/glycine site inhibitor, L-701,324. Collectively, our results suggest that NMDAR/glycine sites might be targeted in the treatment of alcohol abuse in adolescents with early MS, especially females.     

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell’s receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E. coli production of active multi-disulfide-bonded RBD.     

Neuroprotective Effect of Macrophage Migration Inhibitory Factor (MIF) in a Mouse Model of Ischemic Stroke

The mechanism of the neuroprotective effect of the macrophage migration inhibitory factor (MIF) in vivo is unclear. We investigated whether the MIF promotes neurological recovery in an in vivo mouse model of ischemic stroke. Transient middle cerebral artery occlusion (MCAO) surgery was performed to make ischemic stroke mouse model. Male mice were allocated to a sham vehicle, a sham MIF, a middle cerebral artery occlusion (MCAO) vehicle, and MCAO+MIF groups. Transient MCAO (tMCAO) was performed in the MCAO groups, and the vehicle and the MIF were administered via the intracerebroventricular route. We evaluated the neurological functional scale, the rotarod test, and T2-weighted magnetic resonance imaging. The expression level of the microtubule-associated protein 2 (MAP2), Bcl2, and the brain-derived neurotrophic factor (BDNF) were further measured by Western blot assay. The Garcia test was significantly higher in the MCAO+MIF group than in the MCAO+vehicle group. The MCAO+MIF group exhibited significantly better performance on the rotarod test than the MCAO+vehicle group, which further had a significantly reduced total infarct volume on T2-weighted MRI imaging than the MCAO vehicle group. Expression levels of BDNF, and MAP2 tended to be higher in the MCAO+MIF group than in the MCAO+vehicle group. The MIF exerts a neuroprotective effect in an in vivo ischemic stroke model. The MIF facilitates neurological recovery and protects brain tissue from ischemic injury, indicating a possibility of future novel therapeutic agents for stroke patients.     

Characterization in Inhibitory Effectiveness of Carbamazepine in Voltage-Gated Na+ and Erg-Mediated K+ Currents in a Mouse Neural Crest-Derived (Neuro-2a) Cell Line

Carbamazepine (CBZ, Tegretol^®) is an anticonvulsant used in the treatment of epilepsy and neuropathic pain; however, several unwanted effects of this drug have been noticed. Therefore, the regulatory actions of CBZ on ionic currents in electrically excitable cells need to be reappraised, although its efficacy in suppressing voltage-gated Na⁺ current (I_Na) has been disclosed. This study was undertaken to explore the modifications produced by CBZ on ionic currents (e.g., I_Na and erg-mediated K⁺ current [I_K(erg)]) measured from Neuro-2a (N2a) cells. In these cells, we found that this drug differentially suppressed the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of I_Na in a concentration-dependent manner with effective IC₅₀ of 56 and 18 μM, respectively. The overall current–voltage relationship of I_Na(T) with or without the addition of CBZ remained unchanged; however, the strength (i.e., ∆area) in the window component of I_Na (I_Na(W)) evoked by the short ascending ramp pulse (V_ramp) was overly lessened in the CBZ presence. Tefluthrin (Tef), a synthetic pyrethroid, known to stimulate I_Na, augmented the strength of the voltage-dependent hysteresis (Hys_(V)) of persistent I_Na (I_Na(P)) in response to the isosceles-triangular V_ramp; moreover, further application of CBZ attenuated Tef-mediated accentuation of I_Na(P)’s Hys_(V). With a two-step voltage protocol, the recovery of I_Na(T) inactivation seen in Neuro-2a cells became progressively slowed by adding CBZ; however, the cumulative inhibition of I_Na(T) evoked by pulse train stimulation was enhanced during exposure to this drug. Neuro-2a-cell exposure to CBZ (100 μM), the magnitude of erg-mediated K⁺ current measured throughout the entire voltage-clamp steps applied was mildly inhibited. The docking results regarding the interaction of CBZ and voltage-gate Na⁺ (Na_V) channel predicted the ability of CBZ to bind to some amino-acid residues in Na_V due to the existence of a hydrogen bond or hydrophobic contact. It is conceivable from the current investigations that the I_Na (I_Na(T), I_Na(L), I_Na(W), and I_Na(P)) residing in Neuro-2a cells are susceptible to being suppressed by CBZ, and that its block on I_Na(L) is larger than that on I_Na(T). Collectively, the magnitude and gating of Na_V channels produced by the CBZ presence might have an impact on its anticonvulsant and analgesic effects occurring in vivo.