A straightforward screening of a compound library comprising 2439 substances for the identification of new inhibitors for the neurotransmitter transporters GlyT1 and GlyT2 is described. Screening and full-scale competition experiments were performed using recently developed GlyT1 and GlyT2 MS Binding Assays. That way for both targets, GlyT1 and GlyT2, ligands were identified, which exhibited affinities (pKi values) in the low micromolar to sub-micromolar range. The majority of these binders exhibit new chemical scaffolds in the class of GlyT1 and GlyT2 inhibitors, which could be of interest for the development of new ligands with improved affinities for the target proteins. Additionally, compounds with excellent fluorescent properties were found for GlyT2, which renders them promising compounds for future fluorescence-based techniques. All in all, this study demonstrates that MS Binding Assays represent a powerful technology platform also well suited for the screening of compound libraries in a highly reliable and effective manner. 相似文献
In this work, we present label‐free, mass‐spectrometry‐based binding assays (MS Binding Assays), targeting the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT) in simultaneous binding experiments. Using a validated LC–ESI‐MS/MS method for quantification of the selective dopamine transporter inhibitor (R,R)‐4‐(2‐benzhydryloxyethyl)‐1‐(4‐fluorobenzyl)piperidin‐3‐ol ((R,R)‐D‐84), the selective norepinephrine transporter inhibitor (S,S)‐reboxetine, and the selective serotonin reuptake inhibitor (S)‐citalopram, binding affinities at the three monoamine transporters could be characterized simultaneously in a single binding experiment. The performed simultaneous saturation and competition experiments yielded results that are in good accordance with those determined in MS Binding Assays addressing the monoamine transporters individually. The results obtained from this study underscore the potential of MS Binding Assays for simultaneous affinity determination at different targets, which is difficult to accomplish with conventional radioligand binding assays. 相似文献
MS Binding Assays are a label‐free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but an unlabeled reporter ligand is used instead of a radioligand. The study presented herein describes the development of MS Binding Assays that address D1 and D5 dopamine receptors. A highly sensitive, rapid and robust LC–ESI‐MS/MS quantification method for the selective D1 dopamine receptor antagonist SCH23390 ((5R)‐8‐chloro‐3‐methyl‐5‐phenyl‐1,2,4,5‐tetrahydro‐3‐benzazepin‐7‐ol) was established and validated, using its 8‐bromo analogue SKF83566 as an internal standard. This quantification method proved to be suitable for the characterization of SCH23390 binding to human D1 and D5 receptors. Following the concept of MS Binding Assays, saturation experiments for D1 and D5 receptors were performed, as well as competition experiments for D1 receptors. The results obtained are in good agreement with results from radioligand binding assays and therefore indicate that the established MS Binding Assays addressing D1 and D5 receptors are well‐suited substitutes for radioligand binding assays, the technique that has so far dominated affinity determinations toward these targets. 相似文献
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-{(Ra)-1-[4-([1,1′:2′,1′′-terphenyl]-2-yl)buta-2,3-dien-1-yl](3R)-piperidine-3-carboxylic acid} and rac-{(Sa)-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 pKi=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. 相似文献
Drugs targeting type 4 dipeptidyl peptidase (DPP-4) are beneficial for glycemic control, whereas fibroblast activation protein alpha (FAP-α) is a potential target for cancer therapies. Unlike other gliptins, linagliptin displays FAP inhibition. We compared biophysical and structural characteristics of linagliptin binding to DPP-4 and FAP to better understand what differentiates linagliptin from other gliptins. Linagliptin exhibited high binding affinity (KD) and a slow off-rate (koff) when dissociating from DPP-4 (KD 6.6 pM; koff 5.1×10−5 s−1), and weaker inhibitory potency to FAP (KD 301 nM; koff>1 s−1). Co-structures of linagliptin with DPP-4 or FAP were similar except for one second shell amino acid difference: Asp663 (DPP-4) and Ala657 (FAP). pH dependence of enzymatic activities and binding of linagliptin for DPP-4 and FAP are dependent on this single amino acid difference. While linagliptin may not display any anticancer activity at therapeutic doses, our findings may guide future studies for the development of optimized inhibitors. 相似文献
We herein present label‐free, mass‐spectrometry‐based binding assays (MS Binding Assays) for the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT). Using this approach both enantiomers of the triple reuptake inhibitor indatraline as well as its cis‐configured diastereomer were investigated toward hDAT, hNET, and hSERT in saturation experiments. The dissociation rate constants for (1R,3S)‐indatraline binding at hDAT, hNET, and hSERT were determined in kinetic studies. These experiments revealed an allosteric effect of clomipramine on the dissociation of (1R,3S)‐indatraline from hSERT. Finally, a comprehensive set of known monoamine transport inhibitors and substrates was studied in competition experiments at hDAT, hNET, and hSERT, using (1R,3S)‐indatraline as nonlabeled marker. The results are in excellent agreement with those reported for radioligand binding assays. Therefore, the established MS Binding Assays are a promising alternative to the latter for the characterization of new monoamine reuptake inhibitors at DAT, NET, and SERT. 相似文献
In this research, a SiC/ZrB2 coating was produced on graphite by reactive melt infiltration and plasma spraying method. The coating characterization was performed using XRD analysis, electron microscopy equipped with energy dispersive spectrometer (EDS), and supersonic flame ablation test at 2073 K. The results indicated that the dense C/SiC coating with good ablation resistance can be obtained at 1873 K. The coating thickness decreased with increasing infiltration temperature. The results of ablation test showed that by increasing the infiltration temperature and holding time, weight loss and mass ablation rate decreased from 22.63% to 9.83% and 3.63 × 10−3 g cm−2 s−1 to 1.34 × 10−3 g cm−2 s−1, respectively. The results showed that by using the ZrB2 as outer coating the ablation resistance improved remarkably. The weight loss and mass ablation rates for the SiC/ZrB2 coating were 12.79% and 1.857 × 10−3 g cm−2 s−1, respectively. 相似文献
The technique of simultaneous pulse radiolysis and photolysis, PRAP, has been utilized to study the reactions of various radicals with ground state ZnTPPS and the triplet state ZnTPPST in aqueous solutions. The radicals H and OH add to both states with k ∼ 1 × 1010 M−1 s−1. The CH2C(CH3)2OH radical from t-BuOH is relatively inert toward ZnTPPS but reacts rapidly (k = 1.8 × 109 M−1 s−1) with ZnTPPST to form an adduct. Electron transfer reactions are found to be about an order of magnitude faster with the triplet than with the ground state. The (CH3)2COH radical reduces both ZnTPPS (k = 1 × 108 M−1 s−1) and ZnTPPST (k = 3 × 109 M−1 s−1) to the anion radical (ZnTPPS)−. The radical Br−2 oxidizes both states to the cation radical (ZnTPPS)+ with k = 8 × 108 M−1 s−1 for the ground state and 5 × 109 M−1 s−1 for the triplet. The transient cation Cd+ reduces both states with a diffusion-controlled rate (k = 1 × 1010 M−1 s−1) to produce the anion radical. The above mechanisms of radical addition and electron transfer are also supported by the product spectra. 相似文献
The adsorption of telon blue (acid blue 25) dye and deorlene yellow (basic yellow) dye on to carbon has been studied. A two-resistance mass transfer model has been developed based on film resistance and homogeneous solid phase diffusion. The model may be applied over a wider range of operating conditions than previous models since it has more extensive analytical components. The variables investigated were initial dye concentration and solid: liquid ratio, and the experimental and theoretical results were in good agreement. The adsorption of telon blue on to carbon has been described using an external mass transfer coefficient of 2.0 × 10−3 cm s−1 and a homogeneous solid phase diffusion coefficient of 2.0 × 10−9 cm2 s−1. The adsorption of deorlene yellow has been described using an external mass transfer coefficient of 1.0 × 10−3 cm s−1 and a homogeneous solid phase diffusion coefficient of 3.0 × 10−10 cm2 s−1. 相似文献
The branched-pore adsorption model, expressed by an external mass transfer coefficient kf, a solid diffusivity Ds, a lumped micropore diffusion rate parameter kb, and the fraction of macropores f, describes kinetic data from initial contact of adsorbent-adsorbate to the long-term ( > 24 hours) adsorption stages with reasonable accuracy.In this work the model is applied for three basic dye systems, namely Basic Red 22, Basic Yellow 21 and Basic Blue 69, all on carbon. A single value of each parameter describes each dye system. The kf values are 0.18 × 10−2±28%, 0.3 × 10−2±17% and 0.2 × 10−2 ± 20% cm s−1, the Ds values are 0.33 × 10−9 21%, 0.72 × 10−9 ± 9% and 0.72 × 10−9 ± 9% cm2 s−1, the kb values are 0.65 × 10−6 ± 7.7%, 1.8 × 10−6 0.2 × 10−6 1% s−1, while the f values are 0.55 ± 9%, 0.60 ± 10 % and 0.18 ± 11%, each for Basic Red 22, Basic Yellow 21 and Basic Blue 69 respectively.The model is based on the internal structure of the carbon particle being divided into a macropore and a micropore region. The latter has an upper-bound capacity of 241, 245 and 656 mg g−1 for Basic Red 22, Basic Yellow 21 and Basic Blue 69 respectively. A sensitivity analysis for each parameter has been carried out. 相似文献
This paper reports a relatively simple low-temperature non-isothermal oxidative desulphurisation of coal organic sulphur by weakening the CS bond using HgCl2 solution to an inorganic sulphur-free high-sulphur Indian coal. When oxidised from 50°C to 150°C in air under normal atmospheric pressure, there is continuous decrease of organic sulphur content in the samples of the feed and Hg-treated coals. Desulphurisation is more in the Hg-treated coal (4.97–14.53 wt.%) than in the feed coal (3.72–10.93 wt.%). Kinetic study reveals that the oxidative desulphurisation process follows pseudo-first order kinetics and the rate constants have been found to be in the range (3.09–5.06)×10−5 s−1 for feed coal and (4.19–6.80)×10−5 s−1 for Hg-treated coal. The activation energies for the sulphur loss reaction in the oxidative desulphurisation process by using the pseudo-first order kinetic (feed coal: 2.21×102 J mol−1; Hg-treated coal: 1.53×102 J mol−1) have been found to be almost similar to those calculated by applying the Coats and Redfern's equation (feed coal: 2.19×102 J mol−1; Hg-treated coal: 1.53×102 J mol−1). However, the value is higher (feed coal: 3.50×102 J mol−1; Hg-treated coal: 2.70×102 J mol−1) when Horowitz and Metzger's equation is applied. The frequency factors computed by the pseudo-first order kinetics are very low and have been found to be 2.66×10−5 s−1 for feed coal and 3.96×10−5 s−1 for Hg-treated coal, suggesting very low rate of successful collisions for the formation of the activated complex. Evaluation of thermodynamic parameters viz., ΔH, ΔU, ΔS and ΔG, reveals that this oxidative desulphurisation process is non-spontaneous in nature and the degree of non-spontaneity of such a process in the feed coal is more relative to that of the Hg-treated coal. 相似文献
The limited effect of current medications on neuropathic pain (NP) has initiated large efforts to develop effective treatments. Animal studies showed that glycine transporter (GlyT) inhibitors are promising analgesics in NP, though concerns regarding adverse effects were raised. We aimed to study NFPS and Org-25543, GlyT-1 and GlyT-2 inhibitors, respectively and their combination in rat mononeuropathic pain evoked by partial sciatic nerve ligation. Cerebrospinal fluid (CSF) glycine content was also determined by capillary electrophoresis. Subcutaneous (s.c.) 4 mg/kg NFPS or Org-25543 showed analgesia following acute administration (30–60 min). Small doses of each compound failed to produce antiallodynia up to 180 min after the acute administration. However, NFPS (1 mg/kg) produced antiallodynia after four days of treatment. Co-treatment with subanalgesic doses of NFPS (1 mg/kg) and Org-25543 (2 mg/kg) produced analgesia at 60 min and thereafter meanwhile increased significantly the CSF glycine content. This combination alleviated NP without affecting motor function. Test compounds failed to activate G-proteins in spinal cord. To the best of our knowledge for the first time we demonstrated augmented analgesia by combining GlyT-1 and 2 inhibitors. Increased CSF glycine content supports involvement of glycinergic system. Combining selective GlyT inhibitors or developing non-selective GlyT inhibitors might have therapeutic value in NP. 相似文献
Reactions of H atoms with COS, CS2, and H2S were studied behind reflected shock waves at temperatures between 1170 K and 1830 K and pressures around 1.0 bar by applying atomic resonance absorption spectroscopy (ARAS) for time-resolved measurements of H atoms at Lα. The thermal decomposition of a few ppm ethyl iodide (C2H5I) was used as a H-atom source. In the presence of a large excess of the molecular reactant COS, CS2, or H2S, a consumption of H was observed which follows a pseudo first-order rate law. Rate coefficients for the reactions: were determined to be: k1 = 2.4 × 1014exp(–3415 K/T) cm3mol−1s−1k2 = 1.4 × 1015exp(–9250 K/T) cm3mol−1s−1k3 = 2.5 × 1014exp(–2890 K/T) cm3mol−1s−1相似文献
Greenhouse gases emitted into the atmosphere by burning of fossil fuels cause global warming. One option is obtaining biodiesel. Nannochloropsis oculata was cultured under different light intensities and reactors at 25°C for 21 days with f/2 medium to assess their effects on cell density, lipid, and fatty acids (FAs). N. oculata improved cell density on fed-batch glass tubular reactor (7 L) at 200 μmol E m−2 s−1, yielding 3.5 × 108 cells ml−1, followed by fed-batch Erlenmeyer flask (1 L) at 650 μmol E m−2 s−1 with 1.7 × 108 cells ml−1. The highest total lipid contents (% g lipid × g dry biomass−1) were 44.4 ± 0.8% for the reactor (1 L) at 650 μmol E m−2 s−1 and 35.2 ± 0.2% for the tubular reactor (7 L) at 200 μmol E m−2 s−1, until twice as high compared with the control culture (Erlenmeyer flask 1 L, 80 μmol E m−2 s−1) with 21.2 ± 1%. Comparing the total lipid content at 200 μmol E m−2 s−1, tubular reactor (7 L) and reactor 1 L achieved 35.2 ± 0.2% and 28.3 ± 1%, respectively, indicating the effect of shape reactor. The FAs were affected by high light intensity, decreasing SFAs to 2.5%, and increased monounsaturated fatty acids + polyunsaturated fatty acids to 2.5%. PUFAs (20:5n-3) and (20:4n-3) were affected by reactor shape, decreasing by half in the tubular reactor. In the best culture, fed-batch tubular reactor (7 L) at 200 μmol E m−2 s−1 contains major FAs (16:0; 38.06 ± 0.16%), (16:1n-7; 30.74 ± 0.58%), and (18:1n-9; 17.15 ± 0.91%). 相似文献
The interaction of human recombinant sterol carrier protein-2 (SCP-2) with sterols was examined. Two independent ligand binding
methods, Lipidex 1000 binding of [3H]cholesterol and a fluorescent dehydroergosterol binding assay, were used to determine the affinity of SCP-2 for sterols.
Binding analysis indicated SCP-2 bound [3H]cholesterol and dehydroergosterol with aKd of 0.3 and 1.7 μM, respectively, and suggested the presence of a single binding site. Phase fluorometry and circular dichroism
were used to characterize the SCP-2 sterol binding site. Alterations in dehydroergosterol lifetime, SCP-2 tryptophan lifetime,
and SCP-2 tryptophan quenching by acrylamide upon cholesterol binding demonstrated a shielding of the SCP-2 tryptophan from
the aqueous solvent by bound sterol. Differential polarized phase fluorometry revealed decreased SCP-2 tryptophan rotational
correlation time upon cholesterol binding. Circular dichroism of SCP-2 indicated that cholesterol elicited a small decrease
in SCP-2 alpha helical content. The data suggest that SCP-2 binds sterols with affinity consistent with a lipid transfer protein
that may act either as an aqueous carrier or at a membrane surface to enhance sterol desorption. 相似文献