Table of Content

01 December 2019, Volume 28 Issue 11

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Original articles

Membrane binding of the pH sensitive antimicrobial peptide LAH4 studied by EPR spectroscopy

Yina Sheng, Shenlin Wang, Guoquan Liu

2019, 28(11):  749-759.  DOI: 10.5246/jcps.2019.11.071

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Peptide-membrane binding is vital for many biological events, including the bacteria combating by antimicrobial peptides. Using the pH sensitive LAH4 peptide as model, we employed a convenient electron paramagnetic resonance (EPR) method to study the peptide-membrane binding process in artificial phospholipid membranes. Based on spectral changes of the nitroxide radicallabeled to the peptides, we characterized binding kinetics and affinity of peptides to different phospholipid membranes. The binding affinity of LAH4 towards POPG was more than an order of magnitude higher than those towards DMPC and POPC. The binding kinetics showed that LAH4 initially bound to POPG much more quickly than to DMPC and POPC. Additionally, pH also affected the binding kinetics in LAH4-membrane interactions, which helped explain the pH dependent antimicrobial activity of LAH4. The method might be further used to monitor the membrane binding/cell penetration of antimicrobial peptide in living cells.



Engineering the protein targeting two pathways of cerebral ischemia reperfusion injury provides better neuroprotective effect than targeting one pathway

Kuai Yu, Ruyi Li, Yuanjun Zhu, Xiaoyan Liu, Yinye Wang

2019, 28(11):  760-769.  DOI: 10.5246/jcps.2019.11.072

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We hypothesized that neuroprotective agents targeting various pathways involved in cerebral ischemia/reperfusion (I/R) injury might be superior to that targeting single pathway. Here, we prepared a fusion protein (B-I) by combining anti-apoptotic Bcl-xL (B) and anti-inflammatory IL-10 (I). B-I could cross blood brain barrier by its N-terminal TAT domain, and be cleaved into separate B and I by Caspase-1. B-I treatment significantly reduced the cerebral infarct volume, better than B or I treatment alone, and equivalent to B and I treatment (B+I). Treatment with B or B-I significantly attenuated I/R-induced neuronal apoptosis as shown by the decrease in apoptotic rate and the inhibition of caspase-3 activity. Moreover, all recombinant proteins, especially B-I, remarkably attenuated I/R-induced up-regulation of TNF-α. These results suggested that fusion protein B-I inhibiting both inflammation and apoptosis provided better neuroprotective effects than inhibiting either one alone. Our study suggested that multiple pathways targeting brain I-R injury could enhance the neuroprotective effect, and it provided a new idea for the study of neuroprotective drugs for ischemic stroke.



Construction of a phage displayed library based on a lipocalin scaffold and preliminary screening of anticalins with specificity for the FcεRI-α receptor

Hua Peng, Jinkui Ma, Jie Cheng, Chengzhi Huang, Hongbin Zhang

2019, 28(11):  770-777.  DOI: 10.5246/jcps.2019.11.073

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The primers were designed according to the gene sequence of lipocalin protein family, and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR. The random mutation lipocalin library was constructed using phagemid expression vector. Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells, and the lipocalin secondary library binding to mast cells was obtained. Then the lipocalin secondary library was enriched and screened with FcεRI-α receptor protein as target molecule, and specific binding phages were eluted. After three rounds of screening, eight recombinant phage clones were randomly selected from elution clones of the third round. ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-α receptor of mast cells. These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-α receptor, and also lay the foundation for the development of biological small molecule drugs to treat IgE associated allergic diseases.



Apatinib induces apoptosis of breast cancer cells by redistribution of Fas into lipid rafts

Shunchao Yan, Xin Jiao, Na Li, Yangfan Du, Chengyao Jiang

2019, 28(11):  778-785.  DOI: 10.5246/jcps.2019.11.074

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Apatinib, an oral anti-angiogenic agent, has been shown to have anti-cancer effects for several cancer cell types. However, little is known about the direct anti-tumor activity of apatinib for breast cancer cells. Herein, the direct effect of apatinib on breast cancer cells and its mechanism of action were assessed. Cell viability was measured with a Cell Counting Kit-8. Cell apoptosis was assessed by flow cytometry. The expression of caspase-8 and the cleavage of poly ADP ribose polymerase were assessed by Western blotting analysis. Lipid rafts and Fas distribution were determined by immunofluorescence microscopy. Apatinib suppressed breast cancer cell proliferation in a dose-dependent manner. Furthermore, apatinib enhanced the aggregation of lipid rafts and the redistribution of Fas into lipid rafts. Pretreatment with methyl-β-cyclodextrin, a cholesterol-sequestering agent, significantlyreversed Fas redistribution and apatinib-induced apoptosis.In conclusion, these results demonstrated that apatinib induced apoptosis of breast cancer cells partially through recruitment of Fas into lipid rafts.



Synthesis and biological evaluation of indeno[1, 2-b]indole derivatives as dual topoisomerase I & II inhibitors: novel multidrug resistant reversal anticancer agents

Dongbo Lu, Yu Chen, Shan Liu, Chao Guo, Xia Li, Zhongjun Li, Xiangbao Meng

2019, 28(11):  786-801.  DOI: 10.5246/jcps.2019.11.075

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A single compound able to inhibit both Topo I and II may present the advantage of improving anti-proliferative activity, with reduced toxic side effects, with respect to the combination of two inhibitors. We designed and synthesized 28 compounds of indeno[1, 2-b]indole derivatives as a new class of Topo I and II inhibitor and successfully identified compound 2-3j, which showed the most potent cell growth inhibition with IC₅₀ =0.74 μM against HCT-116 cell line. Compound 2-3j was also evaluated as a potent topoisomerase I and II inhibitor and can induce apoptosis in human colon cancer cells. 2-3j showed potency against a small panel of drug sensitive and multidrug resistant (MDR) cell lines, and it reversed the MDR of K562/A02, MCF-7/Adr, and KB/Vcr cells at 0.5 μM, with reversal fold values of 3.2, 10.1, and 5.8, respectively. 2-3j might inhibit the function of ABCG2 to increase intracellular drug accumulation and enhance the sensitivity of conventional chemotherapeutic agents for MDR cells. 2-3j could be a promising lead for the development of a new class of antitumor drug acting as inhibitors of Topo I & II and ABCG2.



An established LC-MS/MS method and a developed PK model for the study of pharmacokinetic properties of benapenem in infected mice

Xiwei Ji, Zisheng Kang, Yun Li, Xiping Yang, Xifeng Ma, Chongtie Shi, Yuan Lv

2019, 28(11):  802-811.  DOI: 10.5246/jcps.2019.11.076

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Benapenem is a new parenteral beta-lactam antibacterial with a broad antibacterial spectrum. In the present study, we developed and validated a simple, rapid and sensitive assay method using D6-benapenem as internal standard (IS) after one-step precipitation with methanol to determine benapenem in the plasma of infected mice. Separation was achieved on a reverse phase C₁₈ column with a mobile phase composed of acetonitrile containing 0.2% formic acid–water (0.2% formic acid) and 10 mmol/L ammonium acetate in gradient elution mode. A triple quadrupole tandem mass spectrometer with electrospray ionization source was used as detector and operated by multiple reaction monitoring (MRM) in the positive ion mode. Calibration curves were linear (r>0.99) between 10 and 2000 ng/mL. The quantitative limit was 10 ng/mL, and the intra- and inter- precisions were <4.85% and <1.47%, respectively. The extraction recovery of benapenem and IS was 97.07%–107.09% and 92.47%–111.59%, respectively. The intra- and inter- accuracies were –9.70%– –11.00%, and the matrix effects of benapenem and IS were 85.68%–92.04% and 83.17%–92.04%, respectively. The method was successfully applied to the preclinical pharmacokinetic (PK) studies of benapenem. We also developed a two-compartment model to characterize the PK profiles of benapenem in infected mice, which could provide a better understanding of the PK properties of benapenem.



Simultaneous determination of the pharmacokinetics of the active components of San-Chen-pill by UPLC-Q-Exactive-MS

Lingling Zhang, Huiwen Zhang, Shikui Wu, Hong Liu, Jingyuan Li, Xin Dong, Huimin Xia, Huanyun Wang

2019, 28(11):  812-824.  DOI: 10.5246/jcps.2019.11.077

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In the present study, we developed a rapid, sensitive and efficient ultra high-performance liquid chromatography-tandemmass spectrometric (UPLC-Q-Exactive-MS) method, and six bioactive constituents in San-Chen-pill (SCP), including cholic acid (CA),hyodeoxycholic acid (HDCA), hydroxysafflor yellow A (HSYA), kaempferol-3-O-rutinoside (KFR), anhydrosafflor yellow B (AHSYB) and syringin (SRG), in rat plasma after oral administration of San-Chen-pill, were simultaneously determined. Plasma samples were pretreated with methanol for protein precipitation. Chromatography separation was performed on a SHIMADZU of shim-pack GIST C₁₈ column using a gradient mobile phase consisting of acetonitrile and 0.1% formic acid aqueous solution. A tandem mass spectrometric detection with an electrospray ion source (HESI) interface was conducted in negative ionization mode. For all the six analytes of interest, the calibration curves were linear within the concentration rage of 0.50–1280 ng/mL with r≥0.99. The intra-day and inter-day precisions (in terms of relative standard deviation, RSD) were all below 12.7% for all six analytes. Extraction recovery, matrix effect and stability data all met the acceptance criteria of FDA guideline for bioanalytical method validation. In addition, San-Chen-pill is a traditional Mongolian herbal medicine that has been prepared with three medicinal herbs,Calculus bovis, Carthamus tinctorius L. and Concretio silicea bambusae, and it shows potent anti-anginal activity in all preparationswith in a ratio of 1:1:1. The pharmacokinetic parameters of San-Chen-pill were reported herein for the first time, and this developed method was successfully used in a pharmacokinetic study for San-Chen-pill.