BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
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2020
·Vol. 84
·期 6
·1153-1159
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IF 1.516
Xue Guangpu,Xie Xie,Zhou Yang,Yuan Cai,Huang Mingdong,Jiang Longguang
Peptidic inhibitors of proteases are attracting increasing interest not only as drug candidates but also for studying the function and regulation mechanisms of these enzymes. Previously, we screened out a cyclic peptide inhibitor of human uPA and found that Ala substitution of P2 residue turns upain-1 to a substrate. To further investigate the effect of P2 residue on the peptide behavior transformation, we constructed upain-1-W3F, which has Phe replacement in the P2 position. We determined KD and Ki of upain-1-W3F and found that upain-1-W3F might still exist as an inhibitor. Furthermore, the high-resolution crystal structure of upain-1-W3F·uPA reveals that upain-1-W3F indeed stays as an intact inhibitor bind to uPA. We thus propose that the P2 residue plays a nonnegligible role in the conversion of upain-1 to a substrate. These results also proposed a strategy to optimize the pharmacological properties of peptide-based drug candidates by hydrophobicity and steric hindrance.Abbreviations : uPA: urokinase-type plasminogen activator; SPD: serine protease domain; S1 pocket: specific substrate-binding pocket
研究领域:
药理学
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生物化学
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分子生物学
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DOI
PubMed
高影响因子
INTERNATIONAL IMMUNOPHARMACOLOGY
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2023
·
IF 5.6
Shixin Duan,Jun Wang,Xinyi Lou,Dongxin Chen,Peiyunfeng Shi,Hongchao Jiang,Zhiming Wang,Wen Li,Feng Qian
As a crucial member of the Interleukin-1 (IL-1) family, IL-33 plays an indispensable role in modulating inflammatory responses. Here, we developed an effective anti-human IL-33 monoclonal antibody (mAb) named 5H8. Importantly, we have identified an epitope (FVLHN) of IL-33 protein as a recognition sequence for 5H8, which plays an important role in mediating the biological activity of IL-33. We observed that 5H8 significantly suppressed IL-33-induced IL-6 expression in bone marrow cells and mast cells in a dose-dependent manner in vitro. Furthermore, 5H8 effectively relieved HDM-induced asthma and PR8-induced acute lung injury in vivo. These findings indicate that targeting the FVLHN epitope is critical for inhibiting IL-33 function. In addition, we detected that the Tm value of 5H8 was 66.47℃ and the KD value was 173.0 pM, which reflected that 5H8 had good thermal stability and high affinity. Taken together, our data suggest that our newly developed 5H8 antibody has potential as a therapeutic antibody for treating inflammatory diseases.
研究领域:
免疫学
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药理学
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炎症
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产品关联
DOI
PubMed
高影响因子
LAB ON A CHIP
·
2020
·Vol. 20
·期 9
·1577-1585
·
IF 6.774
Jingjing Guo,Li Lin,Kaifeng Zhao,Yanling Song,Mengjiao Huang,Zhi Zhu,Leiji Zhou,Chaoyong Yang
The dissociation constant (Kd) is a crucial parameter for characterizing binding affinity in molecular recognition, including antigen–antibody, DNA–protein, and receptor–ligand interactions. However, conventional methods for Kd characterization usually involve a multi-step process and time-consuming operations for incubation, washing, and detection, thus causing problems, such as time delays, microbead loss, degradation of sensitive molecules, and personal errors. Here we demonstrate an automated ligand binding affinity evaluation platform (Auto-affitech) using digital microfluidics (DMF), with individual droplets at the microliter level, programmed to rapidly perform the incubation and separation of target-beads and binding ligands. Because the loss of the beads influences the detection results, we propose a new strategy for magnetic bead separation using DMF, termed the bidirectional separation method. By splitting one droplet into two asymmetric droplets, high bead retention efficiency (89.57% ± 0.05%) and high washing efficiency (99.59% ± 0.17%, with four washings) were obtained. We demonstrate the determination of Kd of an aptamer–protein system (EpCAM and its corresponding aptamer SYL3C) and an antigen–antibody system (H5N1 antigen and antibody), proving the capability and universality of Auto-affitech in various receptor–ligand systems. Integrating all the sample processing procedures, the Auto-affitech not only saves manual labor and minimizes personal errors, but also conserves samples and shortens analysis time. Overall, this platform successfully demonstrates to be an automated approach for dissociation constant evaluation and exhibits great potential for highly efficient screening of ligands.
研究领域:
生物技术
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分析化学
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分子生物学
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微流控
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2022
·
IF 12.545
Xiaojuan Wang,Zefeng Mao,Ruipeng Chen,Shuning Li,Shuyue Ren,Jun Liang,Zhixian Gao
DNA origami technology has great potential for biosensor applications. Here, we described the construction of a self-assembled DNA origami biosensor for the precise localization of fluorescent aptamers . Due to the molecular weight difference between DNA origami and aptamer, centrifugal filters were used to quantitatively detect adenosine triphosphate (ATP). The ATP-specific aptamer labeled with fluorescence reporter 6-carboxyfluorescein FAM (FAM-aptamer) was selected as the recognition element and signal probe . ATP duplexed aptamers bound to triangular DNA origami by base-complementary pairing, resulting in high fluorescence signals on the origami arrays. The competitive binding of ATP toward the FAM-aptamer triggered the release of FAM-aptamer-ATP complexes from the surface of the origami array, resulting in weakened fluorescence signals. For ATP quantification, 100 kD centrifugal filters were employed, followed by measurement of the fluorescence signal trapped on the origami arrays of the filter device. The successful synthesis of origami-aptamer arrays was characterized by atomic force microscopy , laser confocal microscopy , and electrophoresis . Fluorescence measurements exhibited an excellent linear relationship with logarithms of ATP concentrations within 0.1–100 ng mL −1 , with a detection limit of 0.29 ng mL −1 . By replacing aptamers and complementary strands, we demonstrated the potential of this method for 17β-estradiol detection. Considering that the detection mechanism is based on the hybridization and displacement of DNA strands, the detection system had the potential for recharging. Our study provides new insights into applying DNA origami technology in small molecule detection.
研究领域:
生物传感器
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纳米技术
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分子生物学
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分析化学
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产品关联
DOI
PubMed
高影响因子
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