Application of FRET Biosensors in Mechanobiology and Mechanopharmacological Screening

Liu, Longwei; He, Fangchao; Yu, Yiyan; Wang, Yingxiao

doi:10.3389/fbioe.2020.595497

Cited by 55 publications

(33 citation statements)

References 199 publications

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“…However, we found this catalytic site (Cys144) was next to our solution 2 ( Figure 3 B). To further determine whether hypericin could inhibit the enzymatic activity of 3CLpro, we took advantage of an established FRET assay [ 37 , 38 , 39 ] by using the reporter FRET substrate Dabcyl-YNSTLQ↓AGLRKM-E-Edans. As a start, we expressed the recombinant PEDV 3CLpro with conventional methods of prokaryotic expression, followed by purification with Glutathione Sepharose (GSH) 4B column (GE Healthcare, Chicago, IL, USA) ( Figure 4 A).…”

Section: Resultsmentioning

confidence: 99%

Hypericin Inhibit Alpha-Coronavirus Replication by Targeting 3CL Protease

Zhang

Chen

Zou

et al. 2021

Viruses

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The porcine epidemic diarrhea virus (PEDV) is an Alphacoronavirus (α-CoV) that causes high mortality in infected piglets, resulting in serious economic losses in the farming industry. Hypericin is a dianthrone compound that has been shown as an antiviral activity on several viruses. Here, we first evaluated the antiviral effect of hypericin in PEDV and found the viral replication and egression were significantly reduced with hypericin post-treatment. As hypericin has been shown in SARS-CoV-2 that it is bound to viral 3CLpro, we thus established a molecular docking between hypericin and PEDV 3CLpro using different software and found hypericin bound to 3CLpro through two pockets. These binding pockets were further verified by another docking between hypericin and PEDV 3CLpro pocket mutants, and the fluorescence resonance energy transfer (FRET) assay confirmed that hypericin inhibits the PEDV 3CLpro activity. Moreover, the alignments of α-CoV 3CLpro sequences or crystal structure revealed that the pockets mediating hypericin and PEDV 3CLpro binding were highly conserved, especially in transmissible gastroenteritis virus (TGEV). We then validated the anti-TGEV effect of hypericin through viral replication and egression. Overall, our results push forward that hypericin was for the first time shown to have an inhibitory effect on PEDV and TGEV by targeting 3CLpro, and it deserves further attention as not only a pan-anti-α-CoV compound but potentially also as a compound of other coronaviral infections.

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Section: Resultsmentioning

confidence: 99%

Hypericin Inhibit Alpha-Coronavirus Replication by Targeting 3CL Protease

Zhang

Chen

Zou

et al. 2021

Viruses

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“…For many of these sensors, Förster resonance energy transfer (FRET) is used as a detectable output [ 8 , 45 ]. These sensors often use two fluorescent proteins, such as cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), together as an FRET pair, relying on the conformational change of the biosensor protein to bring both proteins into closer proximity in the presence of ligand [ 46 , 47 , 48 , 49 ]. In a recent work by Juárez et al, a synthetic transcription factor biosensor was constructed in the lab, linking ligand binding to DNA transcription through an engineered allosteric interaction [ 50 ].…”

Section: Mechanistic Classes Of Biosensors Within Bacterial Cellsmentioning

confidence: 99%

Strategies for Improving Small-Molecule Biosensors in Bacteria

Miller

Bennett

2022

Biosensors

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In recent years, small-molecule biosensors have become increasingly important in synthetic biology and biochemistry, with numerous new applications continuing to be developed throughout the field. For many biosensors, however, their utility is hindered by poor functionality. Here, we review the known types of mechanisms of biosensors within bacterial cells, and the types of approaches for optimizing different biosensor functional parameters. Discussed approaches for improving biosensor functionality include methods of directly engineering biosensor genes, considerations for choosing genetic reporters, approaches for tuning gene expression, and strategies for incorporating additional genetic modules.

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“…132 Fluorophores, which can be applied in FRET, now embody natural dyes, semiconductor quantum dots, uorescent proteins, metallic chelates, several noble metals and different nanoparticles, and also polymers, basically uorescent amino acids and organic cofactors. 132,133 Recently, a uorescent turn-on sensor for detecting micromolar levels of bacterial LPS has been industrialized. Polydiacetylene liposome surface modied with fabricated pentalysine peptide and histidine have effectively displayed uorescence signal only in the presence of LPS.…”

Section: Optical Biosensors For Detection Of Lpsmentioning

confidence: 99%