isoquercitrin and baicalin

Sortase A (SrtA) has long been recognized as an ideal drug target for therapeutic agents against Gram-positive pathogens. However, the SrtA of Streptococcus suis (Ss-SrtA), an important zoonotic agent, has not been studied. In this study, the enzymatic properties of Ss-SrtA were investigated, and inhibition of Ss-SrtA by natural products was evaluated. Ss-SrtA was expressed and purified. The purified recombinant Ss-SrtA had maximal activity at pH 6.0-7.5, 45°C, and showed a Km of 6.7 μM for the hydrolysis of substrate abz-LPATG-dnp. Different from Staphylococcus aureus SrtA (Sa-SrtA) which is stimulated by Ca2+, Ss-SrtA was observed to be Ca2+ independent. Structural analysis showed that salt bridges formed between K111 and D180 in Ss-SrtA replaced the function of Ca2+ in Sa-SrtA to stabilize the substrate-binding cleft. Site-directed mutagenesis identified H126, C192 and R200 as the key residues of Ss-SrtA active site. To discover potential inhibitors, the percent inhibition of sortase activity by natural products was measured. Among these selected natural products, acteoside, isoquercitrin and baicalin were discovered as novel SrtA inhibitors, with IC50 values of 36.3 ± 1.3 μM, 100.0 ± 1.3 μM and 85.4 ± 1.5 μM, respectively. The inhibitory effects of these three natural products were further confirmed on endogenous Sa-SrtA. Using a previously established S. aureus model with a fluorescent-labeled Sa-SrtA substrate, acteoside, isoquercitrin, and baicalin showed 86%, 28% and 45% inhibition on endogenous Sa-SrtA activity, respectively. Overall, these findings shed new light on enzymatic properties, Ca2+-independent catalytic mechanism and potential inhibitors of Ss-SrtA.

Topics: Amino Acid Sequence; Aminoacyltransferases; Bacterial Proteins; Calcium; Catalytic Domain; Cysteine Endopeptidases; Drug Evaluation, Preclinical; Enzyme Inhibitors; Flavonoids; Glucosides; Hydrogen-Ion Concentration; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Phenols; Quercetin; Streptococcus suis; Temperature

In this study, 2,6-dimethyl-β-cyclodextrin (DM-β-CD) functionalized graphene nanosheets (DM-β-CD-GNs) were successfully synthesized by a simple wet-chemical strategy. The as obtained DM-β-CD-GNs were characterized by UV-vis spectroscopy, Fourier transform Infrared (FT-IR) spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM). The new nanocomposite possesses the unique properties of graphene (large surface area and high conductivity) and DM-β-CD (high supramolecular recognition and enrichment capability). Based on the above properties, a highly sensitive electrochemical sensor was developed to detect two flavonoid drugs (isoquercitrin and baicalin). At the DM-β-CD-GNs modified glassy carbon electrode (DM-β-CD-GNs/GCE), the peak currents of the two drugs increased dramatically compared with that on the bare GCE and GNs/GCE which due to the synergetic effects of GNs and DM-β-CD molecules. The linear response ranges for isoquercitrin and baicalin are 10nM-3.0μM and 0.04μM -3.0μM, with the detection limits of 4nM and 10nM, respectively. The method might open up a new possibility for the widespread use of electrochemical sensors for monitoring of ultra-trace flavonoid drugs owing to its advantages of simple preparation, low cost, high sensitivity, good stability and reproducibility.

Topics: beta-Cyclodextrins; Biosensing Techniques; Conductometry; Electrodes; Equipment Design; Equipment Failure Analysis; Flavonoids; Graphite; Nanoparticles; Quercetin; Surface Properties