DNase-targeted natural product screening based on a sensitive and selective DNase I detecting system

Abstract

As a widely used deoxyribonuclease, DNase I is involved in many physiological processes including tumor cell proliferation, metastasis and apoptosis. Furthermore, the level of this enzyme in serum can act as a functional biomarker for the therapeutic monitoring of systemic lupus erythematosus and other diseases. We report here a low cost and sensitive DNase I detecting system based on the single-stranded fluorogenic substrate and nanographene oxide (NGO) and use it for DNase-targeted natural product screening. The system with a detection limit of 0.005 U was then used to evaluate the effect of external factors on DNase I. The results show that Hg 2+ , As 2+ , Pb 2+ , Cd 2+ and Cu 2+ can inhibit DNase I activity in a concentration-dependent manner with IC 50 values of 0.37 mM (Hg 2+ ), 2.7 mM (As 2+ ), 5 mM (Pb 2+ ), 5.3 mM (Cd 2+ ) and 7.8 mM (Cu 2+ ), respectively. Meanwhile, 10 natural compounds isolated from Cyclocarya paliurus leaves were screened as DNase I inhibitors, while 5 compounds were identified as activators. Finally, the system was used to discriminate DNase activity of serum samples with and without HBV. The results showed that HBV infection significantly decreased the level of DNase I in serum samples. In summary, these data indicate that this method with the advantages of rapidity, low cost and high sensitivity is hopeful for DNase assay in biological samples as well as compound screening in vitro.

Expanding the Toolbox for Label-Free Enzyme Assays: A Dinuclear Platinum(II) Complex/DNA Ensemble with Switchable Near-IR Emission

“…A Lineweaver-Burk double reciprocal plot of 1/ V 0 versus 1/[S] revealed a linear correlation (Figure 6B) with a Michaelis–Menten constant (K m ) of 1.26 ± 0.3 µM. This calculated K m value is in good agreement with previously reported K m values for DNAse I, which fall in the range of 0.4–2.19 µM [21,22]. These results show that the 4 / QIII DNA ensemble is an efficient real-time assay of DNAse I activity and its kinetic parameters.…”

“…These methods are time-consuming, labor intensive, and/or require use of covalently labelled DNA. These limitations are partially addressed by luminescence-based DNAse I assays that feature simplicity, sensitivity and ease of operation [21,22,23]. However, these methods often rely on the use of fluorophore-labelled DNA [24,25,26,27].…”

1,2,3,4‐Tetrahydroisoquinoline Derivatives as a Novel Deoxyribonuclease I Inhibitors

“…In addition, active compounds 2 , 15 , 18 , and 22 exhibited, respectively 2.70‐, 2.45‐, 2.43‐ and 2.44‐times better inhibitory activity in comparison to crystal violet used as a positive control. Observed DNase I inhibitory potential of active 1,2,3,4‐tetrahydroisoquinolines is in line with a multitude of other small organic DNase I inhibitors, whose IC 50 mainly range between 70 and 150 μM, [28–34] whereas the most potent small natural products inhibitors of DNase I are cyclocariol F and schisanlactone E with IC 50 s of 12.5±2 μM and 30±3 μM, respectively [35] …”

Repurposing of 8‐Hydroxyquinoline‐Based Butyrylcholinesterase and Cathepsin B Ligands as Potent Nonpeptidic Deoxyribonuclease I Inhibitors

“…Sixteen identified DNase I inhibitors with IC 50 values below 80 μM ( 7 – 9 , 11 , 12 , 14 – 24 ), exceeded the inhibitory activity of all previously reported small‐molecule DNase I inhibitors [8,19,21–29] . To the best of our knowledge, compounds 8 and 22 are among the most potent synthetic non‐peptide DNase I inhibitors reported to date, with the natural product cyclocariol F inhibiting DNase I with a lower IC 50 value (IC 50 =12.5±2 μM) [30] . However, unlike compounds 8 and 22 , which follow Lipinski's rule of five and have additionally been studied in vitro , cyclocariol F violates this rule (Supporting Information Table S1).…”