Journal: Antimicrobial Agents and Chemotherapy

Article Title: Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities

doi: 10.1128/AAC.01641-20

Figure Lengend Snippet: Effects of nucleotide analogs on the helicase activity of polyomavirus Tags. The helicase activities of the polyomavirus Tags were determined as previously described (83) and measured in the absence or presence of increasing amounts HDP-CDV. The reaction products were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a Fuji FLA-5100 phosphor imager. (A) A representative result. Lanes 1, heat denatured (HD) substrate, and 3, no Tag, served as controls for complete denaturing of the substrate and negative control. Lane 2 presents the unwinding activity of 0.6 µg of SV40 Tag without inhibitor, whereas lanes 4 to 10 show the Tag unwinding activity in the presence of increasing amounts HDP-CDV as indicated. The bands were quantified with ImageQuant analysis software (Fujifilm Europe, Düsseldorf, Germany), and the relative DNA unwinding was calculated by determining the percentage ratio of unwound DNA in analog-treated samples to the solvent-treated sample. (B) The presented data are the mean and standard deviation of 3 experiments of the inhibition of SV40 and BKV Tag helicase activities by HDP-CDV. Both curves were fitted using the program GraphPad and the model “log[inhibitor] versus normalized response” plus the equation Y=1001 + 10(X−LogIC50).

Article Snippet: The bands were quantified with ImageQuant analysis software (Fujifilm Europe, Düsseldorf, Germany), and the relative DNA unwinding was calculated by determining the percentage ratio of unwound DNA in analog-treated samples to the solvent-treated sample. (B) The presented data are the mean and standard deviation of 3 experiments of the inhibition of SV40 and BKV Tag helicase activities by HDP-CDV.

Techniques: Activity Assay, Negative Control, Software, Standard Deviation, Inhibition

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities

doi: 10.1128/AAC.01641-20

Figure Lengend Snippet: ssDNA and dsDNA binding activities for SV40 Tag. The binding of SV40 Tag to ssDNA and dsDNA was analyzed as previously described (51) to optimize Tag concentrations required to investigate the inhibition of Tag-DNA interactions by nucleotide analogs. (A and C) The binding products using (A) ssDNA and (B) SV40 Tag site 2-containing dsDNA were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a phosphor imager. The bands were quantified with ImageQuant analysis software, and the bound DNA was calculated by determining the ratio of the bound to total DNA. The data were fitted using GraphPad and the model “one site-specific binding with Hill slope” to obtain the binding constants (panels B and D). The KD values for Tag binding to ssDNA and dsDNA are 131.9 ± 3.1 nM and 120.8 ± 4.7 nM, respectively.

Article Snippet: The bands were quantified with ImageQuant analysis software (Fujifilm Europe, Düsseldorf, Germany), and the relative DNA unwinding was calculated by determining the percentage ratio of unwound DNA in analog-treated samples to the solvent-treated sample. (B) The presented data are the mean and standard deviation of 3 experiments of the inhibition of SV40 and BKV Tag helicase activities by HDP-CDV.

Techniques: Binding Assay, Inhibition, Software

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities

doi: 10.1128/AAC.01641-20

Figure Lengend Snippet: Effects of HDP-CDV on the DNA binding activity of SV40 Tag. Following the determination of the binding constants for SV40 Tag binding to ssDNA and dsDNA, the effects of HDP-CDV on ssDNA and dsDNA binding function of Tag were determined via electrophoretic mobility shift assay (EMSA) at the indicated analog concentrations in the presence of 300 nM Tag. The reaction Tag-ssDNA and Tag-dsDNA products were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a phosphor imager. (A and C) Representative results. The bands were quantified with ImageQuant software, and the relative DNA binding was calculated by determining the percentage ratio of bound DNA of the HDP-CDV treated samples to the solvent-treated sample. (B and D) The mean and standard deviation from 3 independent experiments are presented. The data were fitted with the model “log[inhibitor] versus response − variable slope” on GraphPad to obtain the binding curves and inhibition constants using the equations Y=−1.09 + 92.81 + 10(X−LogIC50)*3.406 and Y=−7.83 + 96.51 + 10(X−LogIC50)*2.129 (panels B and D, respectively). The IC50 values for the inhibition of binding of Tag to ssDNA and dsDNA by HDP-CDV were determined as 17.4 ± 1.02 µM and 22.1 ± 1.04 µM, respectively.

Article Snippet: The bands were quantified with ImageQuant analysis software (Fujifilm Europe, Düsseldorf, Germany), and the relative DNA unwinding was calculated by determining the percentage ratio of unwound DNA in analog-treated samples to the solvent-treated sample. (B) The presented data are the mean and standard deviation of 3 experiments of the inhibition of SV40 and BKV Tag helicase activities by HDP-CDV.

Techniques: Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Software, Standard Deviation, Inhibition

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Alkoxylalkyl Esters of Nucleotide Analogs Inhibit Polyomavirus DNA Replication and Large T Antigen Activities

doi: 10.1128/AAC.01641-20

Figure Lengend Snippet: HDP-CDV does not influence the DNA binding activity of RPA. (A and B) The effects of HDP-CDV on the binding of RPA to ssDNA were determined via EMSA at the indicated analog concentrations in the presence of 2 nM RPA. The free ssDNA and the RPA-ssDNA complexes were separated on nondenaturing polyacrylamide gels, which were subsequently analyzed using a phosphor imager (panel A). The bands were quantified with ImageQuant software, and the relative DNA binding was calculated by determining the percentage ratio of bound DNA of the HDP-CDV-treated samples to the solvent-treated sample. Then the results from 3 independent experiments were fitted with various nonlinear regression models using GraphPad, but none yielded reliable results. Therefore, a linear regression analysis was performed yielding the trendline with the equation y = − 3.5114×112.6; r2 = 0.9776 (panel B).

Article Snippet: The bands were quantified with ImageQuant analysis software (Fujifilm Europe, Düsseldorf, Germany), and the relative DNA unwinding was calculated by determining the percentage ratio of unwound DNA in analog-treated samples to the solvent-treated sample. (B) The presented data are the mean and standard deviation of 3 experiments of the inhibition of SV40 and BKV Tag helicase activities by HDP-CDV.

Techniques: Binding Assay, Activity Assay, Software