In silico discovery and biological validation of ligands of FAD synthase, a promising new antimicrobial target
Fig 6
In vitro assessment of VSHs ability to bind and to inhibit CaFADS.
A) Thermal denaturation curve for CaFADS (2 μM) observed by differential scanning fluorescence and Tm shifts observed in the presence of the compounds at 250 μM. Thermal stability curves are plotted against the normalized fluorescence signal. Experiments were carried out in 20 mM PIPES, pH 7.0, 10 mM MgCl2, 2% DMSO. B) Dependence of Δ Tm on the VSH concentration and data fit to Eq 2. C) Dose-response curves for the FMNAT activity of CaFADS in the presence of representative VSHs. Experiments performed at 25 °C in 20 mM PIPES, pH 7.0, 10 mM MgCl2, 2% DMSO, with 5 μM FMN and 50 μM ATP. Values derived from these representations are included in Table 2, such as the IC50 and % of remaining activity at 250 μM of the VSH. D) Comparison of the effects of the VSHs on the RFK and FMNAT activities of CaFADS. All the experiments were carried out at 25 °C, in 20 mM PIPES pH 7.0, MgCl2 (10 mM when assaying FMNAT activity and 0.8 mM when assaying RFK activity) at saturating concentrations substrates and in the presence of 250 μM of the VSH (2% DMSO, final concentration). Compound color code: Protein in the absence of VSH is shown in light gray, C3 is violet, C5 is red, C6 is green, C9 is blue, C12 is black (shown as control, neither binder nor inhibitor) and C18 is orange. Note that not all molecules are shown in all panels. In panel D, compounds different from the above mentioned are indicated in dark gray and calculated activity percentages are relative to the corresponding ones in absence of compounds. (n = 3, mean ± SD).