Fig 1.
Main stages of the funnel-like VS protocol.
The protocol consists of four stages: i) flexible pharmacophore-based VS (flexi-pharma) [31], ii) docking and exponential consensus ranking (ECR-docking) [43], iii) MD simulations with consensus ranking -that includes a new Morse-based ligand-flexibility score-, and iv) biological experimental binding and activity assays. At each stage, the compound library was filtered. The protocol was optimized and validated over a library of 1993 compounds, which was previously tested over CaFADS [41]. On the left, we show an example of the reduction of this library going from 100% to 0.25% through the successive steps.
Fig 2.
A score that uses a Morse potential (Eq 3) was implemented for scoring the flexibility of the ligand inside the pocket using MD simulations. The input variable is the standard deviation of the RMSD of the ligand’s atomic positions around the binding site. Ligands that show large RMSD variations are considered very flexible -with dissociation tendencies (i.e., unstable)- and their behavior is penalized (right of vertical black dashed arrow). Ligands with small RMSD fluctuations are considered rigid leading to a conformational penalization (left of black vertical dashed arrow). The Morse potential was implemented with a force constant k = 1 kcal/mol.nm2, a depth of the well of ω = 1 kcal/mol, and the minimum is localized at r0 = 0.242 Å.
Fig 3.
Flexi-pharma VS stage over the Pretswick library.
Average enrichment plot of the Pretswick library using the flexi-pharma stage over MD conformations of ligand-free CaFADS. The affinity-grid threshold value is 0.1% and 600 equidistant frames obtained from an MD of 60 ns were used [42]. The flexi-pharma number of votes for each molecule was used as a score to calculate the EPs. Bootstrapping analysis was performed by sampling with replacement 100 times to obtain the average EP and its standard deviation. The violet line shows the screening threshold (≈30%) for the selection of molecules to be filtered and passed onto the second stage.
Fig 4.
ECR-docking VS stage over the Pretswick library.
Enrichment plot using the ECR (black line) from the best Autodock4.2 pose that is re-scored with four scoring functions (Autodock4.2, Vina, Vinardo and CYscore). The shaded area encloses the best and worst behaviors for the individual scoring functions. The enrichment plot is normalized by the initial database values (39-ligands and 1993 compounds). The violet line shows the threshold for the selection of molecules for the third VS stage.
Fig 5.
MD-ranking 1 VS stage over the Pretswick library.
Enrichment plot obtained using an ECR methodology (black) from the combination of the ECR scoring function-based rank (ECR-SF) (green) and Morse-based rank (blue). 5 ns of MD for 100 complexes were carried out. We used 200 equidistant frames from the last 2 ns of MD simulation. The enrichment plot is normalized to the initial database (39-ligands and 1993 compounds). The violet line shows the threshold for the molecule selection for the MD-ranking 2 stage.
Table 1.
EFs obtained for each VS stages using the Prestwick library.
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).
Table 2.
In vitro performance of VS hits over the FMNAT and RFK+FMNAT CaFADS activities.
Table 3.
In vivo performance of VS hits.
Fig 7.
Docking poses showing the main interactions of the VSHs and CaFADS.
These were obtained with Autodock4.2 and were used as the starting conformation for the MD stage.