Discovery of a non-peptidic inhibitor of west nile virus NS3 protease by high-throughput docking

Abstract

Background: The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections.

Methodology: In this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by 15N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay.

Conclusion: The inhibitor [4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl]-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections.

Figure 1. Comparison of the calculated versus experimental binding free energies for 37 peptidic inhibitors of the WNV protease .

Peptide sequences are listed in Table S-I (found in Text S1). The solid diagonal is the ideal line of perfect prediction. The dashed diagonals delimit the 1 kcal/mol error region.

Figure 2. Schematic picture of the in silico screening campaign.

Docking of the 11715 compounds with one more positive charges or at least five hydrogen bond donors was performed by DAIM/SEED/FFLD using the 2fp7 structure of the WNV protease as explained in the text.

Figure 3. Compound 1, according to ¹H chemical shifts (see Figure 4).

Figure 4. Binding affinity of compound 1 to WNV NS2B(K96A)-NS3pro.

The changes in ¹H chemical shifts of the backbone amide protons of Thr52 and Glu101 were plotted as a function of increasing concentration of compound 1. A 10 mM stock solution of compound 1 was titrated into a 80 µM solution of ¹⁵N/¹³C-labeled NS2B(K96A)-NS3pro containing 20 mM phosphate (pH 7.0) and 2 mM DTT. The ¹⁵N-HSQC spectra were recorded at 25°C on an 800 MHz NMR spectrometer. The value derived from the fitting curves is about 40 µM (30±10 and 50±10 µM for the cross-peaks of Thr52 and Glu101, respectively).

Figure 5. Superimposition of ¹⁵N-HSQC spectra of WNV NS2B(K96A)-NS3pro in the absence (red) and presence (blue) of compound 1 at 25°C.

The resonance assignments are shown for selected peaks. The samples contained 0.9 mM protein in 90% H2O/10% D2O, 20 mM HEPES buffer (pH 7.0), 2 mM DTT. The complex with compound 1 was prepared by titrating the protein solution with a single addition of 15 µl of a DMSO solution of compound 1 to a final concentration of 3 mM. The spectra were recorded at a ¹H NMR frequency of 800 MHz.

Figure 6. Binding mode of compound 1.

(Top) Interactions between compound 1 and WNV protease. The atoms of the protein and ligand are shown as balls and colored as carbon in black, oxygen in red, nitrogen in blue, and sulfur in yellow. Intermolecular hydrogen bonds are shown by green dashed lines with distances in Å. The intermolecular van der Waals contacts interactions (distance ≤4 Å) are shown in red dashed lines. (Figure made by Ligplot [67]). (Bottom) Stereoview of the binding mode of 1. The protease is shown as cartoon and its secondary structure is emphasized by different colors: helices in red, strands in yellow, and loops in green. Atoms of compound 1 are colored as in the (top) with hydrogens in black. This figure was prepared using PyMOL (Delano Scientific, San Carlos, CA).

Figure 7. The protease surface (PDB code 2FP7) is colored according to atomic partial charges.

(Left): Binding mode of the propylamine derivative of compound 1. Compound 1 and its propylamine derivative are in magenta and colored by atom type, respectively. (Right): Binding mode of the nitrophenyl derivative of compound 1. The substrate 2-naphthoyl-KKR-pNA and the nitrophenyl derivative of compound 1 are in white and colored by atom type, respectively. The binding mode of the substrate is modelled based on Ref. 4 and only part of it is shown for clarity. These figures were prepared using PyMOL (Delano Scientific, San Carlos, CA).