Viruses, Vol. 15, Pages 74: Structural Investigations on Novel Non-Nucleoside Inhibitors of Human Norovirus Polymerase

Conceptualization, M.B. and S.F.; methodology, M.B., G.G., G.N., G.P., M.L., N.S.-F., J.V.D., V.N., U.P. and S.F.; validation, G.G., G.N., J.R.-P., S.F. and M.B.; formal analysis, G.G., G.N., G.P., N.S.-F., J.V.D., J.R.-P., S.F. and M.B.; investigation, M.B., S.F., J.R.-P. and A.B.; resources, M.B., S.F., J.R.-P., A.B., J.N., R.S., R.G.-R. and J.R.-D.; data curation, G.G., G.N., G.P., N.S.-F., J.V.D., J.R.-P., S.F. and M.B.; writing—original draft preparation, M.B., G.G., M.L., V.N., G.N. and S.F.; writing—review and editing, all authors; supervision, M.B., S.F. and J.R.-P.; project administration, M.B., S.F. and J.R.-P.; funding acquisition, M.B. and S.F. All authors have read and agreed to the published version of the manuscript.

Figure 1. Chemical structures of previously reported inhibitors of norovirus RdRp [16]. Figure 1. Chemical structures of previously reported inhibitors of norovirus RdRp [16]. Viruses 15 00074 g001

Figure 2. Micromolar inhibitors of norovirus RdRp previously identified by our group through a docking-based virtual screening approach [18]. Figure 2. Micromolar inhibitors of norovirus RdRp previously identified by our group through a docking-based virtual screening approach [18]. Viruses 15 00074 g002

Figure 3. Summary of the planned structural modifications to the scaffolds of hit compounds 1–5.

Figure 4. Previously proposed binding mode for 5 (carbon atoms in purple) to the human norovirus RdRp, overlapped with the co-crystallized ligand PPNDS (atoms in grey) [18,19]. The binding site area is represented as green molecular surface. Human norovirus RdRp is represented as green ribbon, with carbon atoms in green. Black dashed lines represent non-bonded interactions (e.g., hydrogen bonds, electrostatic interactions) between the ligand and the amino acid residues of the protein. Figure 4. Previously proposed binding mode for 5 (carbon atoms in purple) to the human norovirus RdRp, overlapped with the co-crystallized ligand PPNDS (atoms in grey) [18,19]. The binding site area is represented as green molecular surface. Human norovirus RdRp is represented as green ribbon, with carbon atoms in green. Black dashed lines represent non-bonded interactions (e.g., hydrogen bonds, electrostatic interactions) between the ligand and the amino acid residues of the protein. Viruses 15 00074 g004

Figure 5. Proposed binding mode for 1 (carbon atoms in purple) and 1e,f (carbon atoms in lilac) to the human norovirus RdRp (PDB ID 4LQ3). The binding site area is represented as green molecular surface. Human norovirus RdRp is represented as green ribbon, with carbon atoms in green. Black dashed lines represent non-bonded interactions (e.g., hydrogen bonds, electrostatic interactions) between the ligand and the amino acid residues of the protein.

Figure 6. Proposed binding mode for 2 (carbon atoms in purple) and 2g,h (carbon atoms in lilac) to the human norovirus RdRp (PDB ID 4LQ3). The binding site area is represented as green molecular surface. Human norovirus RdRp is represented as green ribbon, with carbon atoms in green. Black dashed lines represent non-bonded interactions (e.g., hydrogen bonds, electrostatic interactions) between the ligand and the amino acid residues of the protein.

Figure 7. Proposed binding mode for 3 (carbon atoms in purple), 3a–d and 3f (carbon atoms in lilac) to the human norovirus RdRp (PDB ID 4LQ3). The binding site area is represented as green molecular surface. Human norovirus RdRp is represented as green ribbon, with carbon atoms in green. Black dashed lines represent non-bonded interactions (e.g., hydrogen bonds, electrostatic interactions) between the ligand and the amino acid residues of the protein.

Figure 8. Proposed binding mode for 4 (carbon atoms in purple) and 4a–c (carbon atoms in lilac) to the human norovirus RdRp (PDB ID 4LQ3). The binding site area is represented as green molecular surface. Human norovirus RdRp is represented as green ribbon, with carbon atoms in green. Black dashed lines represent non-bonded interactions (e.g., hydrogen bonds, electrostatic interactions) between the ligand and the amino acid residues of the protein.

Figure 9. Proposed binding mode for 5 (carbon atoms in purple), 5a and 64 (carbon atoms in lilac) to the human norovirus RdRp (PDB ID 4LQ3). The binding site area is represented as green molecular surface. Human norovirus RdRp is represented as green ribbon, with carbon atoms in green. Black dashed lines represent non-bonded interactions (e.g., hydrogen bonds, electrostatic interactions) between the ligand and the amino acid residues of the protein.

Scheme 1. Preparation of 1 and its novel target analogues 1a–f. Reagents and conditions: (i) H2SO4, MeOH, reflux, o.n. (88%); (ii) NH2NH2*H2O, EtOH, reflux, o.n. (94%); (iii) NH4SCN, EtOH, HCl, reflux, o.n. (58%); (iv) 10% aq. NaOH, reflux, o.n. (72%); (v) K2CO3, Me2CO, r.t., o.n. (72–99%); (vi) Pyr, EtOH, reflux, o.n. (53–71%); (vii) m-CPBA, DCM, r.t., o.n. (34%); (viii) Nb(OEt)5, 30% aq. H2O2, MeOH, 45 °C, 2 h (87%).

Scheme 2. Preparation of 2 and novel target analogues 2a–h and 3a–f. Reagents and conditions: (i) AcOH, 130 °C, o.n. (83–93%); (ii) H2, Pd/C, THF, r.t., o.n. (74–99%); (iii) Pyr, THF, r.t., o.n. or anhydrous DCM, Et3N, r.t., o.n. (36–59%); (iv) H2SO4, MeOH, reflux, o.n. (89%); (v) a. diethyl chlorophosphate, Et3N, anhydrous DCM, 0 °C, 2h (65%), b. TMSBr, Pyr, anhydrous DCM, 0 °C to r.t., 56h (32%); (vi) H2SO4, MeOH, reflux, o.n. or MeI, K2CO3, Me2CO, reflux, 4h (84–99%); (vii) NH2NH2*H2O, EtOH, reflux, o.n. (58–96%); (viii) EtOH, reflux, o.n. (47–88%); (ix) diethylchlorophoshpate, Et3N, anhydrous DCM, 0 °C to r.t., o.n. (67–99%); (x) TMSBr, Pyr, anhydrous DCM, 0 °C to r.t., o.n. (54–95%).

Scheme 3. Preparation of novel analogues 4a–c and 5a. Reagents and conditions: (i) Pd(PPh3)4, K2CO3, H2O, EtOH, PhMe, reflux, o.n. (32–63%); (ii) ß-alanine, AcOH, 100 °C, o.n. (29–67%); (iii) a. diethyl chlorophosphate, Et3N, anhydrous DCM, 0 °C, 5h (89%), b. TMSBr, anhydrous DCM, 0 °C to r.t., 24 h (49%); (iv) Pyr, anhydrous DCM, 0 °C to r.t., 20 h (quant.); (v) H2, Pd/C, THF, r.t., 24 h (97%); (vi) HCl, NaNO2, SnCl2, H2O, 0 °C, 3 h (77%); (vii) ethyl malonyl chloride, Et3N, THF, -10 °C to r.t., 3 h (56%); (viii) 1M NaOH/EtOH, EtOH, r.t., 30 min (73%); (ix) AcOH, 120 °C, 3 h (72%); (x) a. diethyl chlorophosphate, Et3N, anhydrous DCM, 0 °C, o.n. (66%), b. TMSBr, anhydrous DCM, 0 °C to r.t., 3 h (58%).

Figure 10. Inhibitory effect of the test compounds on the human norovirus RdRp activity. Compounds were tested at 20 μM. Percentage of inhibition was normalized to control DMSO (red bar). PPNDS (green bar) was used as positive control, along with parent compounds 1, 2 and 5. The mean values of triplicate datasets with standard error of the mean are shown.

Figure 11. Counter-screen gel-shift assay to confirm HuNoV (GII.4) RdRp inhibitory activity for compounds 1, 1b, 1f, 2, 2a, 3a,b, 4b,c, 5 and 5a. The compounds were examined for inhibition of primed elongation activity. PE44-NoV RNA templates (32 nucleotides) were extended (44 nucleotides) by the RdRp in the absence of any test compounds (0.5% DMSO [vol/vol] negative control) or with test compounds at a fixed concentration of 100 μM. PPNDS was used as positive controls (100 μM) to demonstrate complete inhibition, and no RdRp was used as a negative control.