Structure-guided design and optimization of dipeptidyl inhibitors of norovirus 3CL protease. Structure-activity relationships and biochemical, X-ray crystallographic, cell-based, and in vivo studies

Abstract

Norovirus infection constitutes the primary cause of acute viral gastroenteritis. There are currently no vaccines or norovirus-specific antiviral therapeutics available for the management of norovirus infection. Norovirus 3C-like protease is essential for viral replication, consequently, inhibition of this enzyme is a fruitful avenue of investigation that may lead to the emergence of antinorovirus therapeutics. We describe herein the optimization of dipeptidyl inhibitors of norovirus 3C-like protease using iterative SAR, X-ray crystallographic, and enzyme and cell-based studies. We also demonstrate herein in vivo efficacy of an inhibitor using the murine model of norovirus infection.

Figure 1

General structure of dipeptidyl inhibitor (I)

Figure 2

Interaction of a cysteine protease (E-Cys-SH) with a peptidyl transition state inhibitor.

Figure 3

Hydrogen bonding interactions between the inhibitor and chain B for NV 3CLpro:ligand (precursor aldehyde of inhibitor 44) Side chain residues are colored cyan, inhibitor is colored gray, and water molecules are represented as red spheres. Hydrogen bonds are represented as dashed lines and water mediated contacts as solid lines.

Figure 4

Residues spanning Ala159-Lys162 located near the benzyl ring of precursor aldehyde of inhibitor 44.

Figure 5

Fo – Fc map (green mesh) of precursor aldehyde derived from compound 17 bound to NV 3CLpro contoured at 3σ.

Figure 6

Hydrogen bond interactions between NV 3CLpro and precursor aldehyde derived from compound 17. Hydrogen bonds are represented as dashed lines and water mediated contacts are shown as solid lines.

Figure 7

Surface representation of precursor aldehyde of compound 17 bound to NV 3CLpro. Neighboring residues of NV 3CLpro are colored yellow (nonpolar), white (weakly polar) and cyan (polar).

Figure 8

Protease inhibitor suppresses norovirus replication in the intestinal tract in mice (effects of a 3CLpro inhibitor treatment on murine norovirus titers in the intestinal tracts of mice). Balb/c mice were orally infected with MNV-1 at 2×10⁶ TCID₅₀ and intraperitoneally given compound 16 at 10 mg/kg twice daily with the first dose starting 4 hrs prior to virus infection. At 72 h post virus infection, the intestinal tract tissues were harvested and processed for determination for virus titers by the TCID₅₀ assay. Mean and the standard error of the mean of virus titers in the small intestine (A) or large intestine (B) at 3 day post MNV-1 infection are shown. Each filled circle indicates a control mouse that was given drug vehicle and each empty box indicates a mouse given compound 16. Asterisk indicates P<0.01.

Scheme 1

Synthesis of inhibitors 13–44. ^aCH₃OH/SOCl₂/45 °C/ 3h; ^bCCl₃O(CO)Cl/ dioxane/ reflux/ 12h; ^c ArCH₂OH/ TEA/ acetonitrile/ reflux/ 2h; ^d1M LiOH(aq)/ THF/ RT/ 3h; ^eEDCl/ HOBt/ DIEA/ DMF; ^f2M LiBH₄/ THF/ CH₃OH; ^gDess-Martin periodinane/ DCM; ^hDiethylphosphite/ DCM/ DIEA; ⁱC₂H₅OH/ EtOAc/ HaHSO₃; ^jEtOAc/ HOAc/ cyclopropyl isocyanide/ K₂CO₃(aq)/ CH₃OH/ 18h