Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors

Abstract

Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the US alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED(50) of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters.

Figure 1

Proteolytic cleavage of the nonstructural polyprotein of norovirus (Norwalk virus) encoded in open reading frame 1 (ORF1). The indicated cleavage sites at Q/G, E/A or E/G (corresponding to the P₁-P₁′ scissile bond) are mediated by the virus-encoded 3CL cysteine protease.

Figure 2

General representation of the interaction and interrelationships between 3CL protease (E-Cys-SH), transition state inhibitor, X(C=O)Z, and bisulfite adduct, XZ(OH)SO₃Na.

Scheme 1. Reagents

(a) CCI₃0(C=0)CI/dioxane; (b)Triethylamine/R¹OH; (c) Li0H/THF/H₂0; (cl) EDCI/HOBt/DIEA/DMF; (e) LiBH₄/THF; (f) Dess-Martin periodinane/DCM; (g) NaHS03/EtOAc/EtOH/H₂0; (h) Cyclopropyl isonitrile/HOActhen K2CO3/CH3OH/H2O.