Structure-based design and synthesis of triazole-based macrocyclic inhibitors of norovirus protease: Structural, biochemical, spectroscopic, and antiviral studies

Abstract

Outbreaks of acute gastroenteritis caused by noroviruses constitute a public health concern worldwide. To date, there are no approved drugs or vaccines for the management and prophylaxis of norovirus infections. A potentially effective strategy for the development of norovirus therapeutics entails the discovery of inhibitors of norovirus 3CL protease, an enzyme essential for noroviral replication. We describe herein the structure-based design of the first class of permeable, triazole-based macrocyclic inhibitors of norovirus 3C-like protease, as well as pertinent X-ray crystallographic, biochemical, spectroscopic, and antiviral studies.

Design, synthesis, and evaluation of a novel series of macrocyclic inhibitors of norovirus 3CL protease

“…The aldehyde and aldehyde bisulfite adducts had comparable potency, however, replacement of the warhead with an α-ketoamide (Table 1, compounds 13 and 25 versus α-ketoamide 31 ) diminished activity. These observations are congruent with the results of previous studies with peptidyl and macrocyclic inhibitors of NV 3CLpro [48–49]. Replacement of the P 2 Leu (R 2 ) residue with cyclohexylalanine (Cha) had a minor effect on potency.…”

“…Fluorescence readings were obtained using an excitation wavelength of 360 nm and an emission wavelength of 460 nm on a fluorescence microplate reader (FLx800; Biotec, Winoosk, VT) 1 h following the addition of substrate. Relative fluorescence units (RFU) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously [33,47–49]. The dose-dependent FRET inhibition curves were fitted with a variable slope by using GraphPad Prism software (GraphPad, La Jolla, CA) in order to determine the IC 50 values of the inhibitors.…”

Structural characterization of the Man5 glycoform of human IgG3 Fc

“…Structure solution was conducted by molecular replacement with Phaser (McCoy et al, 2007) using a single subunit form previously determined structure of IgG1 Fc (PDB: 4DZ8) (Strop et al, 2012) as the search model (Weerawarna et al, 2016). The top solution was obtained in the space group P 2 1 that contained one homodimer in the asymmetric unit.…”

Antiviral targets of human noroviruses

“…These studies further suggest that peptido-mimetics with suitable warheads with a Glu-like chemical entity at P1 for optimal interactions with S1 pocket, and an appropriate combination of hydrophobic residues at P2 and P4 that maximizes the interactions with S2 and S4 pockets, are factors to be considered for enhancing the potency of the inhibitors. More recently, based on the observation that protease-bound peptidyl inhibitors typically adopt a β-strand conformation between the P1 and P3 positions, Weerwarna et al, [73**] have designed and synthesized a novel set of triazole-based macrocyclic inhibitors in which these two positions are linked using a suitable linker such that the P1–P3 is pre-organized into a β-strand conformation for optimal interactions with the norovirus protease as demonstrated by structural studies. In addition to potentially higher stability to metabolic enzymes these inhibitors exhibit increased cellular permeability.…”