Novel polymeric inhibitors of HCoV-NL63

Abstract

The human coronavirus NL63 is generally classified as a common cold pathogen, though the infection may also result in severe lower respiratory tract diseases, especially in children, patients with underlying disease, and elderly. It has been previously shown that HCoV-NL63 is also one of the most important causes of croup in children. In the current manuscript we developed a set of polymer-based compounds showing prominent anticoronaviral activity. Polymers have been recently considered as promising alternatives to small molecule inhibitors, due to their intrinsic antimicrobial properties and ability to serve as matrices for antimicrobial compounds. Most of the antimicrobial polymers show antibacterial properties, while those with antiviral activity are much less frequent. A cationically modified chitosan derivative, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and hydrophobically-modified HTCC were shown to be potent inhibitors of HCoV-NL63 replication. Furthermore, both compounds showed prominent activity against murine hepatitis virus, suggesting broader anticoronaviral activity.

Fig. 1

Structures of the polymers showing anticoronaviral properties. In native chitosan the amino groups are partially deacetylated which means that the glucose groups may be substituted with both –NH₂ and CH₃CONH– groups in the same macromolecule. Since the distribution of acetylated and deacetylated NH₂– groups along the chitosan chain is random, this is often reflected by showing that R may be either H or –COCH₃ group. Consequently, since in HM-HTCC polymer some of the NH₂– groups are substituted with –C₁₂H₂₅ groups, some of R groups may be H, some –COCH₃ groups, and some –C₁₂H₂₅ groups in the same chitosan chain. Therefore, in HM-HTCC R may be H or COCH₃ or C₁₂H₂₅.

Fig. 2

Inhibition of HCoV-NL63 and MHV replication by HTCC and HM-HTCC compounds on LLC-MK2 (A) and LR7 cells (B), respectively. Infection was carried on in culture media, media supplemented with HTCC (100 μg/ml) or media supplemented with HM-HTCC (100 μg/ml). Cells were infected with HCoV-NL63 or MHV at TCID₅₀ of 400. Images were taken on days 2 (MHV) or 6 (HCoV-NL63) post-infection with Nikon Eclipse Ti-S microscope. Magnification: 200×. The results shown are representative of at least three independent experiments.

Fig. 3

Inhibition of HCoV-NL63 replication on LLC-MK2 cells by HTCC (A) and HM-HTCC (B). Data are presented as HCoV-NL63 RNA copies/ml (black triangles) and are reflected by values on the left Y-axis. The cell viability is presented on the right Y-axis (black circles). All assays were performed in triplicate and average values with standard errors (error bars) are presented.

Fig. 4

Inhibition of HCoV-NL63 replication in HAE cultures. (A) Inhibition of virus replication in the cell culture, as determined with real-time PCR. Data on virus replication are presented as HCoV-NL63 RNA copies/ml. ^∗∗∗p < 0.001; ^∗p < 0.01; ns, not significant. (B) Cytotoxicity of HTCC and HM-HTCC on HAE cultures. Due to limited availability of HAE cultures, these experiments were conducted with limited number of concentrations. Cell viability was assessed with XTT assay and the data on the Y-axis represent the % of values obtained for control samples. All assays were performed in duplicate in at least two independent experiments and average values with standard errors (error bars) are presented.

Fig. 5

Inhibition of MHV replication on LR7 cells by HTCC (A) and HM-HTCC (B). Data are presented as MHV RNA copies/ml (black triangles) and are reflected by values on the left Y-axis. The cell viability is presented on the right Y-axis (black circles). All assays were performed in triplicate and average values with standard errors (error bars) are presented.

Fig. 6

Decreased fluorescence emission due to Se-NL63 and HTCC interaction. (A) Fluorescence spectra of FITC-HTCC in the presence of 0, 1.0, 2.0, and 2.5 μg/ml of Se-NL63 (the sequence of changes in the spectra for increasing concentration of Se-NL63 is given by an arrow; a.u., arbitrary units) and (B) The concentration-dependent decrease in relative fluorescence intensity (I/I₀) of FITC-HTCC at λ = 513 nm in the presence of Se-NL63 concentration (λ_ex = 494 nm, c_FITC-HTCC = 0.5 μg/ml) (■). Analogical measurements of FITC-HTCC spectra in the presence of bovine serum albumin (BSA) were performed as a control (●). Both assays were performed in triplicate and the representative spectra are presented (Fig. 6A) or average values of fluorescence intensity at the band maximum with standard errors (error bars) (Fig. 6B).