Niacinamide enhances cathelicidin mediated SARS-CoV-2 membrane disruption

Abstract

The continual emergence of SARS-CoV-2 variants threatens to compromise the effectiveness of worldwide vaccination programs, and highlights the need for complementary strategies for a sustainable containment plan. An effective approach is to mobilize the body's own antimicrobial peptides (AMPs), to combat SARS-CoV-2 infection and propagation. We have found that human cathelicidin (LL37), an AMP found at epithelial barriers as well as in various bodily fluids, has the capacity to neutralise multiple strains of SARS-CoV-2. Biophysical and computational studies indicate that LL37's mechanism of action is through the disruption of the viral membrane. This antiviral activity of LL37 is enhanced by the hydrotropic action of niacinamide, which may increase the bioavailability of the AMP. Interestingly, we observed an inverse correlation between LL37 levels and disease severity of COVID-19 positive patients, suggesting enhancement of AMP response as a potential therapeutic avenue to mitigate disease severity. The combination of niacinamide and LL37 is a potent antiviral formulation that targets viral membranes of various variants and can be an effective strategy to overcome vaccine escape.

Keywords: COVID-19; LL37; SARS-CoV-2; antimicrobial peptides; niacinamide; skin immunity; viral membrane.

Figure 1

Antiviral activity of LL37 against SARS-CoV-2 variants (A) ACE2 protein levels in various epithelial cell lines (Western blotting) (B) SARS-CoV-2 E-gene expression in Calu3, Caco2, and HaCaT cells, 24 hr post infection (qRT-PCR) (n=4) (C) Secreted LL37 levels in Calu3, Caco2 and HaCaT cells (ELISA) (n=3) (D) Effect of increasing LL37 concentrations on SARS-CoV-2 neutralization (qRT-PCR) (n=5) (E) Effect of LL37 on the infectivity of various SARS-CoV-2 variants (qRT-PCR) (n=3) (F) Neutralization of Pseudo type virus (spike and VSVG) in the presence of LL37 (FACS) (n=3) [Statistical analysis was done using student's t-test (B, E) one-way ANOVA (C, D), two-way ANOVA (F),*p≤0.05, **p≤0.001, ***p≤0.0001].

Figure 2

Mode of action of LL37 peptide mediating antiviral activity (A) The LL37 peptide gets deeply embedded in the membrane in 200ns (B) Representative membrane thickness averaged over the last 20 ns of MD simulations in absence (left) and presence (right) of the LL37 peptide. (C) LL37 causes a reduction in lipid ordering. Both the saturated (sn-1) and the unsaturated (sn-2) lipid acyl chains exhibit reduced ordering in presence of the LL37 peptide, quantified (bottom) using the lipid order parameter (S_CD) (mean ± SD from last 20ns of 5 independent simulations) (D) Charge neutralization of vesicles in the presence of LL37 (n=3) (E) Membrane disruption assay using virus-like vesicles, mimicking viral membrane and PS concentration (FRET) (n=3) (F) Particle size analysis of SARS-CoV-2 in the presence of LL37 (DLS) (n=3) (G) Particle size analysis of Pseudovirus (Spike and VSVG) in the presence of LL37 (DLS) (n=3) [Statistical analysis was done using student t-test for (D, G), two-way ANOVA for (E), and one-way ANOVA(F) *p≤0.05, **p≤0.001, ***p≤0.0001].

Figure 3

Effect of niacinamide on LL37 (A) Viral gene expression at different concentrations of LL37 in the presence of niacinamide (qRT-PCR) (n=3) (B) Effect of niacinamide combined with LL37 on the TCID50/ml of various SARS-CoV-2 strains (n=3) (C) Partial sequence of the amphipathic LL37 peptide is represented with an idealized helical wheel (left) with residues colored according to their nature (hydrophobic, grey; polar, green; acidic, red; basic, blue). Right, two views of the LL37 peptide (color scheme same as the wheel; sidechains, black) overlaid with niacinamide molecules (grey) within 6 Å of the peptide (over 10 ns). The 10 ns overlay emphasizes the solvent-like encapsulation of the peptide by niacinamide with greater residence over the hydrophobic face of the helix than the polar/charged face (D) Contribution of each residue of LL37 towards interaction with niacinamide from MD simulations (mean of 3 independent simulations, errors propagated from each replicate). Residues contributing >1 kJ mol-1 are labelled (E) Niacinamide in the membrane forms hydrogen bonds with the Lys (K) and Arg (R) residues of LL37. Whereas, the aliphatic residues of LL37 which preferentially interacted with niacinamide in solution (D) are now predominantly engaged with the hydrophobic acyl chains and so, are unable to contribute much to bind niacinamide (F) Representative snapshot (left) of niacinamide penetrating deeper than water into the hydrophobic core of the membrane (LL37, black peptide; lipid acyl chains, grey lines; niacinamide, space filling; water, orange) which is quantified on the right, averaging the molecular density (membrane, black; niacinamide, green; water, orange) over the entire 200 ns trajectory (5 replicates, mean ± SD) [Statistical analysis was done using student t-test for (A) and two-way ANOVA for (B), *p≤0.05, **p≤0.001, ***p≤0.0001].

Figure 4

Niacinamide enhances antiviral activity of LL37 (A) Membrane disruption assay on addition of LL37 and niacinamide (FRET) (n=3) (B) Measurement of LL37 in various patient cohorts (ELISA) (A total of 41 individuals were subdivided into negative (16), positive symptomatic (13), and positive asymptomatic (12) cohorts (C) Effect of niacinamide addition to saliva on SARS-CoV-2 neutralisation (TCID50) (n=4) (D) Effect of skin scrub and its combination with niacinamide on SARS-CoV-2 viral gene expression (RT-qPCR) (Control, B3: n=3, Skin scrub, Skin scrub + B3: n=6).[Statistical analysis was done using two-way ANOVA for (A), Kruskal Wallis test (non-parametric ANOVA) for (B), one-way ANOVA for (C), and student t-test for (D)].