Phloretin Exerts Anti-Tuberculosis Activity and Suppresses Lung Inflammation

An increase in the prevalence of the drug-resistant Mycobacteria tuberculosis necessitates developing new types of anti-tuberculosis drugs. Here, we found that phloretin, a naturally-occurring flavonoid, has anti-mycobacterial effects on H37Rv, multi-drug-, and extensively drug-resistant clinical isolates, with minimum inhibitory concentrations of 182 and 364 μM, respectively. Since Mycobacteria cause lung inflammation that contributes to tuberculosis pathogenesis, anti-inflammatory effects of phloretin in interferon-γ-stimulated MRC-5 human lung fibroblasts and lipopolysaccharide (LPS)-stimulated dendritic cells were investigated. The release of interleukin (IL)-1β, IL-12, and tumor necrosis factor (TNF)-α was inhibited by phloretin. The mRNA levels of IL-1β, IL-6, IL-12, TNF-α, and matrix metalloproteinase-1, as well as p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, were suppressed. A mouse in vivo study of LPS-stimulated lung inflammation showed that phloretin effectively suppressed the levels of TNF-α, IL-1β, and IL-6 in lung tissue with low cytotoxicity. Phloretin was found to bind M. tuberculosis β-ketoacyl acyl carrier protein synthase III (mtKASIII) with high affinity (7.221 × 107 M−1); a binding model showed hydrogen bonding of A-ring 2′-hydroxy and B-ring 4-hydroxy groups of phloretin with Asn261 and Cys122 of mtKASIII, implying that mtKASIII can be a potential target protein. Therefore, phloretin can be a useful dietary natural product with anti-tuberculosis benefits

Role of phenylalanine and valine10 residues in the antimicrobial activity and cytotoxicity of piscidin-1.

Piscidin-1 (Pis-1) is a linear antibacterial peptide derived from mast cells of aquacultured hybrid striped bass that comprises 22 amino acids with a phenylalanine-rich amino-terminus. Pis-1 exhibits potent antibacterial activity against pathogens but is not selective for distinguishing between bacterial and mammalian cells. To determine the key residues for its antibacterial activity and those for its cytotoxicity, we investigated the role of each Phe residue near the N-terminus as well as the Val10 residue located near the boundary of the hydrophobic and hydrophilic sectors of the helical wheel diagram. Fluorescence dye leakage and tryptophan fluorescence experiments were used to study peptide-lipid interactions, showing comparable depths of insertion of substituted peptides in different membranes. Phe2 was found to be the most deeply inserted phenylalanine in both bacterial- and mammalian-mimic membranes. Each Phe was substituted with Ala or Lys to investigate its functional role. Phe2 plays key roles in the cytotoxicity as well as the antibacterial activities of Pis-1, and Phe6 is essential for the antibacterial activities of Pis-1. We also designed and synthesized a piscidin analog, Pis-V10K, in which Lys was substituted for Val10, resulting in an elevated amphipathic α-helical structure. Pis-V10K showed similar antibacterial activity (average minimum inhibitory concentration (MIC)  = 1.6 µM) to Pis-1 (average MIC  = 1.5 µM). However, it exhibited much lower cytotoxicity than Pis-1. Lys10-substituted analogs, Pis-F1K/V10K, Pis-F2K/V10K, and Pis-F6K/V10K in which Lys was substituted for Phe retained antibacterial activity toward standard and drug-resistant bacterial strains with novel bacterial cell selectivity. They exert anti-inflammatory activities via inhibition of nitric oxide production, TNF-α secretion, and MIP-1 and MIP-2 production. They may disrupt the binding of LPS to toll-like receptors, eventually suppressing MAPKs-mediated signaling pathways. These peptides may be good candidates for the development of peptide antibiotics with potent antibacterial activity but without cytotoxicity

Antimicrobial activity of Pis-1 and its analogs against standard bacterial strains.

a<p>Minimum inhibitory concentrations(MICs)were determined in three independent experiments performed in triplicate with a standard deviation of 14.0%.</p>b<p>The minimal peptide concentration that produced hemolysis. When no detectable hemolysis was observed at 100 µM, a value of 200 µM was used to calculate the therapeutic index.</p>c<p>The ratio of the MHC (µM) over the averageMIC (µM). Larger values indicate greater cell selectivity.</p><p>Antimicrobial activity of Pis-1 and its analogs against standard bacterial strains.</p

Enhancement of the intensity of FITC-labeled LPS as a function of Pis-1, Pis-V10K, Pis-F1K/V10K, Pis-F2K/V10K, Pis-F6K/V10K, and LL37 concentration.

<p><i>AU</i>, absorbance unit.</p

Peptide-induced permeabilization of lipid vesicles.

<p>Dose-response curves for calcein leakage from EYPC/EYPG (7∶3, w/w) LUVs (A), and EYPC/cholesterol (10∶1, w/w) LUVs (B) induced by the peptides.</p

Dose-response curves for the cytotoxicity of peptides toward cells of the mouse embryonic fibroblast-derived NIH 3T3 cell line.

<p>Dose-response curves for the cytotoxicity of peptides toward cells of the mouse embryonic fibroblast-derived NIH 3T3 cell line.</p

CD spectra of Pis-1 and its analogues in (A) H₂O, (B) 50 mM DPC, (C) 100 mM SDS micelles, (D) 1 mM SUVs of PC:PG (7∶3, w/w), and (E) 1 mg/mL LPS solution.

<p>CD spectra of Pis-1 and its analogues in (A) H₂O, (B) 50 mM DPC, (C) 100 mM SDS micelles, (D) 1 mM SUVs of PC:PG (7∶3, w/w), and (E) 1 mg/mL LPS solution.</p

The α-helical wheel diagram for Pis-1.

<p>The arrows indicate the position of Val¹⁰ substituted with Lys. The Pis-1 wheel is amphipathic, with the hydrophobic residues in the lower part and the hydrophilic residues in the upper part.</p