Understanding the Mechanism of Action of NAI-112, a Lanthipeptide with Potent Antinociceptive Activity

Abstract

NAI-112, a glycosylated, labionine-containing lanthipeptide with weak antibacterial activity, has demonstrated analgesic activity in relevant mouse models of nociceptive and neuropathic pain. However, the mechanism(s) through which NAI-112 exerts its analgesic and antibacterial activities is not known. In this study, we analyzed changes in the spinal cord lipidome resulting from treatment with NAI-112 of naive and in-pain mice. Notably, NAI-112 led to an increase in phosphatidic acid levels in both no-pain and pain models and to a decrease in lysophosphatidic acid levels in the pain model only. We also showed that NAI-112 can form complexes with dipalmitoyl-phosphatidic acid and that Staphylococcus aureus can become resistant to NAI-112 through serial passages at sub-inhibitory concentrations of the compound. The resulting resistant mutants were phenotypically and genotypically related to vancomycin-insensitive S. aureus strains, suggesting that NAI-112 binds to the peptidoglycan intermediate lipid II. Altogether, our results suggest that NAI-112 binds to phosphate-containing lipids and blocks pain sensation by decreasing levels of lysophosphatidic acid in the TRPV1 pathway.

Keywords: TPRV1; VISA strains; lanthipeptide; lipid II; lysophosphatidic acid; untargeted lipidomics.

Figure 1

Structures of NAI-112 (upper) and labyrinthopeptin A2 (lower). In pink, the amino acids involved in the labionin bridges are highlighted.

Figure 2

Lipidomic study. (A) Representative chromatogram of HR-LC-MS analysis of untargeted lipidomics. (B) Feature clustering using principle component analysis (PCA). Vehicle group in red shows clear separation from the NAI-112-treated group. (C) Orthogonal projection to latent structures–discriminant analysis (OPLS-DA) was used to identify lipids differentially expressed between vehicle and NAI-112 groups.

Figure 3

Effect on levels of phosphatidic acid (PA) and lysophosphatidic acid (Lyso PA or LPA). The levels of PA (18:1/20:4) and PA (18:0/18:0) increased by NAI-112 treatment in both naive and in-pain mice (A,B). No significant effects were observed on LPA levels in no-pain mice (C), while LPA (18:0) reduced after treatment with NAI-112 in in-pain mice (D). In all panels, values are expressed as means ± SEM. The two-tailed t-test was used to assess statistical significance: * p < 0.05; ** p < 0.01; n.s., not significant.

Figure 4

Mass spectra of the sample containing DPPA and NAI-112 in negative ionization mode (upper spectrum) and in positive ionization mode (lower spectrum). Addition of Tris to improve DPPA solubility led to the formation of Tris adducts, visible in positive ionization mode. The orange circle indicates the lanthipeptide alone, and green and blue circles show the lanthipeptides complexed with LPA and DPPA, respectively.

Figure 5

NAI-112-resistant mutants. (A) Relative change in NAI-112 MIC after serial passages of S. aureus ATCC 6538P. The starting MIC was 32 µg/mL. (B) Growth of the wild type (wt) and two mutants selected after the 8th (R8.1) and the 15th (R15.5) passage from the experiment in panel (A). See the Isolation of NAI-112-Resistant Mutants section for details.

Figure 6

Number of mutations (SNPs or INDELs) observed in each mutant strain in comparison with the parental strain and their distribution in comparison with the other mutants.