Two distinct amphipathic peptide antibiotics with systemic efficacy

Abstract

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibiotic-resistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-state ¹⁵N NMR data. While horine was potent primarily against gram-positive pathogens, verine showed broad-spectrum antimicrobial activity. Both peptides protected greater than 80% mice from infection-caused deaths. Moreover, horine and verine also displayed significant systemic efficacy in different murine models comparable to conventional antibiotics. In addition, they could eliminate resistant pathogens and preformed biofilms. Significantly, the peptides showed no nephrotoxicity to mice after intraperitoneal or intravenous administration for 1 wk. Our study underscores the significance of horine and verine in fighting drug-resistant pathogens.

Keywords: NMR; antibiotic resistance; nephrotoxicity; peptide antibiotics; systemic efficacy.

Fig. 1.

Relationships between averaged net charge and hydrophobic residue content. (A) A linear correlation between hydrophobic residue content (Pho) and arginine content (R) of AMPs based on 3,177 peptides in the current APD (6). (B) There is no such correlation between lysine contents and Pho. The peptides in the APD (6) were separated into 10 bins based on hydrophobic contents (0–10, 11–20, 21–30, 31–40, 41–50, 51–60, 61–70, 71–80, 81–90, and 91–100%), which are represented as 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 on the x axis of this plot. These relationships remained the same from 2017 to 2020. Data were obtained from the APD in March 2020.

Fig. 2.

Design and NMR characterization of the two distinct amphipathic peptides. (A) The amino acid sequence of WW291 is distributed evenly on the wheel. Reading from position 1 (W1) clockwise generated WW291. Reading from position 2 (W2) in the same manner produced WW292. Repeating this reading from position 1 to position 8 (W8) led to eight peptides (B). From this sequence permutation, we identified WW295 with increased activity against Klebsiella (SI Appendix, Table S1). NMR structural determination revealed two distinct amphipathic models: Classic helix of WW291 with a horizontal axis (C and D) and nonclassic spiral structure of WW295 with a vertical axis (E and F), all relative to the hydrophobic surface. As depicted in the potential surfaces (G–J), both structures possess positive charges (cyan) for recognition of the negative bacterial surfaces and hydrophobic surfaces (white) for membrane anchoring: G and H for WW291 and I and J for WW295. NMR sample conditions are given in the legend to SI Appendix, Table S2. (K and L) Peptide membrane orientation determination by solid-state NMR spectroscopy. The solid-state NMR ¹⁵N chemical shift (<100 ppm) of a single-site ¹⁵N-labeled leucine in horine (K) and verine-L (L) indicates an H-N vector (red) parallel to membrane surface. This enables us to position the 3D structure of horine (M) and verine-L (N) on the lipid bilayer so that the H-N vector (in ball-and-stick) is approximately parallel to the bacterial membranes.

Fig. 3.

In vitro potential of horine (green) and verine (gold). (A) Horine and verine (2 × MIC) killed the exponential phase S. aureus USA300 LAC in 120 min. (B) The two peptides (2 × MIC) also killed nafcillin-induced persisters of S. aureus. (C) Verine, but not doripenem, inhibited the attachment of K. pneumoniae E406-17 in a dose-dependent manner. (D) Horine disrupted the 48-h established biofilms of S. aureus. In the confocal images, live bacteria in the untreated control are in green (E) and dead bacteria (F) treated at 16 µM of horine are in red. (G) Verine was effective in disrupting 48-h established Klebsiella biofilms. In the confocal images, live cells are green in untreated K. pneumoniae (H), and dead cells are red in verine-treated K. pneumoniae (I).

Fig. 4.

Peptide toxicity in vitro and in vivo. Toxicity of horine and verine to mammalian cells in vitro (A) or mice (B–G). (A) Peptide toxicity to liver, kidney, lung, and spleen cells determined by the XTT method. (B) Scheme of the peptide toxicity study in mice. C57BL/6 mice (n = 3) were intraperitoneally administered with vehicle or horine (10 mg/kg) daily for a week. Shown are (C) body weight change percentages, kidney histology images (H&E stained) for (D) vehicle control- and (E) horine-treated, (F) kidney chemistry profile, and (G) whole-blood cell analysis. The abbreviations for blood cells can be found in SI Appendix, Additional Methods. The data represent the mean ± SD. This figure indicates no significant kidney histologic differences between the vehicle control (D) and treatment group (E). There is no significant acute tubular injury or interstitial inflammation. D and E amplified by 400-fold.

Fig. 5.

Systemic efficacy of horine and verine in neutropenic mice administered intraperitoneally (IP) or intravenously (IV). (A) Mouse survival after S. aureus USA300 LAC infection (∼2 × 10⁷ CFU) without (black) and with a single dose treatment (intraperitoneally, 2 h postinfection) of horine (green) or vancomycin (vanco, blue) at 10 mg/kg. (B) Mouse survival after K. pneumoniae E406-17 infection (∼1 × 10⁷ CFU) without (black) and with a single dose of verine (orange) and doripenem (dori, blue) at 15 mg/kg. Survival of the C57BL/6 mice (n = 8 in each group) was observed for 5 d postinfection. (C–F) The CFU burden in spleen, lung, kidney, and liver 24 h after infection of neutropenic mice treated with the peptide via either the intraperitoneally (C and E) or intravenously (D and F) route. (C and D) Mice were intraperitoneally infected with S. aureus USA300 LAC at 2 × 10⁶ CFU per animal without (black) and with horine (green) or vancomycin (blue) treatment at 10 mg/kg 2 h postinfection (n = 8, C57BL/6 mice intraperitoneally, and n = 5, BALB/c mice intravenously). (E and F) Mice were intraperitoneally infected with K. pneumoniae E406-17 at 5 × 10⁵ CFU per animal without (black) and with verine (orange) or doripenem (blue) treatment at 15 mg/kg 2 h postinfection, n = 5 (C57BL/6 mice intraperitoneally and BALB/c mice intravenously). The bacterial loads from each mouse were plotted as individual points and error bars represent the deviation within the experimental group. *P < 0.05; **P < 0.01; and ***P < 0.001 (determined by t test); n.s., not significant.