Synthetic antibacterial discovery of symbah-1, a macrocyclic β-hairpin peptide antibiotic

Abstract

The rapid development and spread of antibiotic resistance necessitate the development of novel strategies for antibiotic discovery. Symbah-1, a synthetic peptide antibiotic, was identified in a high-throughput antibacterial screen of random peptide sequences. Symbah-1 functions through membrane disruption and contains broad spectrum bactericidal activity against several drug-resistant pathogens. Circular dichroism and high-resolution mass spectrometry indicate symbah-1 has a β-hairpin structure induced by lipopolysaccharide and is cyclized via an intramolecular disulfide bond. Together these data classify symbah-1 as an uncommon synthetic member of the β-hairpin antimicrobial peptide class. Symbah-1 displays low hemolysis but loses activity in human serum. Characterization of a symbah-1 peptide library identified two variants with increased serum activity and protease resistance. The method of discovery and subsequent characterization of symbah-1 suggests large synthetic peptide libraries bias toward macrocyclic β-hairpin structure could be designed and screened to rapidly expand and better understand this rare peptide antibiotic class.

Keywords: Biochemistry; Microbiology; Structural biology.

Graphical abstract

Figure 1

Symbah-1 is a peptide antibiotic functioning via membrane disruption (A) Diagram of SLAY peptide display system in the outer membrane (OM). (B) Symbah-1 peptide sequence (blue = charged, yellow = hydrophobic, green = polar, purple = cysteine). (C) Growth curve with and without induction of peptide display (+/−). Data represented by mean +/- one standard deviation of triplicate samples (n = 3). (D) Table showing E. coli W3110 MICs. (E) Killing curve of E. coli W3110 cells treated with different concentration of symbah-1. Data are represented by the mean +/- one standard deviation of triplicate samples (n = 3). Error bars represent one standard deviation of triplicate samples (n = 3). (F) PI uptake in relative fluorescence units (RFU) of E. coli W3110 cells challenged with different peptides and antibiotics. Data are represented by mean +/− one standard deviation of triplicate samples (n = 3).

Figure 2

Symbah-1 is a synthetic member of the β-AMP class (A) AlphaFold v2 model of symbah-1 tertiary structure. (B) Circular dichroism spectra of symbah-1 with addition of various concentrations of LPS. (C) High-resolution mass spectra of the +5 charge state isotope peaks of cyclic symbah-1 in MH, cyclic symbah-1 in MH + 10 mM DTT, and symbah-1 in MH. The measured M_mi (monoisotopic mass) of each peptide is inset top left.

Figure 3

Macrocyclic structure is critical for activity due to reduced protease lability (A) Symbah-1 variant peptide MICs against E. coli W3110. (B) Circular dichroism spectra for symbah-1 variants with 0.8 mg/mL LPS. (C) MBCs for symbah-1 variants against E. coli KS292, HM130, and HM130 with 160 ng/mL trypsin protease in MH media. Single dots represent individual replicates. Bars represent the median value (n = 3).

Figure 4

Symbah-1 has broad bactericidal activity against drug resistant pathogens (A) Table of symbah-1 MICs against various bacterial strains in MH media and RPMI media. (B) Table of clinically relevant antibiotics against A. baumannii AB5075 in MH media and RPMI media. (C) CFUs of the kidneys and liver resulting from injection of 1 × 10⁶A. baumannii AB5075 cells in the mouse intraperitoneal space with and without treatment with 5 mg/kg symbah-1. Filled circles or triangles represent CFUs from a single replicate; lines represent the mean of all replicates from one condition (∗ two-tailed t test p value ≤ 0.05) (n ≥ 4).

Figure 5

Symbah-1 has low hemolysis but loses activity in human serum (A) Percent hemolysis of serial dilutions of symbah-1, protegrin-1 (PG-1), tachyplesin-1 (TP-1), and thanatin. Error bars represent one standard deviation (n = 3). (B) MBCs of β-AMPs with MH and MH with 40% human serum (HS). Single dots represent single replicates of triplicate experiments. Bars represent the median value. (C) Table of M_mi (monoisotopic mass) for β-AMPs in PBS as determined by high-resolution LC/MS. Data are represented by mean +/− one standard deviation of triplicate samples (n = 3).

Figure 6

Optimized peptides are more resistant to protease digestion (A) Representative images of five microliters of overnight culture from MIC assays spotted on LB agar. Columns have 2-fold increasing concentrations of trypsin protease and rows have 2-fold dilutions of peptide. (B) Model diagrams of symbah-1, SBH-15, and SBH-22 residues in a β-hairpin conformation (blue = charged, yellow = hydrophobic, green = polar, purple = cysteine). (C) Charge and hydrophobicity surface distributions based on AlphaFold v2 predicted structures (yellow = hydrocarbons with no polar substitutions, blue = nitrogen atoms of lysine and arginine, white = all remaining atoms and polar backbone).