Exploring Structure-Activity Relationships and Modes of Action of Laterocidine

Abstract

A significant increase in life-threatening infections caused by Gram-negative "superbugs" is a serious threat to global health. With a dearth of new antibiotics in the developmental pipeline, antibiotics with novel mechanisms of action are urgently required to prevent a return to the preantibiotic era. A key strategy to develop novel anti-infective treatments is to discover new natural scaffolds with distinct mechanisms of action. Laterocidine is a unique cyclic lipodepsipeptide with activity against multiple problematic multidrug-resistant Gram-negative pathogens, including Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacterales. Here, we developed a total chemical synthesis methodology for laterocidine and undertook systematic structure-activity relationship studies with chemical biology and NMR. We discovered important structural features that drive the antimicrobial activity of laterocidine, leading to the discovery of an engineered peptide surpassing the efficacy of the original peptide. This engineered peptide demonstrated complete inhibition of the growth of a polymyxin-resistant strain of Pseudomonas aeruginosa in static time-kill experiments.

Figure 1

Amino acid sequences of laterocidine (1), brevicidine (2), relacidine (3), paenibacterin A₃ (4), polymyxin B₁ (5), and teixobactin (6).

Scheme 1. Total Synthesis of Laterocidine

(a) Fmoc-AA-OH (3 equiv), HCTU (3 equiv), DIPEA (6 equiv), in DMF 50 min; (b) 20% piperidine/DMF (1 × 5 min, 1 × 10 min); (c) Alloc-Gly-OH (5 equiv), DIC (5 equiv), DMAP (0.3 equiv) in DMF; (d) palladium tetrakis(triphenylphosphine) (0.1 equiv), PhSiH₃ (10 equiv), in DCM 40 min; (e) 10% HFIP in DCM (1 × 30 min, 1 × 5 min); (f) DPPA, DIEA in DMF 6 h; (g) TFA:TIPS:DODT:H₂O (92.5:2.5:2.5:2.5) 90 min. HPLC and mass spectrum of peptides 1, 8, and 10. O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), 1,1,1,3,3,3-hexafluoro isopropanol (HFIP), dichloromethane (DCM) diphenylphosphoryl azide (DPPA), di-isopropyl carbodiimide (DIC), dimethylaminopyridine (DMAP), diisopropylethylamine (DIEA), dimethylformamide (DMF), triisopropylsilane (TIPS), 3,6-dioxa-1,8-octanedithiol (DODT), and trifluoroacetic acid (TFA).

Figure 2

Structure–activity relationship exploration underpinned by solid-phase peptide synthesis protocol. Colored residues are important determinants of antimicrobial activity. Aromatic and hydrophobic residues are highlighted in brown, cationic residues are shown in blue, and ester bond in red.

Figure 3

Time-kill studies with polymyxin B (PMB), laterocidine (peptide 1), and peptide 11 against (A) polymyxin-susceptible P. aeruginosa PAO1 and (B) polymyxin-resistant P. aeruginosa PAO1R. All peptides were used at 1, 4, and 8× MIC.

Figure 4

Flow cytometry analysis of P. aeruginosa PAO1 with no treatment (control) or following 1 h of treatment with laterocidine (peptide 1) at 1 × or 8 × MIC (equivalent to 2 and 16 μg/mL, respectively). (A) Membrane integrity (PI), (B) respiration (CTC), (C) membrane polarity (DiBAC), and (D) oxidative stress (CellROX Green). PI, propidium iodide; CTC, 5-cyano-2,3-ditolyl tetrazolium chloride; and DiBAC, bis(1,3-dibutylbarbituric acid) trimethine oxonol.

Figure 5

(A) Part of the amide and aromatic (6.7–8.6 ppm) region of the 600 MHz ¹H NMR spectrum during titration of 1.5 mM laterocidine (black) with 16 μL of 10 mg/mL E. coli O111:B4 LPS (red) showing line broadening due to binding to the LPS micelle. (B) Superposed, stereo view of the 41 XplorNIH trNOE NMR structures calculated from a total of 100 as viewed from the top and side. Positively charged ornithine side chains are shown in green, hydrophobic side chains in magenta, and the backbone in gray. Thr, Asn, and _D-Ser side chains are shown in orange. (C) Superposition of a portion of the 2D NOESY spectra of 1.5 mM laterocidine (blue) and after the addition of E. coli LPS0111:B4 (red) both recorded at 23 °C in 50 mM acetate buffer (d₃), 10% D₂O, pH 4.5. The 2D NOESY mixing times were 140 and 350 ms for with and without LPS added. (D) Schematic showing a proposed interaction with LPS highlighting the amphipathicity of the laterocidine and complementary separation of the positively charged Orn5 and Orn7 side chains with the sugar phosphates of lipid A in LPS. The backbone of laterocidine is represented as a yellow ribbon. The LPS coordinates was extracted from PDB 6S8H.

Figure 6

(A) Maximum tolerable dose for peptide 1 (Native) and peptide 11, PMB was calculated using Swiss female mice (weighing 22–28 g and aged 7 weeks). All mice were administered an intravenous bolus of the peptides (mg/kg free base) via a lateral tail vein (≤0.1 mL). (B) Efficacy of laterocidine (peptide 1) and peptide 11 in a bloodstream infection murine model by P. aeruginosa FADDI-PA070. Laterocidine analogues (8 mg/kg, N = 3, 4 h time point).