Developing a Gram-Negative Selective Peptide-Drug Conjugate

Abstract

Resistance to fluoroquinolone antibiotics has serious implications for healthcare; here, we conjugate the widely used fluoroquinolone ciprofloxacin to a proline-rich antimicrobial peptide (PrAMP) oncocin to improve oncocin's potency in ciprofloxacin-sensitive and ciprofloxacin-resistant strains of Escherichia coli. The conjugate molecule (oncocin-cipro-c) is ∼3× more potent than the parent oncocin, as determined by MIC, while retaining Gram-negative selectivity. We have characterized oncocin-cipro-c's interactions with three intracellular targets, two from oncocin (DnaK and 70S ribosome) and a third from ciprofloxacin (gyrase). Oncocin-cipro-c is also able to facilitate mast cell degranulation at a lower concentration than the parent peptide. The development of multimode antibiotics like oncocin-cipro-c is essential in the coming decades of antibiotic resistance.

1

Generation and antimicrobial testing of oncocin and the ciprofloxacin conjugates. (A) Ciprofloxacin can be N-protected with Fmoc to enable use in SPPS to generate N-cipro-oncocin and oncocin-cipro-C. (B–D) Antimicrobial potency of oncocin, the ciprofloxacin conjugate, coadministration of oncocin and ciprofloxacin, and ciprofloxacin alone was assessed against (B) E. coli ATCC 259220, (C) BAA 3051, and (D) S. aureus ATCC 29213. The red triangle represents the activity of oncocin-cipro-C, and the star represents the activity of ciprofloxacin alone. The C-terminal acid variant of oncocin-cipro-c could not be generated.

2

Structures of ciprofloxacin and oncocin bound to targets. (A) Structure of gyrase with ciprofloxacin bound highlighted in panel (B) showing that the amino termini of ciprofloxacin are solvent-exposed. (C) Structure of ciprofloxacin with the binding-occupied areas is indicated with a green line, and key interactions are noted. (D) Structure of DnaK with oncocin bound; highlighted in panel (E) shows the binding domain, oncocin threads through DnaK with the occupied amino acids indicated in panel (H) with the orange bar. (F) Transverse view of the structure of the 70S ribosome, with oncocin bound; highlighted in panel (G) shows the binding domain, with the occupied amino acids indicated in panel (H) with the pink bar. Together, the structural data support C-terminal conjugation of ciprofloxacin to oncocin to permit targeting of each of the enzymes. PDB IDs: (A, B) 2XCT; (D, E) 3QNJ; and (F, G) 4Z8C.

3

Conjugate-mediated degranulation in LAD2 mast cells. (A) Mast cells were treated with compounds (40 uM) and degranulation measured through quantification of b-hexosaminidase release. responses in MRGPRX2 knock-down cells are shown in striped bars. (B) More complete concentration-degranulation relationships are shown. Responses in MRGPRX2 knock-down cells are shown as dashed lines.

4

CD spectra of 50 μM oncocin and ciprofloxacin conjugates. (A) CD spectra of oncocin, conjugates, ciprofloxacin, and nonconjugated control in buffer. (B–F) CD spectra of oncocin and conjugates in DPC micelles at L/P of 100:1 (blue line), SDS micelles at L/P of 100:1 (green line), POPG/TOCL (9:1) LUV at L/P of 100:1 (green line), and POPE/POPG LUV at L/P of 100:1 (red line). Lines are averaged of three repeats performed at 298 K.

5

Calcein-leakage assays of 100 μM LUV in the presence of oncocin and conjugates at increasing concentrations: (A) POPC LUV, (B) POPE/POPG (7:3) LUV, and (C) POPG/TOCL LUV incubated with oncocin (circles), Oncocin and ciprofloxacin (triangles), ciprofloxacin (squares), oncocin-cipro-c (filled squares), and N-cipro-oncocin (filled triangles). Error bars are SD from three replicates performed at 298 K.

6

Membrane permeabilization of E. coli ATCC25922 in the presence of oncocin, cipro, and the conjugates. Each compound was tested at 2×, 1×, 5×, 25×, and 125× MIC to assess permeabilization. Oncocin and oncocin-cipro-C showed similar profiles with 25× MIC showing the greatest permeabilization, while Oncocin and free cipro showed no dose-dependent effect, while N-cipro-oncocin showed reduced permeabilization in a dose-dependent effect.