Theranostic Gallium Siderophore Ciprofloxacin Conjugate with Broad Spectrum Antibiotic Potency

Abstract

Pathogenic bacteria scavenge ferric iron from the host for survival and proliferation using small-molecular chelators, siderophores. Here, we introduce and assess the gallium(III) complex of ciprofloxacin-functionalized desferrichrome (D2) as a potential therapeutic for bacterial infection using an in vitro assay and radiochemical, tracer-based approach. Ga-D2 exhibits a minimum inhibitory concentration of 0.23 μM in Escherichia coli, in line with the parent fluoroquinolone antibiotic. Competitive and mutant strain assays show that Ga-D2 relies on FhuA-mediated transport for internalization. Ga-D2 is potent against Pseudomonas aeruginosa (3.8 μM), Staphylococcus aureus (0.94 μM), and Klebsiella pneumoniae (12.5 μM), while Fe-D2 is inactive in these strains. Radiochemical experiments with E. coli reveal that ⁶⁷Ga-D2 is taken up more efficiently than ⁶⁷Ga-citrate. In naive mice, ⁶⁷Ga-D2 clears renally and is excreted 13% intact in the urine. These pharmacokinetic and bacterial growth inhibitory properties qualify Ga-D2 for future investigations as a diagnosis and treatment tool for infection.

Figure 1.

Chemical structures of structurally related hydroxamate siderophore–antibiotic conjugates synthesized and investigated previously, as well as subject of studies in this work: D1, D2 and albomycin (Fe-D3), a “Trojan horse” conjugate produced by Streptomyces. Black: metal ion, red: siderophore, green: linker, blue: antibiotic.

Figure 2.

MIC assays of non-functionalized siderophores (A,B), D1 (C) and D2 (D) in E. coli K12 show that the Ga-D2 complex exhibits greatest potency when compared to ciprofloxacin, while non-functionalized Fe–siderophores act as growth promoters at concentrations above 10⁻⁵ M (n = 3) and Fe–sideromycin complexes exhibit attenuated growth inhibition.

Figure 3.

(A) MIC challenge assay of Ga-D1 in the presence of 100× excess Fe–DFO and Ga–DFO and (B) Ga-D2 in the presence of 100× excess Fe–LDFC and Ga–LDFC in E. coli K12 (n = 3). Parent siderophore Ga- and Fe-complexes compete effectively for transporter-mediated internalization.

Figure 4.

MIC assays of apo D1, Fe-D1, Ga-D1 complexes and apo D2, Fe-D2, Ga-D2 in E. coli AN193 (FhuA mutant). Growth inhibitory activity of the LDFC based conjugates was attenuated, whereas the activity of DFO-based conjugates was not significantly affected.

Figure 5.

Representative radiolabeling HPLC-trace for the characterization of ⁶⁷Ga-D2 (R_t = 10.56 min, left axis, ⁶⁷Ga counts per second), in comparison with HPLC characterization of the apo D2 ligand (R_t = 10.30 min, right axis, absorbance at 280 nm) is shown. Free ⁶⁷Ga elutes at 0.7 min.

Figure 6.

(A) Time-dependent, radiochemical bacterial uptake studies in E. coli K12 of ⁶⁷Ga–siderophore–ciprofloxacin conjugates in iron sufficient (striped bars) and iron-depleted, DP-treated media pH = 7.4 (solid bars) show that uptake is enhanced in comparison with ⁶⁷Ga-citrate. (B) In the presence of 120× excess Fe–LDFC, the uptake of ⁶⁷Ga-D2 is attenuated significantly after 1 h (P < 0.0001), corroborating results obtained in challenge MIC experiments.

Figure 7.

(A) Comparative biodistribution of ⁶⁷Ga-D1, ⁶⁷Ga-D2, ⁶⁷Ga-D3 and ⁶⁷Ga-citrate in naive mice (n = 3) shows predominantly renal clearance of all compounds. (B) Metabolite analysis of ⁶⁷Ga-D2 (open circles) shows detectable intact complex (13%) in the urine 1 h post injection. Radioanalytical HPLC trace of the ⁶⁷Ga-D2 dose formulation prior to administration is shown as a reference (red line).

Scheme 1.

Synthesis Scheme for apo-D1 Adapted from refs 41 and

Scheme 2.

Functionalization of Ciprofloxacin, Subsequent Conjugation to Protected Hydroxamate Fragment P5 Followed by Sequential Deprotection Steps Yields the Conjugate D2

Scheme 3.

Synthesis Scheme for LDFC adapted from ref 51