Exploitation of Antibiotic Resistance as a Novel Drug Target: Development of a β-Lactamase-Activated Antibacterial Prodrug

Abstract

Expression of β-lactamase is the single most prevalent determinant of antibiotic resistance, rendering bacteria resistant to β-lactam antibiotics. In this article, we describe the development of an antibiotic prodrug that combines ciprofloxacin with a β-lactamase-cleavable motif. The prodrug is only bactericidal after activation by β-lactamase. Bactericidal activity comparable to ciprofloxacin is demonstrated against clinically relevant E. coli isolates expressing diverse β-lactamases; bactericidal activity was not observed in strains without β-lactamase. These findings demonstrate that it is possible to exploit antibiotic resistance to selectively target β-lactamase-producing bacteria using our prodrug approach, without adversely affecting bacteria that do not produce β-lactamase. This paves the way for selective targeting of drug-resistant pathogens without disrupting or selecting for resistance within the microbiota, reducing the rate of secondary infections and subsequent antibiotic use.

Figure 1

Selected representative examples of cephalosporin prodrugs (PROTAX 1,2, and BMY-46633 3(35)) and co-drugs (MCO 4(38) and Ro 23-9424 5(39)).

Figure 2

(A) Mechanism of β-lactamase triggered cephalothin hydrolysis. (B) General structure of proposed cephalosporin–ciprofloxacin prodrug 6.

Figure 3

Plot of log of percentage hydrolysis in CFT073 + CTX-M-1 cells against calculated log P (cLogP) for synthesized compounds (filled circle) and cephalothin 7 (open circle): linear regression (dashed line), R² = 0.60 (GraphPad Prism 7).

Scheme 1. Synthesis of Prodrug 35

Reagents and conditions: (i) acetic anhydride, NaHCO₃, H₂O, acetone, 0 °C, 30 min; (ii) TBTA, DCM, 60 °C, 16 h; (iii) TMSI, DCM, rt, 2 h; (iv) Boc₂O, 1 M NaOH, THF, rt, 16 h; (v) 0.1 M NaOH, MeOH, rt, 30 min; (vi) 3:1 1,4-dioxane/DMF, rt, 4 h; (vii) 1:1 TFA/DCM, anisole, 0 °C to rt.

Figure 4

Activity of prodrug 35 and ciprofloxacin 31 against recombinant DNA gyrase ± CTX-M-15. (A) DNA was separated by agrose gel electrophoresis with 2 log DNA ladder: oc, open circle DNA; rel, relaxed DNA; sc, supercoiled DNA. (B) Quantification of gel bands corresponding to supercoiled DNA and normalized to no gyrase and gyrase only activity (ImageJ 1.52a): Gyr, DNA gyrase; cip, ciprofloxacin 31; PD, prodrug 35; CTX, CTX-M-15. Error bars represent SEM (n = 4); prodrug vs prodrug + CTX-M-15 was analyzed by unpaired t-test, p = 0.0004 (GraphPad Prism 7.03).

Figure 5

Antibacterial activities for prodrug 35 (blue) and ciprofloxacin 31 (red) against CFT073 E. coli cells WT and expressing empty plasmid (pEMP), CTX-M-15 (pCTX), NDM1 (pNMD1), and KPC (pKPC): (A) dose–response curves, where each point represents the mean ± SEM, n = 3; (B) summary of MIC values.

Figure 6

Effect of prodrug 35 (blue) or ciprofloxacin 31 (red) against six uropathogenic E. coli clinical isolates. Each point represents the mean ± SEM, n = 3.

Figure 7

Survival of CFT073 pEMP (open circle) and pCTX (filled circle) with no treatment (green), ciprofloxacin 31 (78 nM) (red), or prodrug 35 (78 nM) (blue): Cipro, ciprofloxacin; PD, prodrug 35.