Evaluation of Heterocyclic Carboxamides as Potential Efflux Pump Inhibitors in Pseudomonas aeruginosa

Abstract

The ability to rescue the activity of antimicrobials that are no longer effective against bacterial pathogens such as Pseudomonas aeruginosa is an attractive strategy to combat antimicrobial drug resistance. Herein, novel efflux pump inhibitors (EPIs) demonstrating strong potentiation in combination with levofloxacin against wild-type P. aeruginosa ATCC 27853 are presented. A structure activity relationship of aryl substituted heterocyclic carboxamides containing a pentane diamine side chain is described. Out of several classes of fused heterocyclic carboxamides, aryl indole carboxamide compound 6j (TXA01182) at 6.25 µg/mL showed 8-fold potentiation of levofloxacin. TXA01182 was found to have equally synergistic activities with other antimicrobial classes (monobactam, fluoroquinolones, sulfonamide and tetracyclines) against P. aeruginosa. Several biophysical and genetic studies rule out membrane disruption and support efflux inhibition as the mechanism of action (MOA) of TXA01182. TXA01182 was determined to lower the frequency of resistance (FoR) of the partner antimicrobials and enhance the killing kinetics of levofloxacin. Furthermore, TXA01182 demonstrated a synergistic effect with levofloxacin against several multidrug resistant P. aeruginosa clinical isolates.

Keywords: Pseudomonas aeruginosa; RND efflux pumps; antimicrobial drug resistance; efflux pump inhibitors; heterocyclic carboxamides.

Figure 1

Chemical structures of representative lead efflux pump inhibitors.

Figure 2

Structures of the two-aryl alkyl diaminopentanamide potentiators.

Figure 3

General structure of improved aryl hydrophobic head groups as potential EPIs.

Figure 4

Outer- and inner-membrane permeabilization studies with TXA01182. Basal levels of NCF hydrolysis (A,B) or PI fluorescence (C) are observed upon addition of TXA01182 at concentrations below 12.5 and 25 μg/mL, respectively, indicating intact outer and inner membranes.

Figure 5

(A) TXA01182 concentration-dependent inhibition of EtBr efflux. (B) polymyxin B does not inhibit EtBr efflux under the same conditions.

Figure 6

TXA01182 enhances the killing kinetics of levofloxacin (LVX). Time-kill kinetics of LVX alone and in combination with different concentrations of TXA01182 on P. aeruginosa. Values expressed as mean log₁₀ of CFU/mL. Error bars represent standard deviation.

Figure 7

General synthetic scheme for compounds 6a–n. Reagents and conditions: (i) Pd(dppf)Cl₂, Na₂CO₃, toluene/EtOH/H₂O, 100 °C; (ii) NaOH, THF/EtOH/H₂O, r.t. to 60 °C; (iii) EDCI, HOBt, DIPEA, DMF, r.t.; (iv) 4 M HCl in dioxane/MeOH. * stereo centers.

Figure 8

Synthetic scheme for compounds 6j (TXA01182).