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.

Evaluation of a Conformationally Constrained Indole Carboxamide as a Potential Efflux Pump Inhibitor in Pseudomonas aeruginosa

“…The impact of TXA09155 on NCF hydrolysis is minimal at concentrations below 25 μg/mL, suggesting that TXA09155 does not interact with the outer membrane of P. aeruginosa at these concentrations ( Figure 2 A). This result is comparable to what was previously reported for TXA01182 [ 29 ] suggesting that incorporation of the N -2-(4-aminomethylpyrrolidin-2-yl) methyl moiety that produced TXA09155 did not introduce membrane disruption properties to the compound. Polymyxin B was used as a positive control ( Figure 2 B).…”

“…These results suggest that the killing kinetics for the combination of TXA09155/moxifloxacin are faster than those of moxifloxacin alone. The potentiation of a minimally bactericidal concentration of moxifloxacin (1×-MIC) by TXA09155 seen in Figure 6 is consistent with similar studies performed with a TXA01182/levofloxacin combination in P. aeruginosa [ 29 ].…”

“…Efflux of EtBr from P. aeruginosa in the presence of TXA09155 was carried out as described previously [ 29 ].…”

“…Time-kill studies were carried out as described previously [ 29 ] with the following changes. When indicated, moxifloxacin was added to the prepared bacterial suspensions at one time the MIC (2 μg/mL).…”

“…Hence, the discovery of novel EPI scaffolds active against Gram-negative pathogens, particularly P. aeruginosa , with the potential of clinical use is still needed. TAXIS Pharmaceuticals is committed [ 26 ] to finding solutions to multidrug-resistant (MDR) bacterial infections and has previously published preliminary efforts on a diaminopentanamide class of potentiators [ 27 , 28 ] and more recently on a heterocyclic carboxamide class of potentiators that yielded TXA01182 [ 29 ]. TXA01182 potentiates monobactam, fluoroquinolones, sulfonamides, and tetracyclines against P. aeruginosa .…”