Potentiation of Antibacterial Activity of the MB-1 Siderophore-Monobactam Conjugate Using an Efflux Pump Inhibitor

Abstract

Preliminary enthusiasm over the encouraging spectrum and in vitro activities of siderophore conjugates, such as MB-1, was recently tempered by unexpected variability in in vivo efficacy. The need for these conjugates to compete for iron with endogenously produced siderophores has exposed a significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux for siderophore secretion in Pseudomonas aeruginosa and provide evidence that efflux inhibition may circumvent this in vivo-relevant resistance liability.T he extent and severity of antibiotic resistance in Gram-negative pathogens require the development of innovative therapeutic interventions that not only circumvent existing clinically relevant resistance mechanisms but will also result in limited spontaneous resistance emergence. Although standard in vitro methodologies exist for predicting resistance development, translational deficiencies that can stem from their lack of in vivo physiological relevance are becoming increasingly apparent. We previously described the siderophore-monobactam conjugate MB-1 ( Fig. 1 [1]), which has broad-spectrum in vitro activities against multidrug-resistant (MDR) Gram-negative pathogens, including Pseudomonas aeruginosa, when tested using standard methods established by the Clinical and Laboratory Standards Institute (CLSI) (1). Unfortunately, we have also demonstrated that despite these encouraging in vitro activities, the efficacy of MB-1 in a neutropenic murine infection model was highly variable and not directly predictable from traditional in vitro activity assays (2). Instead, the development of a modified in vitro resistance frequency assay, which relies on the utilization of more in vivo-relevant iron conditions, has proven to be more predictive of the efficacy of MB-1 in vivo. Additionally, this assay has demonstrated a role for endogenous siderophores, such as pyoverdine, in mediating the adaptive cellular response that enables P. aeruginosa to transiently resist MB-1 activity (2). While these results suggest that the use of MB-1 as monotherapy is not a viable option, we have speculated about alternative strategies that may mitigate this adaptive response and resurrect MB-1 as a legitimate antibacterial agent. Among these potentiation-type approaches, we considered the idea that inhibiting endogenous siderophore functionality should circumvent the adaptation phenotype mediated by the competition for iron by native siderophore systems. Siderophore secretion into the extracellular milieu has been shown to be dependent on functional efflux pumps in Gram-negative bacteria (3-5). In the case of P. aeruginosa, the secretion of newly synthesized pyoverdine is accomplished by the ABC transporter-type efflux system encoded by pvdR, pvdT, and opmQ (6). Additionally, this system is responsible for pyoverdine recycling after successive rounds of iron acquisition and delivery (7).To prove that inhibiting the activity of this pump can potentiate the activity of MB-1, we generated a pvdRT-op...

Humanized Exposures of Cefiderocol, a Siderophore Cephalosporin, Display Sustained in vivo Activity against Siderophore-Resistant <b><i>Pseudomonas aeruginosa</i></b>

“…Under extremely low iron conditions in vivo , bacterial cells will not depend on less efficient siderophore for iron acquisition, thus diminishing the MB-1 activity [11]. A subsequent study demonstrated that using an inhibitor of the efflux pump that exports pyoverdine out of cells potentiated the in vivo activity of MB-1; thus, these data substantiate the hypothesis of importance of iron-binding affinity [24]. Similarly, SMC-3176 was vulnerable to the development of the same type of resistance that is reversible with drug exposure cessation.…”

Antibiotic Hybrids: the Next Generation of Agents and Adjuvants against Gram-Negative Pathogens?

“…Through the attachment of the ironchelating siderophore adjuvant, significantly higher intracellular drug concentrations can be achieved by hijacking the bacterial iron transport system. Several of these ␤-lactam-siderophore hybrid drugs have entered preclinical/clinical trials (232)(233)(234)(235)(236), notably the cephalosporin-catechol hybrid cefiderocol, which has advanced to and is currently in phase 3 clinical trials.…”

Siderophore Cephalosporin Cefiderocol Utilizes Ferric Iron Transporter Systems for Antibacterial Activity against Pseudomonas aeruginosa

“…Recently, siderophore-conjugated monobactam antibacterial agents, BAL30072, MB-1, MC-1, and SMC-3176 with a hydroxypyridone moiety, have been reported to show potent antibacterial activities against some MDR Gram-negative bacteria, including P. aeruginosa (19, 21, 22), but the development of MB-1 and SMC-3176 has been problematic due to a lack of correlation between in vitro activity and in vivo efficacy (21–23). …”