Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections

Abstract

One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies. IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.

The chemotherapeutic drug methotrexate selects for antibiotic resistance

“…We initially determined the MICs of MTX in clinical [35] and laboratory strains of E. coli (Table S1). Initial experiments revealed variable, but high MTX MICs, ranging from 4 to 32 mg/mL in the different genetic backgrounds, with the exception of <0.25 mg/mL for E. coli W3110 Δ7NR tolC (Tables S4-S5) [36] . This being consistent with previous reports demonstrating that E. coli displays intrinsic resistance towards MTX due to AcrAB-TolC mediated efflux [ 2 , 17 ].…”

Engineering the Escherichia coli Nitroreductase NfsA to Create a Flexible Enzyme-Prodrug Activation System

“…To discover improved prodrug-converting nitroreductases we screened two previously-generated nfsA gene libraries: 1) a randomized codon mutagenesis library (“7RCM”) that uses NDT or NNK degenerate codons to randomize seven key residues in the active site of NfsA (S41, F42, F83, S224, R225, F227 and K222; Copp et al, 2020 ); and 2) a site-directed mutagenesis library (“10SDM”) that encodes all possible combinations of ten NfsA substitutions previously found to improve activation of the dinitrobenzamide mustard prodrug PR-104A (I5T, S41Y, E99G, L103M, K222E, R225A, R225G, R225P, F227S, L229V; Copp et al, 2017 ) ( Supplementary Figure S1 ). Here we sought to probe both libraries for variants with an improved capacity to activate nitro-CBI-DEI, CB1954 and metronidazole.…”

“…Instead, a pre-selection strategy was employed to remove 7RCM library clones comprising low- or non-functioning nitroreductase variants, or empty pUCX plasmid. Pre-selection involved plating transformed cells on agar plates supplemented with 0.5 or 5 µM niclosamide, a nitroaromatic antibiotic that is toxic to E. coli: Δ tolC cells but can be detoxified by functional nitroreductases ( Copp et al, 2020 ).…”

“…As an initial selection for niclosamide detoxification has been shown to greatly enrich for NfsA variants that have an improved capacity to reduce metronidazole ( Copp et al, 2020 ), we began by screening for improvement in this activity, followed by counter-screening to identify variants that were also improved with nitro-CBI-DEI and CB1954 (screening workflow summarized in Supplementary Figure S2 ). In total, 1750 10SDM library clones (a number predicted by GLUE ( Firth and Patrick, 2008 ) to provide >95% library coverage) and 114 niclosamide pre-selected 7RCM library clones (57 from each niclosamide concentration) were screened for improved metronidazole activity via E. coli growth inhibition assays as previously described ( Prosser et al, 2013 ).…”

Intracellular complexities of acquiring a new enzymatic function revealed by mass-randomisation of active-site residues

“…We have previously conducted several different mutagenesis studies on nfsA , seeking to enhance activity with prodrugs and/or positron emission tomography (PET) imaging probes for cancer gene therapy applications ( Williams, 2013 ; Copp et al, 2017 ; Rich, 2017 ), or to assess potential collateral sensitivities between niclosamide and the antibiotics metronidazole and nitrofurantoin ( Copp et al, 2020 ). Based on this previous work we empirically identified eight active-site residues (S41, L43, H215, T219, K222, S224, R225, and F227; Figure 1A ) as being individually mutable and having the potential to contribute to generically improved nitroreductase activity.…”