This review examines the vast catalytic and therapeutic potential offered by type I (i.e. oxygen-insensitive) nitroreductase enzymes in partnership with nitroaromatic prodrugs, with particular focus on gene-directed enzyme prodrug therapy (GDEPT; a form of cancer gene therapy). Important first indications of this potential were demonstrated over 20 years ago, for the enzyme-prodrug pairing of Escherichia coli NfsB and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, it has become apparent that both the enzyme and the prodrug in this prototypical pairing have limitations that have impeded their clinical progression. Recently, substantial advances have been made in the biodiscovery and engineering of superior nitroreductase variants, in particular development of elegant high-throughput screening capabilities to enable optimization of desirable activities via directed evolution. These advances in enzymology have been paralleled by advances in medicinal chemistry, leading to the development of second- and third-generation nitroaromatic prodrugs that offer substantial advantages over CB1954 for nitroreductase GDEPT, including greater dose-potency and enhanced ability of the activated metabolite(s) to exhibit a local bystander effect. In addition to forging substantial progress towards future clinical trials, this research is supporting other fields, most notably the development and improvement of targeted cellular ablation capabilities in small animal models, such as zebrafish, to enable cell-specific physiology or regeneration studies.

Engineering of enzymes to more efficiently activate genotoxic prodrugs holds great potential for improving anticancer gene or antibody therapies. We report the development of a new, GFP-based, high-throughput screening platform to enable engineering of prodrug-activating enzymes by directed evolution. By fusing an inducible SOS promoter to an engineered GFP reporter gene, we were able to measure levels of DNA damage in intact Escherichia coli and separate cell populations by fluorescence activating cell sorting (FACS). In two FACS iterations, we were able to achieve a 90,000-fold enrichment of a functional prodrug-activating nitroreductase from a null library background.

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.

Gene-directed enzyme prodrug therapy (GDEPT) uses tumor-tropic vectors to deliver prodrug-converting enzymes such as nitroreductases specifically to the tumor environment. The nitroreductase NfsB from Escherichia coli (NfsB_Ec) has been a particular focal point for GDEPT and over the past 25 years has been the subject of several engineering studies seeking to improve catalysis of prodrug substrates. To facilitate clinical development, there is also a need to enable effective non-invasive imaging capabilities. SN33623, a 5-nitroimidazole analogue of 2-nitroimidazole hypoxia probe EF5, has potential for PET imaging exogenously delivered nitroreductases without generating confounding background due to tumor hypoxia. However, we show here that SN33623 is a poor substrate for NfsB_Ec. To address this, we used assayguided sequence and structure analysis to identify two conserved residues that block SN33623 activation in NfsB_Ec and close homologues. Introduction of the rational substitutions F70A and F108Y into NfsB_Ec conferred high levels of SN33623 activity and enabled specific labeling of E. coli expressing the engineered enzyme. Serendipitously, the F70A and F108Y substitutions also substantially improved activity with the anticancer prodrug CB1954 and the 5-nitroimidazole antibiotic prodrug metronidazole, which is a potential biosafety agent for targeted ablation of nitroreductase-expressing vectors.

Preparation of ISEs often requires long and complicated conditioning protocols limiting their application as tools for in field measurements. Herein, we eliminated the need for conditioning by loading the membrane cocktail with primary ion solution. This protocol significantly shortens the preparation time of ISEs yielding functional electrodes with submicromolar detection limits.The scientific research in ion-selective electrodes (ISEs) has gained momentum within the last years due to improvements in the limits of detection and selectivity, becoming now applicable for trace-level measurements by understanding transmembrane ion fluxes. 1 The response of ISEs can be described by the phase boundary potential, E PB , according the following equation:Here a I (aq) and a I (org) are the activities of primary ion (I) of charge z in aqueous and organic phases respectively, while E 0 , R, T, and F are the standard potential, gas constant, temperature and Faraday constant, respectively. When a I (org) is kept constant, the equation 1 reduces to the well-known Nernst equation:In order to render an ion-selective membrane functional, the ionophore and lipophilic ionic sites are required. One of the major roles of ionophore is to make relatively strong complexes with the primary ion, thereby establishing their constant activity in the membrane. 2 For more details see Equations SI1-SI5 in the supporting information. The role of the lipophilic ionic sites is to provide ion-exchange properties. For cation selective membrane, this process could be described by the following equilibrium:where L is a ligand (ionophore) that forms ion-ionophore complex with ion I of stoichiometry n. + − is a lipophilic ion exchanger composed of lipophilic anion R -and its counterion M + . Partitioning of I from aqueous sample into the membrane results in its exchange with M + . Anion − remains in the membrane thereby rendering the membrane permselective while preserving the charge balance. 3 In a typical experimental protocol for the preparation of ion-selective membranes the ion-exchange process is obtained by conditioning (soaking) the membrane in an aqueous solution containing the ion I (traditional protocol). 4 Significant effort in ISEs field has been spent on researching ways to miniaturize 5-9 and optimize/simplify the preparation of ISEs. [10][11][12] Reducing or eliminating the need for the conditioning step prior to the use of the electrodes is an important step for devising a simple, practical protocol for ISEs applications. 12 This would enable nontrained personnel to use ISEs quickly and reliably. In this work we propose a simple alteration of the sensor's conditioning protocol. Instead of placing the ISEs in a solution of primary ions I, solution is added directly into the membrane cocktail prior to its casting. The concentration of that solution is calculated to allow for stoichiometric exchange of I and M. Consequently, ions I are present in the membrane facilitating the formation of ion-ionophore complex according to the ...