A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ∼5-fold more effective than the canonical nitroreductase NfsB.

Deciphering the biosynthesis of a novel lipid in Mycobacterium tuberculosis expands the known roles of the nitroreductase superfamily.

Mycobacterium tuberculosis's (Mtb) success as a pathogen is due in part to its sophisticated lipid metabolic programs, both catabolic and biosynthetic. Several of Mtb lipids have specific roles in pathogenesis, but the identity and roles of many are unknown. Here, we demonstrated that the tyz gene cluster in Mtb, previously implicated in resistance to oxidative stress and survival in macrophages, encodes the biosynthesis of acyl-oxazolones. Heterologous expression of tyzA (Rv2336), tyzB (Rv2338c) and tyzC (Rv2337c) resulted in the biosynthesis of C12:0-tyrazolone as the predominant compound, and the C12:0-tyrazolone was identified in Mtb lipid extracts. TyzA catalyzed the N-acylation of l-amino acids, with highest specificity for l-Tyr and l-Phe and lauroyl-CoA (kcat/KM = 5.9 ± 0.8 × 103 M-1s-1). In cell extracts, TyzC, a flavin-dependent oxidase (FDO) of the nitroreductase (NTR) superfamily, catalyzed the O2-dependent desaturation of the N-acyl-L-Tyr produced by TyzA, while TyzB, a ThiF homolog, catalyzed its ATP-dependent cyclization. The substrate preference of TyzB and TyzC appear to determine the identity of the acyl-oxazolone. Phylogenetic analyses revealed that the NTR superfamily includes a large number of broadly distributed FDOs, including five in Mtb that likely catalyze the desaturation of lipid species. Finally, TCA1, a molecule with activity against drug-resistant and persistent tuberculosis, failed to inhibit the cyclization activity of TyzB, the proposed secondary target of TCA1. Overall, this study identifies a novel class of Mtb lipids, clarifies the role of a potential drug target, and expands our understanding of the NTR superfamily.

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening.

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ~5-fold more effective than the canonical nitroreductase NfsB.

A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype.

We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Discovery of Highly Trimethoprim-Resistant DfrB Dihydrofolate Reductases in Diverse Environmental Settings Suggests an Evolutionary Advantage Unrelated to Antibiotic Resistance.

Type B dihydrofolate reductases (DfrB) are intrinsically highly resistant to the widely used antibiotic trimethoprim, posing a threat to global public health. The ten known DfrB family members have been strongly associated with genetic material related to the application of antibiotics. Several dfrB genes were associated with multidrug resistance contexts and mobile genetic elements, integrated both in chromosomes and plasmids. However, little is known regarding their presence in other environments. Here, we investigated the presence of dfrB beyond the traditional areas of enquiry by conducting metagenomic database searches from environmental settings where antibiotics are not prevalent. Thirty putative DfrB homologues that share 62 to 95% identity with characterized DfrB were identified. Expression of ten representative homologues verified trimethoprim resistance in all and dihydrofolate reductase activity in most. Contrary to samples associated with the use of antibiotics, the newly identified dfrB were rarely associated with mobile genetic elements or antibiotic resistance genes. Instead, association with metabolic enzymes was observed, suggesting an evolutionary advantage unrelated to antibiotic resistance. Our results are consistent with the hypothesis that multiple dfrB exist in diverse environments from which dfrB were mobilized into the clinically relevant resistome. Our observations reinforce the need to closely monitor their progression.

Interrogation of the Structure-Activity Relationship of a Lipophilic Nitroaromatic Prodrug Series Designed for Cancer Gene Therapy Applications.

PR-104A is a dual hypoxia/nitroreductase gene therapy prodrug by virtue of its ability to undergo either one- or two-electron reduction to its cytotoxic species. It has been evaluated extensively in pre-clinical GDEPT studies, yet off-target human aldo-keto reductase AKR1C3-mediated activation has limited its use. Re-evaluation of this chemical scaffold has previously identified SN29176 as an improved hypoxia-activated prodrug analogue of PR-104A that is free from AKR1C3 activation. However, optimization of the bystander effect of SN29176 is required for use in a GDEPT setting to compensate for the non-uniform distribution of therapeutic gene transfer that is often observed with current gene therapy vectors. A lipophilic series of eight analogues were synthesized from commercially available 3,4-difluorobenzaldehyde. Calculated octanol-water partition coefficients (LogD7.4) spanned > 2 orders of magnitude. 2D anti-proliferative and 3D multicellular layer assays were performed using isogenic HCT116 cells expressing E. coli NfsA nitroreductase (NfsA_Ec) or AKR1C3 to determine enzyme activity and measure bystander effect. A variation in potency for NfsA_Ec was observed, while all prodrugs appeared AKR1C3-resistant by 2D assay. However, 3D assays indicated that increasing prodrug lipophilicity correlated with increased AKR1C3 activation and NfsA_Ec activity, suggesting that metabolite loss from the cell of origin into media during 2D monolayer assays could mask cytotoxicity. Three prodrugs were identified as bono fide AKR1C3-negative candidates whilst maintaining activity with NfsA_Ec. These were converted to their phosphate ester pre-prodrugs before being taken forward into in vivo therapeutic efficacy studies. Ultimately, 2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl dihydrogen phosphate possessed a significant 156% improvement in median survival in mixed NfsA_Ec/WT tumors compared to untreated controls (p = 0.005), whilst still maintaining hypoxia selectivity comparable to PR-104A.

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

Selection for a promiscuous enzyme activity provides substantial opportunity for competition between endogenous and newly-encountered substrates to influence the evolutionary trajectory, an aspect that is often overlooked in laboratory directed evolution studies. We selected the Escherichia coli nitro/quinone reductase NfsA for chloramphenicol detoxification by simultaneously randomising eight active-site residues and interrogating ~250,000,000 reconfigured variants. Analysis of every possible intermediate of the two best chloramphenicol reductases revealed complex epistatic interactions. In both cases, improved chloramphenicol detoxification was only observed after an R225 substitution that largely eliminated activity with endogenous quinones. Error-prone PCR mutagenesis reinforced the importance of R225 substitutions, found in 100% of selected variants. This strong activity trade-off demonstrates that endogenous cellular metabolites hold considerable potential to shape evolutionary outcomes. Unselected prodrug-converting activities were mostly unaffected, emphasising the importance of negative selection to effect enzyme specialisation, and offering an application for the evolved genes as dual-purpose selectable/counter-selectable markers.

In the cell, most tasks are performed by big molecules called proteins, which behave like molecular machines. Although proteins are often described as having one job each, this is not always true, and many proteins can perform different roles. Enzymes are a type of protein that facilitate chemical reactions. They are often specialised to one reaction, but they can also accelerate other side-reactions. During evolution, these side-reactions can become more useful and, as a result, the role of the enzyme may change over time. The main role of the enzyme called NfsA in Escherichia coli bacteria is thought to be to convert molecules called quinones into hydroquinones, which can protect the cell from toxic molecules produced in oxidation reactions. As a side-reaction, NfsA has the potential to protect bacteria from an antibiotic called chloramphenicol, but it generally does this with such low efficacy that the effects are negligible. Producing hydroquinones is helpful to the cell in some situations, but if bacteria are regularly exposed to chloramphenicol, NfsA's role aiding antibiotic resistance could become more important. Over time, the enzyme could evolve to become better at neutralising chloramphenicol. Therefore, NfsA provides an opportunity to study the evolution of proteins and how bacteria adapt to antibiotics. To see how evolution might affect the activity of NfsA, Hall et al. generated 250 million E. coli with either random or targeted changes to the gene that codes for the NfsA enzyme. The resulting variants of NfsA that were most effective against chloramphenicol all had a change that eliminated the enzyme's ability to convert quinones. This result demonstrates a key trade-off between roles for NfsA, where one must be lost for the other to improve. These results demonstrate the interplay between a protein's different roles and provide insight into bacterial drug resistance. Additionally, the experiments showed that the bacteria with improved resistance to chloramphenicol also became more sensitive to another antibiotic, metronidazole. These findings could inform the fight against drug-resistant bacterial infections and may also be helpful in guiding the design of proteins with different roles.

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

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.

Engineering Escherichia coli NfsB To Activate a Hypoxia-Resistant Analogue of the PET Probe EF5 To Enable Non-Invasive Imaging during Enzyme Prodrug Therapy.

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 assay-guided 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.

Exploring the sequence, function, and evolutionary space of protein superfamilies using sequence similarity networks and phylogenetic reconstructions.

Integrative computational methods can facilitate the discovery of new protein functions and enzymatic reactions by enabling the observation and investigation of complex sequence-structure-function and evolutionary relationships within protein superfamilies. Here, we highlight the use of sequence similarity networks (SSNs) and phylogenetic reconstructions to map the functional divergence and evolutionary history of protein superfamilies. We exemplify this approach using the nitroreductase (NTR) flavoenzyme superfamily, demonstrating that SSN investigations can provide a rapid and effective means to classify groups of proteins, expose sequence similarity relationships across the global scale of a protein superfamily, and efficiently support detailed phylogenetic analyses. Integration of such approaches with systematic experimental characterization will expand our understanding of the functional diversity of enzymes, their evolution, and their associated physiological roles.

Evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative vector for bacterial-directed enzyme-prodrug therapy.

Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses and bacteria to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to the prodrug. Nitroreductases, able to activate a range of promising nitroaromatic prodrugs to genotoxic metabolites, are of great interest for GDEPT. The bystander effect (cell-to-cell transfer of activated prodrug metabolites) has been quantified for some nitroaromatic prodrugs in mixed multilayer human cell cultures, however while these provide a good model for viral DEPT (VDEPT) they do not inform on the ability of these prodrug metabolites to exit bacterial vectors (relevant to bacterial-DEPT (BDEPT)). To investigate this we grew two Escherichia coli strains in co-culture; an activator strain expressing the nitroreductase E. coli NfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activator to the recipient cells. We used this to investigate five clinically relevant prodrugs: metronidazole, CB1954, nitro-CBI-DEI, and two dinitrobenzamide mustard prodrug analogues, PR-104A and SN27686. Consistent with the bystander efficiencies previously measured in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. However, in contrast with observations in human cell multilayers, the nitrogen mustard prodrug metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, we further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products, consistent with their relative bystander efficiencies in human cell culture. Overall, our data suggest that prodrugs may differ in their suitability for VDEPT versus BDEPT applications and emphasise the importance of evaluating an enzyme-prodrug partnership in an appropriate context for the intended vector.

Revealing Unexplored Sequence-Function Space Using Sequence Similarity Networks.

The rapidly expanding number of protein sequences found in public databases can improve our understanding of how protein functions evolve. However, our current knowledge of protein function likely represents a small fraction of the diverse repertoire that exists in nature. Integrative computational methods can facilitate the discovery of new protein functions and enzymatic reactions through the observation and investigation of the complex sequence-structure-function relationships within protein superfamilies. Here, we highlight the use of sequence similarity networks (SSNs) to identify previously unexplored sequence and function space. We exemplify this approach using the nitroreductase (NTR) superfamily. We demonstrate that SSN investigations can provide a rapid and effective means to classify groups of proteins, therefore exposing experimentally unexplored sequences that may exhibit novel functionality. Integration of such approaches with systematic experimental characterization will expand our understanding of the functional diversity of enzymes and their associated physiological roles.

Evolutionary and molecular foundations of multiple contemporary functions of the nitroreductase superfamily.

Insight regarding how diverse enzymatic functions and reactions have evolved from ancestral scaffolds is fundamental to understanding chemical and evolutionary biology, and for the exploitation of enzymes for biotechnology. We undertook an extensive computational analysis using a unique and comprehensive combination of tools that include large-scale phylogenetic reconstruction to determine the sequence, structural, and functional relationships of the functionally diverse flavin mononucleotide-dependent nitroreductase (NTR) superfamily (>24,000 sequences from all domains of life, 54 structures, and >10 enzymatic functions). Our results suggest an evolutionary model in which contemporary subgroups of the superfamily have diverged in a radial manner from a minimal flavin-binding scaffold. We identified the structural design principle for this divergence: Insertions at key positions in the minimal scaffold that, combined with the fixation of key residues, have led to functional specialization. These results will aid future efforts to delineate the emergence of functional diversity in enzyme superfamilies, provide clues for functional inference for superfamily members of unknown function, and facilitate rational redesign of the NTR scaffold.

Engineering a Multifunctional Nitroreductase for Improved Activation of Prodrugs and PET Probes for Cancer Gene Therapy.

Gene-directed enzyme-prodrug therapy (GDEPT) is a promising anti-cancer strategy. However, inadequate prodrugs, inefficient prodrug activation, and a lack of non-invasive imaging capabilities have hindered clinical progression. To address these issues, we used a high-throughput Escherichia coli platform to evolve the multifunctional nitroreductase E. coli NfsA for improved activation of a promising next-generation prodrug, PR-104A, as well as clinically relevant nitro-masked positron emission tomography-imaging probes EF5 and HX4, thereby addressing a critical and unmet need for non-invasive bioimaging in nitroreductase GDEPT. The evolved variant performed better in E. coli than in human cells, suggesting optimal usefulness in bacterial rather than viral GDEPT vectors, and highlighting the influence of intracellular environs on enzyme function and the shaping of promiscuous enzyme activities within the "black box" of in vivo evolution. We provide evidence that the dominant contribution to improved PR-104A activity was enhanced affinity for the prodrug over-competing intracellular substrates.

Rational design of an AKR1C3-resistant analog of PR-104 for enzyme-prodrug therapy.

The clinical stage anti-cancer agent PR-104 has potential utility as a cytotoxic prodrug for exogenous bacterial nitroreductases expressed from replicating vector platforms. However substrate selectivity is compromised due to metabolism by the human one- and two-electron oxidoreductases cytochrome P450 oxidoreductase (POR) and aldo-keto reductase 1C3 (AKR1C3). Using rational drug design we developed a novel mono-nitro analog of PR-104A that is essentially free of this off-target activity in vitro and in vivo. Unlike PR-104A, there was no biologically relevant cytotoxicity in cells engineered to express AKR1C3 or POR, under aerobic or anoxic conditions, respectively. We screened this inert prodrug analog, SN34507, against a type I bacterial nitroreductase library and identified E. coli NfsA as an efficient bioactivator using a DNA damage response assay and recombinant enzyme kinetics. Expression of E. coli NfsA in human colorectal cancer cells led to selective cytotoxicity to SN34507 that was associated with cell cycle arrest and generated a robust 'bystander effect' at tissue-like cell densities when only 3% of cells were NfsA positive. Anti-tumor activity of SN35539, the phosphate pre-prodrug of SN34507, was established in 'mixed' tumors harboring a minority of NfsA-positive cells and demonstrated marked tumor control following heterogeneous suicide gene expression. These experiments demonstrate that off-target metabolism of PR-104 can be avoided and identify the suicide gene/prodrug partnership of E. coli NfsA/SN35539 as a promising combination for development in armed vectors.

Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility.

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.

Error-prone PCR and effective generation of gene variant libraries for directed evolution.

Any single-enzyme directed evolution strategy has two fundamental requirements: the need to efficiently introduce variation into a gene of interest and the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very large areas of sequence space. Here we present comprehensive protocols and tips for using epPCR to generate gene variants that exhibit a relatively balanced spectrum of mutations and for capturing as much diversity as possible through effective cloning of those variants. The detailed library preparation methods that we describe are generally applicable to any directed evolution strategy that uses restriction enzymes to clone gene variants into an expression plasmid.

Site-saturation mutagenesis by overlap extension PCR.

Site-saturation mutagenesis is a proven strategy for generating high-quality variant gene libraries of a defined size. Variation is introduced via incorporation of degenerate base combinations at specific codon locations, giving rise to a precise series of amino acid substitutions in the encoded protein. Here we describe a simple and efficient overlap PCR protocol for the introduction of degenerate bases at either single or multiple codon locations. The resulting libraries can then be directly screened for improved protein function as either an independent directed evolution study or an adjunct to random mutagenesis strategies (such as error-prone PCR) that are, in isolation, unlikely to access the full repertoire of possible amino acid substitutions at any given position.

The Flavin Reductase MsuE Is a Novel Nitroreductase that Can Efficiently Activate Two Promising Next-Generation Prodrugs for Gene-Directed Enzyme Prodrug Therapy.

Bacterial nitroreductase enzymes that can efficiently catalyse the oxygen-independent reduction of prodrugs originally developed to target tumour hypoxia offer great potential for expanding the therapeutic range of these molecules to aerobic tumour regions, via the emerging cancer strategy of gene-directed enzyme prodrug therapy (GDEPT). Two promising hypoxia prodrugs for GDEPT are the dinitrobenzamide mustard PR-104A, and the nitrochloromethylbenzindoline prodrug nitro-CBI-DEI. We describe here use of a nitro-quenched fluorogenic probe to identify MsuE from Pseudomonas aeruginosa as a novel nitroreductase candidate for GDEPT. In SOS and bacteria-delivered enzyme prodrug cytotoxicity assays MsuE was less effective at activating CB1954 (a first-generation GDEPT prodrug) than the "gold standard" nitroreductases NfsA and NfsB from Escherichia coli. However, MsuE exhibited comparable levels of activity with PR-104A and nitro-CBI-DEI, and is the first nitroreductase outside of the NfsA and NfsB enzyme families to do so. These in vitro findings suggest that MsuE is worthy of further evaluation in in vivo models of GDEPT.

Creation and screening of a multi-family bacterial oxidoreductase library to discover novel nitroreductases that efficiently activate the bioreductive prodrugs CB1954 and PR-104A.

Two potentially complementary approaches to improve the anti-cancer strategy gene-directed enzyme prodrug therapy (GDEPT) are discovery of more efficient prodrug-activating enzymes, and development of more effective prodrugs. Here we demonstrate the utility of a flexible screening system based on the Escherichia coli SOS response to evaluate novel nitroreductase enzymes and prodrugs in concert. To achieve this, a library of 47 candidate genes representing 11 different oxidoreductase families was created and screened to identify the most efficient activators of two different nitroaromatic prodrugs, CB1954 and PR-104A. The most catalytically efficient nitroreductases were found in the NfsA and NfsB enzyme families, with NfsA homologues generally more active than NfsB. Some members of the AzoR, NemA and MdaB families also exhibited low-level activity with one or both prodrugs. The results of SOS screening in our optimised E. coli reporter strain SOS-R2 were generally predictive of the ability of nitroreductase candidates to sensitise E. coli to CB1954, and of the kcat/Km for each prodrug substrate at a purified protein level. However, we also found that not all nitroreductases express stably in human (HCT-116 colon carcinoma) cells, and that activity at a purified protein level did not necessarily predict activity in stably transfected HCT-116. These results highlight a need for all enzyme-prodrug partners for GDEPT to be assessed in the specific context of the vector and cell line that they are intended to target. Nonetheless, our oxidoreductase library and optimised screens provide valuable tools to identify preferred nitroreductase-prodrug combinations to advance to preclinical evaluation.