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Summary The broad-spectrum antibiotic d-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analogue of d-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and d-Ala:d-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5’-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.

Mycobacterium tuberculosis resides in the lungs in various lesion types with unique microenvironmental conditions. This diversity is in line with heterogeneous disease progression and divergent drug efficiency. Fluorescent reporter strains can be used to decipher the micromilieu and to guide future treatment regimens. Current reporters using replicating plasmids, however, are not suitable for long-term mouse infections or studies in non-human primates. Using a combination of recombinant DNA and protein optimization techniques, we have developed reporter strains based on integrative plasmids, which exhibit stimulus-response characteristics and fluorescence intensities comparable to those based on replicating plasmids. We successfully applied the concepts by constructing a multi-color reporter strain able to detect simultaneous changes in environmental pH, Mg2+ concentrations, and protein expression levels.

Tuberculosis (TB) is one of the primary causes of deaths due to infectious diseases. The current TB regimen is long and complex, failing of which leads to relapse and/or the emergence of drug resistance. There is a critical need to understand the mechanisms of resistance development. With increasing drug pressure, Mycobacterium tuberculosis (Mtb) activates various pathways to counter drug-related toxicity. Signaling modules steer the evolution of Mtb to a variant that can survive, persist, adapt, and emerge as a form that is resistant to one or more drugs. Recent studies reveal that about 1/3rd of the annotated Mtb proteome is modified post-translationally, with a large number of these proteins being essential for mycobacterial survival. Post-translational modifications (PTMs) such as phosphorylation, acetylation, and pupylation play a salient role in mycobacterial virulence, pathogenesis, and metabolism. The role of many other PTMs is still emerging. Understanding the signaling pathways and PTMs may assist clinical strategies and drug development for Mtb. In this review, we explore the contribution of PTMs to mycobacterial physiology, describe the related cellular processes, and discuss how these processes are linked to drug resistance. A significant number of drug targets, InhA, RpoB, EmbR, and KatG, are modified at multiple residues via PTMs. A better understanding of drug-resistance regulons and associated PTMs will aid in developing effective drugs against TB.

Mycobacterium tuberculosis is a facultative anaerobe and its characteristic pathological hallmark, the granuloma, exhibits hypoxia in humans and in most experimental models. Thus the host and bacillary adaptation to hypoxia is of central importance in understanding pathogenesis and thereby to derive new drug treatments and vaccines.

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for six decades without significant appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We therefore investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question, we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low rate of emergence of D-cycloserine resistance mutations is the dominant biological factor delaying the appearance of clinical resistance to this antibiotic. Furthermore, we also identified potential compensatory mechanisms able to minimize the severe fitness costs of primary D-cycloserine resistance conferring mutations., D-cycloserine (DCS) has been used for decades to treat Mycobacterium tuberculosis (Mtb) but resistance is rarely observed in clinical isolates. Here, the authors report ultra-low rate of emergence of resistance mutations as the underlying mechanism of DCS resistance evasion in Mtb.

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for decades without appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We investigated whyMycobacterium tuberculosisfails to become resistant to D-cycloserine. To address this question we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysisin vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low mutation frequency associated with D-cycloserine resistance is the dominant factor delaying the appearance of clinical resistance to this antibiotic in bacteria infecting humans, and not lack of potential compensatory mechanisms.One Sentence SummaryWe show that the lack of D-cycloserine resistance inMycobacterium tuberculosisis due its ultra-low mutation frequency rather than lack of compensatory mechanisms.

Directed enzyme evolution is now a routine approach to improve desirable biocatalytic properties. When only a low-throughput screen is available to detect improved variants from a mutant gene library, it is imperative that cloning efficiency be maximized during library synthesis to avoid wasting effort screening empty plasmids. To achieve this we developed pUCXKT, a gain-of-function positive selection expression vector. Insertion of genes amplified using a specialized downstream PCR primer restores key regulatory and genetic elements necessary for co-expression of a kanamycin resistance marker adjacent to the pUCXKT cloning region. We show that pUCXKT enables 100 % cloning efficiency as well as high-level expression of inserted genes. Unlike previous positive selection expression plasmids, the strategy we used to design pUCXKT is readily adaptable to different vector backbones, antibiotic marker genes, and multiple cloning regions.

Recent technological advances in accurate mass spectrometry and data analysis have revolutionized metabolomics experimentation. Activity-based and global metabolomic profiling methods allow simultaneous and rapid screening of hundreds of metabolites from a variety of chemical classes, making them useful tools for the discovery of novel enzymatic activities and metabolic pathways. By using the metabolome of the relevant organism or close species, these methods capitalize on biological relevance, avoiding the assignment of artificial and non-physiological functions. This review discusses state-of-the-art metabolomic approaches and highlights recent examples of their use for enzyme annotation, discovery of new metabolic pathways, and gene assignment of orphan metabolic activities across diverse biological sources.

Stable isotope-mass spectrometry (MS)-based metabolomic profiling is a powerful technique for following changes in specific metabolite pool sizes and metabolic flux under various experimental conditions in a test organism or cell type. Here, we use a metabolomics approach to interrogate the mechanism of antibiotic action of d-cycloserine (DCS), a second line antibiotic used in the treatment of multidrug resistant Mycobacterium tuberculosis infections. We use doubly labeled 13C α-carbon-2H l-alanine to allow tracking of both alanine racemase and d-alanine:d-alanine ligase activity in M. tuberculosis challenged with DCS and reveal that d-alanine:d-alanine ligase is more strongly inhibited than alanine racemase at equivalent DCS concentrations. We also shed light on mechanisms surrounding d-Ala-mediated antagonism of DCS growth inhibition and provide evidence for a postantibiotic effect for this drug. Our results illustrate the potential of metabolomics in cellular drug-target engagement studies and consequently have broad implications in future drug development and target validation ventures.

d-Cycloserine is a second-line drug approved for use in the treatment of patients infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. The unique mechanism of action of d-cycloserine, compared with those of other clinically employed antimycobacterial agents, represents an untapped and exploitable resource for future rational drug design programs. Here, we show that d-cycloserine is a slow-onset inhibitor of MtDdl and that this behavior is specific to the M. tuberculosis enzyme orthologue. Furthermore, evidence is presented that indicates d-cycloserine binds exclusively to the C-terminal d-alanine binding site, even in the absence of bound d-alanine at the N-terminal binding site. Together, these results led us to propose a new model of d-alanine:d-alanine ligase inhibition by d-cycloserine and suggest new opportunities for rational drug design against an essential, clinically validated mycobacterial target.

The increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except in Mycobacterium tuberculosis , where it has so far been underexploited. d -Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within the d -alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies of M. tuberculosis . During our studies on alternative inhibitors of the d -alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro– d -alanine (BCDA) is a very poor inhibitor of recombinant M. tuberculosis Alr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit the d -alanine pathway in intact cells, consistent with its poor in vitro activity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI in M. tuberculosis and thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.

Gene-directed enzyme prodrug therapy (GDEPT) aims to achieve highly selective tumor-cell killing through the use of tumor-tropic gene delivery vectors coupled with systemic administration of otherwise inert prodrugs. Nitroaromatic prodrugs such as CB1954 hold promise for GDEPT as they are readily reduced to potent DNA alkylating agents by bacterial nitroreductase enzymes (NTRs). Transfection with the nfsB gene from Escherichia coli can increase the sensitivity of tumor cells to CB1954 by greater than 1000-fold. However, poor catalytic efficiency limits the activation of CB1954 by NfsB at clinically relevant doses. A lack of flexible, high-throughput screening technology has hindered efforts to discover superior NTR candidates. Here we demonstrate how the SOS chromotest and complementary screening technologies can be used to evaluate novel enzymes that activate CB1954 and other bioreductive and/or genotoxic prodrugs. We identify the major E. coli NTR, NfsA, as 10-fold more efficient than NfsB in activating CB1954 as purified protein (k(cat)/K(m)) and when over-expressed in an E. coli nfsA(-)/nfsB(-) gene deleted strain. NfsA also confers sensitivity to CB1954 when expressed in HCT-116 human colon carcinoma cells, with similar efficiency to NfsB. In addition, we identify two novel E. coli NTRs, AzoR and NemA, that have not previously been characterized in the context of nitroaromatic prodrug activation.

Enzymes fuel the biochemical activities of all cells. Their substrates and products thus represent a potential window into the physiologic state of a cell. Metabolomics focuses on the global, or systems-level, study of small molecules in a given biological system and has thus provided an experimental tool with which to study cellular physiology, including the biochemistry within pathogenic microorganisms. While metabolomic studies of Mycobacterium tuberculosis are still in their infancy, recent studies have begun to deliver unique insights into the composition, organization, activity, and regulation of the bacterium's physiologic network not accessible by other approaches. Here, we outline practical methods for the culture, collection, and analysis of metabolomic samples from M. tuberculosis that emphasize minimally perturbing sample handling, broad and native metabolite recovery, and sensitive, biologically agnostic metabolite detection.

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.

D-cycloserine (DCS) is an antibiotic that is currently used in second-line treatment of tuberculosis. DCS is a structural analogue of D-alanine, and targets two enzymes involved in the cytosolic stages of peptidoglycan synthesis: alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). The mechanisms of inhibition of DCS have been well-assessed using Alr and Ddl enzymes from various bacterial species, but little is known regarding the interactions of DCS with the mycobacterial orthologues of these enzymes. We have over-expressed and purified recombinant Mycobacterium tuberculosis Ddl (MtDdl; Rv2981c), and report a kinetic examination of the enzyme with both its native substrate and DCS. MtDdl is activated by K(+), follows an ordered ter ter mechanism and displays distinct affinities for D-Ala at each D-Ala binding site (K(m,D-Ala1) = 0.075 mm, K(m,D-Ala2) = 3.6 mm). ATP is the first substrate to bind and is necessary for subsequent binding of D-alanine or DCS. The pH dependence of MtDdl kinetic parameters indicate that general base chemistry is involved in the catalytic step. DCS was found to competitively inhibit D-Ala binding at both MtDdl D-Ala sites with equal affinity (K(i,DCS1) = 14 μm, K(i,DCS2) = 25 μm); however, each enzyme active site can only accommodate a single DCS molecule at a given time. The pH dependence of K(i,DCS2) revealed a loss of DCS binding affinity at high pH (pK(a) = 7.5), suggesting that DCS binds optimally in the zwitterionic form. The results of this study may assist in the design and development of novel Ddl-specific inhibitors for use as anti-mycobacterial agents.

CB1954 is an anti-cancer prodrug that can be reduced at either of two nitro groups to form cytotoxic metabolites. We describe here two efficient and previously uncharacterized nitroreductases, YfkO from Bacillus subtilis which reduces CB1954 exclusively at the 4-NO(2) position, and NfsA from Klebsiella pneumoniae which preferentially reduces the 2-NO(2) group. Utilizing these novel enzymes, together with three previously characterized nitroreductases, we demonstrate that the Escherichia coli SOS-chromotest assay can differentially detect the 4-nitro versus 2-nitro reduction products of CB1954 following deletion of the nucleotide excision repair gene uvrB, but not mismatch repair (mutS) or methyltransferase (ada/ogt) genes. These findings may hold significance for identification and selection of nitroreductases for CB1954-mediated gene therapy, particularly when targeting tumors that are deficient in nucleotide excision repair. Moreover, we demonstrate that comparative SOS chromotest analysis in wild type and uvrB mutant strains can be used to determine whether or not nucleotide excision repair plays a significant role in processing DNA damage resulting from activation of different nitroaromatic prodrugs.

d-Cycloserine is a second-line drug approved for use in the treatment of patients infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. The unique mechanism of action of d-cycloserine, compared with those of other clinically employed antimycobacterial agents, represents an untapped and exploitable resource for future rational drug design programs. Here, we show that d-cycloserine is a slow-onset inhibitor of MtDdl and that this behavior is specific to the M. tuberculosis enzyme orthologue. Furthermore, evidence is presented that indicates d-cycloserine binds exclusively to the C-terminal d-alanine binding site, even in the absence of bound d-alanine at the N-terminal binding site. Together, these results led us to propose a new model of d-alanine:d-alanine ligase inhibition by d-cycloserine and suggest new opportunities for rational drug design against an essential, clinically validated mycobacterial target.

Tumor-targeting viruses and bacteria hold great promise as anti-cancer agents. They kill cells by entirely different mechanisms to radio- and chemotherapies, and have potential to synergize with these treatments without overlapping toxicities. Furthermore, these agents can be ‘armed’ with genes that encode enzymes that activate prodrugs - compounds that are deactivated in their administered form, but become highly toxic upon metabolic activation. This not only improves killing of infected cells, but also neighboring non-infected cells, as the prodrug metabolites can diffuse locally and exert a bystander effect. A highly efficient activating enzyme in partnership with a prodrug that has a strong bystander effect can address some of the historical limitations of cancer gene therapy including the inability of biological vectors to reach every target cell. Phase I/II trials of the first-generation nitroaromatic prodrug CB1954 in conjunction with the prototype gene therapy nitroreductase, Escherichia coli NfsB, have been conducted in prostate and liver cancer. These trials provided some evidence of anti-tumor activity but, due to dose-limiting hepatotoxicity, the highest achievable plasma concentration of CB1954 was less than 1% of NfsB's Km. As well as highlighting a need for more efficient nitroreductase enzymes, this has fuelled a search for superior nitroaromatic prodrugs. The next-generation dinitrobenzamide mustard prodrug PR-104A is not only 5–50 fold more dose-potent upon activation, but also better tolerated in humans (MTD 1100 mg/m2 vs 24 mg/m2; q3w, iv). However, E. coli NfsB also has relatively poor (millimolar) affinity for this substrate. To discover more efficient nitroreductases we developed screens for genotoxic prodrug activation, based on their ability to induce reporter genes linked to the E. coli SOS (DNA damage repair) response. We used these to screen a large library of candidate enzymes for DNBM activity, and selected E. coli NfsA as a top candidate for further improvement by random and targeted mutagenesis. High throughput screening of large error prone PCR libraries coupled with medium throughput screening of targeted mutagenesis libraries revealed 10 individual mutations that significantly increased NfsA activity. These mutations were then combined in a synthetic “smart” library, from which eight poly-mutant enzymes were selected for kinetic analysis. Relative to wild type the engineered variants display an 18–40 fold improvement in PR-104A Km with respect to E. coli NfsA, and are 860–1700 fold better than NfsB. Importantly they also retain, or are improved in, their ability to co-metabolize preferred 2-nitroimidazole probes with PET-imaging capabilities (see abstract: Patterson et al, “Molecular imaging using bacterial nitroreductase reporter genes by repurposing the clinical stage hypoxia PET probe EF5”). The enhanced prodrug activation and in vivo imaging potential of these enzymes is now being evaluated in human gene therapy models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B88.

Oncolytic viruses and tumor-tropic bacteria offer promise as cancer therapeutics of the future. There is a need to develop technologies to monitor the spatio-temporal distribution of these live vectors in a manner that is predictive of normal tissue toxicity and antitumor efficacy. Positron emission tomography (PET) is the preferred non-invasive imaging modality (biomarker) but suitable advanced reporter gene/PET probe combinations are lacking. A range of 2-nitroimidazole (2-NI) probes are currently in clinical use for the detection of hypoxia (EF5, FMISO, HX4, FAZA) and thus have already attained a high level of clinical validation. We hypothesized that (2-NI) PET agents might be repurposed to monitor the biodistribution of replicating biological agents, thereby leveraging two decades of research efforts to optimise hypoxia PET probe pharmacology. Bacterial nitroreductases (NTRs; type I) are efficient O2-independent enzymes that provide the necessary catalytic flexibility to achieve this goal. Historically E. coli NfsB has been the focus of gene therapy applications. We cloned eleven candidate NTRs from E. coli namely; AzoR, KefF, MdaB, NemA, NfsA, NfsB, WrbA, YcaK, YcdI, YdjA and Yief. Using HCT116 cells engineered to express each NTR, we showed NfsA alone was able to metabolise a range of 2-NI probe molecules, including EF5 (pentafluoroetanidazole), leading to efficient cellular retention. We confirmed catalytic efficiency of EF5 reduction by recombinant NfsA (kcat/Km 97 s−1/mM). In contrast NfsB only weakly metabolised EF5 (kcat/Km 0.24 s−1/mM). We compared EF5 adduct retention in HCT116 cells, detected by mAb using flow cytometry, either under anoxia (< 10ppm O2) or engineered to express NfsA or NfsB. Following exposure to EF5 in monolayer, median fluorescence was >100-fold greater in NfsA expressing cells than anoxic cells, whereas NfsB cells were negative. Mixed Wt : NfsA multicellular layers containing various ratios of cells demonstrated the capacity to detect, with precision, single NfsA-positive cells in mixed cell populations. HCT116 xenografts composed of increasing proportions of NfsA cells (0% to 25%) were established in nude mice and analysed by immunohistochemistry and flow cytometry. Ex-vivo pimonidazole-binding confirmed all NfsA positive cells were detected in vivo following administration of EF5. NfsA, like NfsB, can bioactivate the clinical-stage prodrug PR-104. We determined the relationship between EF5 retention and PR-104 cytotoxicity in HCT116 xenografts harbouring variable proportions of NfsA cells (0%-25%). A correlation was observed between total EF5 retention and global clonogenic cell kill (r2 = 0.828; p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B89.