Clostridium Bacteria: Harnessing Tumour Necrosis for Targeted Gene Delivery.

Necrosis is a common feature of solid tumours that offers a unique opportunity for targeted cancer therapy as it is absent from normal healthy tissues. Tumour necrosis provides an ideal environment for germination of the anaerobic bacterium Clostridium from endospores, resulting in tumour-specific colonisation. Two main species, Clostridium novyi-NT and Clostridium sporogenes, are at the forefront of this therapy, showing promise in preclinical models. However, anti-tumour activity is modest when used as a single agent, encouraging development of Clostridium as a tumour-selective gene delivery system. Various methods, such as allele-coupled exchange and CRISPR-cas9 technology, can facilitate the genetic modification of Clostridium, allowing chromosomal integration of transgenes to ensure long-term stability of expression. Strains of Clostridium can be engineered to express prodrug-activating enzymes, resulting in the generation of active drug selectively in the tumour microenvironment (a concept termed Clostridium-directed enzyme prodrug therapy). More recently, Clostridium strains have been investigated in the context of cancer immunotherapy, either in combination with immune checkpoint inhibitors or with engineered strains expressing immunomodulatory molecules such as IL-2 and TNF-α. Localised expression of these molecules using tumour-targeting Clostridium strains has the potential to improve delivery and reduce systemic toxicity. In summary, Clostridium species represent a promising platform for cancer therapy, with potential for localised gene delivery and immunomodulation selectively within the tumour microenvironment. The ongoing clinical progress being made with C. novyi-NT, in addition to developments in genetic modification techniques and non-invasive imaging capabilities, are expected to further progress Clostridium as an option for cancer treatment.

Tumor-targeting bacteria as immune stimulants - the future of cancer immunotherapy?

Cancer immunotherapies have been widely hailed as a breakthrough for cancer treatment in the last decade, epitomized by the unprecedented results observed with checkpoint blockade. Even so, only a minority of patients currently achieve durable remissions. In general, responsive patients appear to have either a high number of tumor neoantigens, a preexisting immune cell infiltrate in the tumor microenvironment, or an 'immune-active' transcriptional profile, determined in part by the presence of a type I interferon gene signature. These observations suggest that the therapeutic efficacy of immunotherapy can be enhanced through strategies that release tumor neoantigens and/or produce a pro-inflammatory tumor microenvironment. In principle, exogenous tumor-targeting bacteria offer a unique solution for improving responsiveness to immunotherapy. This review discusses how tumor-selective bacterial infection can modulate the immunological microenvironment of the tumor and the potential for combination with cancer immunotherapy strategies to further increase therapeutic efficacy. In addition, we provide a perspective on the clinical translation of replicating bacterial therapies, with a focus on the challenges that must be resolved to ensure a successful outcome.

Design and Biological Evaluation of Piperazine-Bearing Nitrobenzamide Hypoxia/GDEPT Prodrugs: The Discovery of CP-506.

Off-target aerobic activation of PR-104A by human aldo-keto reductase 1C3 (AKR1C3) has confounded the development of this dual hypoxia/gene therapy prodrug. Previous attempts to design prodrugs resistant to AKR1C3 activation have resulted in candidates that require further optimization. Herein we report the evaluation of a lipophilic series of PR-104A analogues in which a piperazine moiety has been introduced to improve drug-like properties. Octanol-water partition coefficients (LogD7.4) spanned >2 orders of magnitude. 2D antiproliferative and 3D multicellular clonogenic assays using isogenic HCT116 and H1299 cells confirmed that all examples were resistant to AKR1C3 metabolism while producing an E. coli NfsA nitroreductase-mediated bystander effect. Prodrugs 16, 17, and 20 demonstrated efficacy in H1299 xenografts where only a minority of tumor cells express NfsA. These prodrugs and their bromo/mesylate counterparts (25-27) were also evaluated for hypoxia-selective cell killing in vitro. These results in conjunction with stability assays recommended prodrug 26 (CP-506) for Phase I/II clinical trial.

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.

Use of an optimised enzyme/prodrug combination for Clostridia directed enzyme prodrug therapy induces a significant growth delay in necrotic tumours.

Necrosis is a typical histological feature of solid tumours that provides a selective environment for growth of the non-pathogenic anaerobic bacterium Clostridium sporogenes. Modest anti-tumour activity as a single agent encouraged the use of C. sporogenes as a vector to express therapeutic genes selectively in tumour tissue, a concept termed Clostridium Directed Enzyme Prodrug Therapy (CDEPT). Here, we examine the ability of a recently identified Neisseria meningitidis type I nitroreductase (NmeNTR) to metabolise the prodrug PR-104A in an in vivo model of CDEPT. Human HCT116 colon cancer cells stably over-expressing NmeNTR demonstrated significant sensitivity to PR-104A, the imaging agent EF5, and several nitro(hetero)cyclic anti-infective compounds. Chemical induction of necrosis in human H1299 xenografts by the vascular disrupting agent vadimezan promoted colonisation by NmeNTR-expressing C. sporogenes, and efficacy studies demonstrated moderate but significant anti-tumour activity of spores when compared to untreated controls. Inclusion of the pre-prodrug PR-104 into the treatment schedule provided significant additional activity, indicating proof-of-principle. Successful preclinical evaluation of a transferable gene that enables metabolism of both PET imaging agents (for vector visualisation) and prodrugs (for conditional enhancement of efficacy) is an important step towards the prospect of CDEPT entering clinical evaluation.

Bioreductive prodrug PR-104 improves the tumour distribution and titre of the nitroreductase-armed oncolytic adenovirus ONYX-411NTR leading to therapeutic benefit.

Advances in the field of cancer immunotherapy have stimulated renewed interest in adenoviruses as oncolytic agents. Clinical experience has shown that oncolytic adenoviruses are safe and well tolerated but possess modest single-agent activity. One approach to improve the potency of oncolytic viruses is to utilise their tumour selectivity to deliver genes encoding prodrug-activating enzymes. These enzymes can convert prodrugs into cytotoxic species within the tumour; however, these cytotoxins can interfere with viral replication and limit utility. In this work, we evaluated the activity of a nitroreductase (NTR)-armed oncolytic adenovirus ONYX-411NTR in combination with the clinically tested bioreductive prodrug PR-104. Both NTR-expressing cells in vitro and xenografts containing a minor population of NTR-expressing cells were highly sensitive to PR-104. Pharmacologically relevant prodrug exposures did not interfere with ONYX-411NTR replication in vitro. In vivo, prodrug administration increased virus titre and improved virus distribution within tumour xenografts. Colonisation of tumours with high ONYX-411NTR titre resulted in NTR expression and prodrug activation. The combination of ONYX-411NTR with PR-104 was efficacious against HCT116 xenografts, whilst neither prodrug nor virus were active as single agents. This work highlights the potential for future clinical development of NTR-armed oncolytic viruses in combination with bioreductive prodrugs.

Restoring Tumour Selectivity of the Bioreductive Prodrug PR-104 by Developing an Analogue Resistant to Aerobic Metabolism by Human Aldo-Keto Reductase 1C3.

PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes 'off-target' two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues. AKR1C3 expression in human myeloid progenitor cells probably accounts for the dose-limiting myelotoxicity of PR-104 documented in clinical trials, resulting in human PR-104A plasma exposure levels 3.4- to 9.6-fold lower than can be achieved in murine models. Structure-based design to eliminate AKR1C3 activation thus represents a strategy for restoring the therapeutic window of this class of agent in humans. Here, we identified SN29176, a PR-104A analogue resistant to human AKR1C3 activation. SN29176 retains hypoxia selectivity in vitro with aerobic/hypoxic IC50 ratios of 9 to 145, remains a substrate for POR and triggers γH2AX induction and cell cycle arrest in a comparable manner to PR-104A. SN35141, the soluble phosphate pre-prodrug of SN29176, exhibited superior hypoxic tumour log cell kill (>4.0) to PR-104 (2.5-3.7) in vivo at doses predicted to be achievable in humans. Orthologues of human AKR1C3 from mouse, rat and dog were incapable of reducing PR-104A, thus identifying an underlying cause for the discrepancy in PR-104 tolerance in pre-clinical models versus humans. In contrast, the macaque AKR1C3 gene orthologue was able to metabolise PR-104A, indicating that this species may be suitable for evaluating the toxicokinetics of PR-104 analogues for clinical development. We confirmed that SN29176 was not a substrate for AKR1C3 orthologues across all four pre-clinical species, demonstrating that this prodrug analogue class is suitable for further development. Based on these findings, a prodrug candidate was subsequently identified for clinical trials.

Selectively Targeting Tumor Hypoxia With the Hypoxia-Activated Prodrug CP-506.

Hypoxia-activated prodrugs (HAP) are a promising class of antineoplastic agents that can selectively eliminate hypoxic tumor cells. This study evaluates the hypoxia-selectivity and antitumor activity of CP-506, a DNA alkylating HAP with favorable pharmacologic properties. Stoichiometry of reduction, one-electron affinity, and back-oxidation rate of CP-506 were characterized by fast-reaction radiolytic methods with observed parameters fulfilling requirements for oxygen-sensitive bioactivation. Net reduction, metabolism, and cytotoxicity of CP-506 were maximally inhibited at oxygen concentrations above 1 µmol/L (0.1% O2). CP-506 demonstrated cytotoxicity selectively in hypoxic 2D and 3D cell cultures with normoxic/anoxic IC50 ratios up to 203. Complete resistance to aerobic (two-electron) metabolism by aldo-keto reductase 1C3 was confirmed through gain-of-function studies while retention of hypoxic (one-electron) bioactivation by various diflavin oxidoreductases was also demonstrated. In vivo, the antitumor effects of CP-506 were selective for hypoxic tumor cells and causally related to tumor oxygenation. CP-506 effectively decreased the hypoxic fraction and inhibited growth of a wide range of hypoxic xenografts. A multivariate regression analysis revealed baseline tumor hypoxia and in vitro sensitivity to CP-506 were significantly correlated with treatment response. Our results demonstrate that CP-506 selectively targets hypoxic tumor cells and has broad antitumor activity. Our data indicate that tumor hypoxia and cellular sensitivity to CP-506 are strong determinants of the antitumor effects of CP-506.

Tumour Hypoxia-Mediated Immunosuppression: Mechanisms and Therapeutic Approaches to Improve Cancer Immunotherapy.

The magnitude of the host immune response can be regulated by either stimulatory or inhibitory immune checkpoint molecules. Receptor-ligand binding between inhibitory molecules is often exploited by tumours to suppress anti-tumour immune responses. Immune checkpoint inhibitors that block these inhibitory interactions can relieve T-cells from negative regulation, and have yielded remarkable activity in the clinic. Despite this success, clinical data reveal that durable responses are limited to a minority of patients and malignancies, indicating the presence of underlying resistance mechanisms. Accumulating evidence suggests that tumour hypoxia, a pervasive feature of many solid cancers, is a critical phenomenon involved in suppressing the anti-tumour immune response generated by checkpoint inhibitors. In this review, we discuss the mechanisms associated with hypoxia-mediate immunosuppression and focus on modulating tumour hypoxia as an approach to improve immunotherapy responsiveness.

Use of a Luciferase-Expressing Orthotopic Rat Brain Tumor Model to Optimize a Targeted Irradiation Strategy for Efficacy Testing with Temozolomide.

Glioblastoma multiforme (GBM) is a common and aggressive malignant brain cancer with a mean survival time of approximately 15 months after initial diagnosis. Currently, the standard-of-care (SOC) treatment for this disease consists of radiotherapy (RT) with concomitant and adjuvant temozolomide (TMZ). We sought to develop an orthotopic preclinical model of GBM and to optimize a protocol for non-invasive monitoring of tumor growth, allowing for determination of the efficacy of SOC therapy using a targeted RT strategy combined with TMZ. A strong correlation (r = 0.80) was observed between contrast-enhanced (CE)-CT-based volume quantification and bioluminescent (BLI)-integrated image intensity when monitoring tumor growth, allowing for BLI imaging as a substitute for CE-CT. An optimized parallel-opposed single-angle RT beam plan delivered on average 96% of the expected RT dose (20, 30 or 60 Gy) to the tumor. Normal tissue on the ipsilateral and contralateral sides of the brain were spared 84% and 99% of the expected dose, respectively. An increase in median survival time was demonstrated for all SOC regimens compared to untreated controls (average 5.2 days, p < 0.05), but treatment was not curative, suggesting the need for novel treatment options to increase therapeutic efficacy.

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.

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.

Advancing Clostridia to Clinical Trial: Past Lessons and Recent Progress.

Most solid cancers contain regions of necrotic tissue. The extent of necrosis is associated with poor survival, most likely because it reflects aggressive tumour outgrowth and inflammation. Intravenously injected spores of anaerobic bacteria from the genus Clostridium infiltrate and selectively germinate in these necrotic regions, providing cancer-specific colonisation. The specificity of this system was first demonstrated over 60 years ago and evidence of colonisation has been confirmed in multiple tumour models. The use of "armed" clostridia, such as in Clostridium Directed Enzyme Prodrug Therapy (CDEPT), may help to overcome some of the described deficiencies of using wild-type clostridia for treatment of cancer, such as tumour regrowth from a well-vascularised outer rim of viable cells. Successful preclinical evaluation of a transferable gene that metabolises both clinical stage positron emission tomography (PET) imaging agents (for whole body vector visualisation) as well as chemotherapy prodrugs (for conditional enhancement of efficacy) would be a valuable early step towards the prospect of "armed" clostridia entering clinical evaluation. The ability to target the immunosuppressive hypoxic tumour microenvironment using CDEPT may offer potential for synergy with recently developed immunotherapy strategies. Ultimately, clostridia may be most efficacious when combined with conventional therapies, such as radiotherapy, that sterilise viable aerobic tumour cells.

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.

Bioreductive prodrugs as cancer therapeutics: targeting tumor hypoxia.

Hypoxia, a state of low oxygen, is a common feature of solid tumors and is associated with disease progression as well as resistance to radiotherapy and certain chemotherapeutic drugs. Hypoxic regions in tumors, therefore, represent attractive targets for cancer therapy. To date, five distinct classes of bioreactive prodrugs have been developed to target hypoxic cells in solid tumors. These hypoxia-activated prodrugs, including nitro compounds, N-oxides, quinones, and metal complexes, generally share a common mechanism of activation whereby they are reduced by intracellular oxidoreductases in an oxygen-sensitive manner to form cytotoxins. Several examples including PR-104, TH-302, and EO9 are currently undergoing phase II and phase III clinical evaluation. In this review, we discuss the nature of tumor hypoxia as a therapeutic target, focusing on the development of bioreductive prodrugs. We also describe the current knowledge of how each prodrug class is activated and detail the clinical progress of leading examples.

Pseudomonas aeruginosa NfsB and nitro-CBI-DEI--a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy.

BACKGROUND: The nitro-chloromethylbenzindoline prodrug nitro-CBI-DEI appears a promising candidate for the anti-cancer strategy gene-directed enzyme prodrug therapy, based on its ability to be converted to a highly cytotoxic cell-permeable derivative by the nitroreductase NfsB from Escherichia coli. However, relative to some other nitroaromatic prodrugs, nitro-CBI-DEI is a poor substrate for E. coli NfsB. To address this limitation we evaluated other nitroreductase candidates from E. coli and Pseudomonas aeruginosa. FINDINGS: Initial screens of candidate genes in the E. coli reporter strain SOS-R2 identified two additional nitroreductases, E. coli NfsA and P. aeruginosa NfsB, as being more effective activators of nitro-CBI-DEI than E. coli NfsB. In monolayer cytotoxicity assays, human colon carcinoma (HCT-116) cells transfected with P. aeruginosa NfsB were >4.5-fold more sensitive to nitro-CBI-DEI than cells expressing either E. coli enzyme, and 23.5-fold more sensitive than untransfected HCT-116. In three dimensional mixed cell cultures, not only were the P. aeruginosa NfsB expressing cells 540-fold more sensitive to nitro-CBI-DEI than pure cultures of untransfected HCT-116, the activated drug that they generated also displayed an unprecedented local bystander effect. CONCLUSION: We posit that the discrepancy in the fold-sensitivity to nitro-CBI-DEI between the two and three dimensional cytotoxicity assays stems from loss of activated drug into the media in the monolayer cultures. This emphasises the importance of evaluating high-bystander GDEPT prodrugs in three dimensional models. The high cytotoxicity and bystander effect exhibited by the NfsB_Pa/nitro-CBI-DEI combination suggest that further preclinical development of this GDEPT pairing is warranted.

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.

Molecular and cellular pharmacology of the hypoxia-activated prodrug TH-302.

TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxia-selective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH:cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of γH2AX phosphorylation, DNA cross-linking, and cell-cycle arrest. We used Chinese hamster ovary cell-based DNA repair mutant cell lines and established that lines deficient in homology-dependent repair, but not lines deficient in base excision, nucleotide excision, or nonhomologous end-joining repair, exhibited marked sensitivity to TH-302 under hypoxia. Consistent with this finding, enhanced sensitivity to TH-302 was also observed in lines deficient in BRCA1, BRCA2, and FANCA. Finally, we characterized TH-302 activity in the three-dimensional tumor spheroid and multicellular layer models. TH-302 showed much enhanced potency in H460 spheroids compared with H460 monolayer cells under normoxia. Multicellular layers composed of mixtures of parental HCT116 cells and HCT116 cells engineered to express an oxygen-insensitive bacterial nitroreductase showed that TH-302 exhibits a significant bystander effect.