Janine N. Copp

Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954

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.

Targeted mutagenesis of the Vibrio fischeri flavin reductase FRase I to improve activation of the anticancer prodrug CB1954

Phase I/II cancer gene therapy trials of the Escherichia coli nitroreductase NfsB in partnership with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] have indicated that CB1954 toxicity is dose-limiting at concentrations far below the enzyme K(M). Here we report that the flavin reductase FRase I from Vibrio fischeri is also a CB1954 nitroreductase, which has a substantially lower apparent K(M) than E. coli NfsB. To enhance the activity of FRase I with CB1954 we used targeted mutagenesis and an E. coli SOS reporter strain to engineer single- and multi-residue variants that possess a substantially reduced apparent K(M) and an increased k(cat)/K(M) relative to the wild type enzyme. In a bacteria-delivered model for enzyme prodrug therapy, the engineered FRase I variants were able to kill human colon carcinoma (HCT-116) cells at significantly lower CB1954 concentrations than wild type FRase I or E. coli NfsB.

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.

Rapid and flexible biochemical assays for evaluating 4'-phosphopantetheinyl transferase activity.

PPTases (phosphopantetheinyl transferases) are of great interest owing to their essential roles in activating fatty acid, polyketide and non-ribosomal peptide synthetase enzymes for both primary and secondary metabolism, as well as an increasing number of biotechnological applications. However, existing techniques for PPTase characterization and development are cumbersome and technically challenging. To address this, we have developed the indigoidine-synthesizing non-ribosomal peptide synthetase BpsA as a reporter for PPTase activity. Simple co-transformation allows rapid assessment of the ability of a PPTase candidate to activate BpsA in vivo. Kinetic parameters with respect to either CoA or BpsA as variable substrate can then be derived in vitro by continuously measuring the rate of indigoidine synthesis as the PPTase progressively converts BpsA from its apo into holo form. Subsequently, a competition assay, in which BpsA and purified carrier proteins compete for a limited pool of CoA, enables elucidation of kinetic parameters for a PPTase with those carrier proteins. We used this system to conduct a rapid characterization of three different PPTase enzymes: Sfp of Bacillus subtilis A.T.C.C.6633, PcpS of Pseudomonas aeruginosa PAO1, and the putative PPTase PP1183 of Ps. putida KT2440. We also demonstrate the utility of this system for discovery and characterization of PPTase inhibitors.

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.

Directed Evolution Library Creation: methods and protocols

Part I. Point Mutagenesis (i) Random Mutagenesis Methods 1. Error-Prone PCR and Effective Generation of Gene Variant Libraries for Directed Evolution Janine N. Copp, Paulina Hanson-Manful, David F. Ackerley, and Wayne M. Patrick 2. Error-Prone Rolling Circle Amplification Greatly Simplifies Random Mutagenesis Ryota Fujii, Motomitsu Kitaoka, and Kiyoshi Hayashi 3. Random Mutagenesis by Error-Prone Pol Plasmid Replication in Escherichia coli David L. Alexander, Joshua Lilly, Jaime Hernandez, Jillian Romsdahl, Christopher J. Troll, and Manel Camps 4. The Sequence Saturation Mutagenesis (SeSaM) Method Anna Joelle Ruff, Tsvetan Kardashliev, Alexander Dennig, and Ulrich Schwaneberg 5. Generation of Effective Libraries by Neutral Drift Miriam Kaltenbach and Nobuhiko Tokuriki (ii) Saturation Mutagenesis Methods 6. Site Saturation Mutagenesis Elsie M. Williams, Janine N. Copp, and David F. Ackerley 7. Iterative Saturation Mutagenesis (ISM): A Powerful Approach to Engineer Proteins by Systematically Simulating Darwinian Evolution Carlos G. Acevedo-Rocha, Sabrina Killeand, and Manfred T. Reetz 8. Generating Targeted Libraries by the Combinatorial Incorporation of Synthetic Oligonucleotides During Gene Shuffling (ISOR) Liat Rockah-Shmuel, Dan S Tawfik, and Moshe Goldsmith 9. Omni Change: Simultaneous Site-Saturation of up to Five Codons Alexander Dennig, Jan Marienhagen, Anna Joelle Ruff, and Ulrich Schwaneberg (iii) Insertional/Deletional Methods 10. Random Insertional-Deletional Strand Exchange Mutagenesis (RAISE): A Simple Method for Generating Random Insertion and Deletion Mutations Ryota Fujii, Motomitsu Kitaoka, and Kiyoshi Hayashi 11. Transposon-Based Approaches for Generating Novel Molecular Diversity during Directed Evolution Dafydd Jones, James A.J. Arpino, Amy J. Baldwin, and Matthew C. Edmundson Part II. Recombinatorial Methods (i)Homology-Dependent Methods 12. Restriction Enzyme-Mediated DNA Family Shuffling James B.Y.H. Behrendorff, Wayne A. Johnston, and Elizabeth M. J. Gillam 13. Assembly of Designed Oligonucleotides (ADO): A Useful Tool in Synthetic Biology for Creating High Quality Combinatorial DNA Libraries Carlos G. Acevedo-Rocha and Manfred T. Reetz 14. One-Pot, Simple Methodology for Cassette Randomization and Recombination for Focused Directed Evolution (OSCARR) Aurelio Hidalgo Anna Schliessmann, and Uwe T. Bornscheuer 15. USER Friendly DNA Recombination (USERec): Gene Library Construction Requiring Minimal Sequence Homology Benoit Villiers and Florian Hollfelder (ii) Homology-Independent Methods 16. ITCHY: Incremental Truncation for the Creation of Hybrid Enzymes Wayne M. Patrick and Monica L. Gerth 17. Generating Random Circular Permutation Libraries Stefan Lutz, Ashley B. Daugherty, Ying Yu, and Zhen Qian Part III. Structure Guided Methods and Computational Tools for the Design and Analysis of Mutant Libraries 18. Probabilistic Methods in Directed Evolution: Library Size, Mutation Rate, and Diversity Yuval Nov 19. The Mutagenesis Assistant Program Rajni Verma, Tuck Seng Wong, Ulrich Schwaneberg, and Danilo Roccatano 20. Computational Tools for Designing Smart Libraries Eva Sebestova, Jaroslav Bendl, Jan Brezovsky, and Jiri Damborsky 21. Computational Tools for Directed Evolution: A Comparison of Prospective and Retrospective Strategies Julian Zaugg, Yosephine Gumulya, Elizabeth M.J. Gillam, and Mikael Boden 22. Designing Libraries of Chimeric Proteins Using SCHEMA Recombination and RASPP Matthew A. Smith & Frances H. Arnold 23. Non-Contiguous SCHEMA Protein Recombination Matthew A. Smith and Frances H. Arnold 24. Engineering Proteins by Reconstructing Evolutionary Adaptive Paths Vanessa E. Cox and Eric A. Gaucher

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

&NA; 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.

Abstract B88: Discovery, characterization, and engineering of bacterial nitroreductases for gene-directed enzyme prodrug therapy.

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 NfsB9s K m . 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/m 2 vs 24 mg/m 2 ; 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 K m 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.

Abstract B89: Molecular imaging using bacterial nitroreductase reporter genes by repurposing the clinical stage hypoxia PET probe EF5.

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 ( 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.