The YfkO Nitroreductase from Bacillus Licheniformis on Gold-Coated Superparamagnetic Nanoparticles: Towards a Novel Directed Enzyme Prodrug Therapy Approach.
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SUBMITTER: Ball P
PROVIDER: S-EPMC8070144 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:As one targeting strategy of prodrug delivery, gene-directed enzyme prodrug therapy (GDEPT) promises to realize the targeting through its three key features in cancer therapy-cell-specific gene delivery and expression, controlled conversion of prodrugs to drugs in target cells, and expanded toxicity to the target cells' neighbors through bystander effects. After over 20 years of development, multiple GDEPT systems have advanced into clinical trials. However, no GDEPT product is currently marketed as a drug, suggesting that there are still barriers to overcome before GDEPT becomes a standard therapy. In this review, we first provide a general introduction of this prodrug targeting strategy. Then, we utilize the four most thoroughly studied systems to illustrate components, mechanisms, preclinical and clinical results, and further development directions of GDEPT. These four systems are herpes simplex virus thymidine kinase/ganciclovir, cytosine deaminase/5-fluorocytosine, cytochrome P450/oxazaphosphorines, and nitroreductase/CB1954 system. Later, we focus our discussion on bystander effects including local and distant bystander effects. Lastly, we discuss carriers that are used to deliver genes for GDEPT including virus carriers and non-virus carriers. Among these carriers, the stem cell-based gene delivery system represents one of the newest carriers under development, and may brought about a breakthrough to the gene delivery issue of GDEPT.
| S-EPMC4287286 | biostudies-literature
Project description:Nitroreductases (NTR) are a family of bacterial enzymes used in gene directed enzyme prodrug therapy (GDEPT) that selectively activate prodrugs containing aromatic nitro groups to exert cytotoxic effects following gene transduction in tumours. The clinical development of NTR-based GDEPT has, in part, been hampered by the lack of translational imaging modalities to assess gene transduction and drug cytotoxicity, non-invasively. This study presents translational preclinical PET imaging to validate and report NTR activity using the clinically approved radiotracer, 18F-FMISO, as substrate for the NTR enzyme. Methods: The efficacy with which 18F-FMISO could be used to report NfsB NTR activity in vivo was investigated using the MDA-MB-231 mammary carcinoma xenograft model. For validation, subcutaneous xenografts of cells constitutively expressing NTR were imaged using 18F-FMISO PET/CT and fluorescence imaging with CytoCy5S, a validated fluorescent NTR substrate. Further, examination of the non-invasive functionality of 18F-FMISO PET/CT in reporting NfsB NTR activity in vivo was assessed in metastatic orthotopic NfsB NTR expressing xenografts and metastasis confirmed by bioluminescence imaging. 18F-FMISO biodistribution was acquired ex vivo by an automatic gamma counter measuring radiotracer retention to confirm in vivo results. To assess the functional imaging of NTR-based GDEPT with 18F-FMISO, PET/CT was performed to assess both gene transduction and cytotoxicity effects of prodrug therapy (CB1954) in subcutaneous models. Results: 18F-FMISO retention was detected in NTR+ subcutaneous xenografts, displaying significantly higher PET contrast than NTR- xenografts (p < 0.0001). Substantial 18F-FMISO retention was evident in metastases of orthotopic xenografts (p < 0.05). Accordingly, higher 18F-FMISO biodistribution was prevalent ex vivo in NTR+ xenografts. 18F-FMISO NfsB NTR PET/CT imaging proved useful for monitoring in vivo NTR transduction and the cytotoxic effect of prodrug therapy. Conclusions: 18F-FMISO NfsB NTR PET/CT imaging offered significant contrast between NTR+ and NTR- tumours and effective resolution of metastatic progression. Furthermore, 18F-FMISO NfsB NTR PET/CT imaging proved efficient in monitoring the two steps of GDEPT, in vivo NfsB NTR transduction and response to CB1954 prodrug therapy. These results support the repurposing of 18F-FMISO as a readily implementable PET imaging probe to be employed as companion diagnostic test for NTR-based GDEPT systems.
| S-EPMC8058731 | biostudies-literature
Project description:BackgroundEngineered inorganic nanoparticles (NPs) are essential components in the development of nanotechnologies. For applications in nanomedicine, particles need to be functionalized to ensure a good dispersibility in biological fluids. In many cases however, functionalization is not sufficient: the particles become either coated by a corona of serum proteins or precipitate out of the solvent. We show that by changing the coating of magnetic iron oxide NPs using poly-L-lysine (PLL) polymer the colloidal stability of the dispersion is improved in aqueous solutions including water, phosphate buffered saline (PBS), PBS with 10% fetal bovine serum (FBS) and cell culture medium, and the internalization of the NPs toward living mammalian cells is profoundly affected.MethodsA multifunctional magnetic NP is designed to perform a near-infrared (NIR)-responsive remote control photothermal ablation for the treatment of breast cancer. In contrast to the previously reported studies of gold (Au) magnetic (Fe3O4) core-shell NPs, a Janus-like nanostructure is synthesized with Fe3O4 NPs decorated with Au resulting in an approximate size of 60 nm mean diameter. The surface of trisoctahedral Au-Fe3O4 NPs was coated with a positively charged polymer, PLL to deliver the NPs inside cells. The PLL-Au-Fe3O4 NPs were characterized by transmission electron microscopy (TEM), XRD, FT-IR and dynamic light scattering (DLS). The unique properties of both Au surface plasmon resonance and superparamagnetic moment result in a multimodal platform for use as a nanothermal ablator and also as a magnetic resonance imaging (MRI) contrast agent, respectively. Taking advantage of the photothermal therapy, PLL-Au-Fe3O4 NPs were incubated with BT-474 and MDA-MB-231 breast cancer cells, investigated for the cytotoxicity and intracellular uptake, and remotely triggered by a NIR laser of ~ 808 nm (1 W/cm2 for 10 min).ResultsThe PLL coating increased the colloidal stability and robustness of Au-Fe3O4 NPs (PLL-Au-Fe3O4) in biological media including cell culture medium, PBS and PBS with 10% fetal bovine serum. It is revealed that no significant (< 10%) cytotoxicity was induced by PLL-Au-Fe3O4 NPs itself in BT-474 and MDA-MB-231 cells at concentrations up to 100 μg/ml. Brightfield microscopy, fluorescence microscopy and TEM showed significant uptake of PLL-Au-Fe3O4 NPs by BT-474 and MDA-MB-231 cells. The cells exhibited 40 and 60% inhibition in BT-474 and MDA-MB-231 cell growth, respectively following the internalized NPs were triggered by a photothermal laser using 100 μg/ml PLL-Au-Fe3O4 NPs. The control cells treated with NPs but without laser showed < 10% cell death compared to no laser treatment control CONCLUSION: Combined together, the results demonstrate a new polymer gold superparamagnetic nanostructure that integrates both diagnostics function and photothermal ablation of tumors into a single multimodal nanoplatform exhibiting a significant cancer cell death.
| S-EPMC6186064 | biostudies-literature
Project description:Targeted drug delivery to selected sites allows reduced toxicity, enhanced efficiency and interchangeable target potential [Langer, R. (2001) Science 293, 58-59 and Molema, G. & Meijer, D. K. F., eds. (2001) Drug Targeting (Wiley-VCH, Weinheim, Germany)]. We describe a bipartite drug-delivery system that exploits (I) endogenous carbohydrate-to-lectin binding to localize glycosylated enzyme conjugates to specific, predetermined cell types followed by (II) administration of a prodrug activated by that predelivered enzyme at the desired site. The carbohydrate structure of an alpha-L-rhamnopyranosidase enzyme was specifically engineered through enzymatic deglycosylation and chemical reglycosylation. Combined in vivo and in vitro techniques (gamma scintigraphy, microautoradiography and confocal microscopy) determined organ and cellular localization and demonstrated successful activation of alpha-L-rhamnopyranoside prodrug. Ligand competition experiments revealed enhanced, specific localization by endocytosis and a strongly carbohydrate-dependent, 60-fold increase in selectivity toward target cell hepatocytes that generated a >30-fold increase (from 0.02 to 0.66 mg) in protein delivered. Furthermore, glycosylation engineering enhanced the serum-uptake rate and enzyme stability. This created enzyme activity (0.2 units in hepatocytes) for prodrug therapy, the target of which was switched simply by sugar-type alteration. The therapeutic effectiveness of lectin-directed enzyme-activated prodrug therapy was shown through the construction of the prodrug of doxorubicin, Rha-DOX, and its application to reduce tumor burden in a hepatocellular carcinoma (HepG2) disease model.
| S-EPMC521935 | biostudies-literature
Project description: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.
| S-EPMC3795375 | biostudies-literature
Project description:Cytosine deaminase (CDA) is a non-mammalian enzyme with powerful activity in mediating the prodrug 5-fluorcytosine (5-FC) into toxic drug 5-fluorouracil (5-FU), as an alternative directed approach for the traditional chemotherapies and radiotherapies of cancer. This enzyme has been frequently reported and characterized from various microorganisms. The therapeutic strategy of 5-FC-CDA involves the administration of CDA followed by the prodrug 5-FC injection to generate cytotoxic 5-FU. The antiproliferative activity of CDA-5-FC elaborates from the higher activity of uracil pathway in tumor cells than normal ones. The main challenge of the therapeutic drug 5-FU are the short half-life, lack of selectivity and emergence of the drug resistance, consistently to the other chemotherapies. So, mediating the 5-FU to the tumor cells by CDA is one of the most feasible approaches to direct the drug to the tumor cells, reducing its toxic effects and improving their pharmacokinetic properties. Nevertheless, the catalytic efficiency, stability, antigenicity and targetability of CDA-5-FC, are the major challenges that limit the clinical application of this approach. Thus, exploring the biochemical properties of CDA from various microorganisms, as well as the approaches for localizing the system of CDA-5-FC to the tumor cells via the antibody directed enzyme prodrug therapy (ADEPT) and gene directed prodrug therapy (GDEPT) were the objectives of this review. Finally, the perspectives for increasing the therapeutic efficacy, and targetability of the CDA-5-FC system were described.
| S-EPMC9508425 | biostudies-literature
Project description:Au- and iron-based magnetic nanoparticles (NPs) are promising NPs for biomedical applications due to their unique properties. The combination of a gold coating over a magnetic core puts together the benefits from adding the magnetic properties to the robust chemistry provided by the thiol functionalization of gold. Here, the use of Au-coated magnetic NPs for molecular detection of a single methylation in DNA aptamer is described. Binding of ?-thrombin to two aptamers conjugated to these NPs causes aggregation, a phenomenon that can be observed by UV, DLS and MRI. These techniques discriminate a single methylation in one of the aptamers, preventing aggregation due to the inability of ?-thrombin to recognize it. A parallel study with gold and ferromagnetic NPs is detailed, concluding that the Au coating of FexOy NP does not affect their performance and that they are suitable as complex biosensors. These results prove the high detection potency of Au-coated SPIONs for biomedical applications especially for DNA repair detection.
| S-EPMC4661904 | biostudies-other
Project description:BackgroundIndustrial fermentations can generally be described as dynamic biotransformation processes in which microorganisms convert energy rich substrates into a desired product. The knowledge of active physiological pathways, reflected by corresponding gene activities, allows the identification of beneficial or disadvantageous performances of the microbial host. Whole transcriptome RNA-Seq is a powerful tool to accomplish in-depth quantification of these gene activities, since the low background noise and the absence of an upper limit of quantification allow the detection of transcripts with high dynamic ranges. Such data enable the identification of potential bottlenecks and futile energetic cycles, which in turn can lead to targets for rational approaches to productivity improvement. Here we present an overview of the dynamics of gene activity during an industrial-oriented fermentation process with Bacillus licheniformis, an important industrial enzyme producer. Thereby, valuable insights which help to understand the complex interactions during such processes are provided.ResultsWhole transcriptome RNA-Seq has been performed to study the gene expression at five selected growth stages of an industrial-oriented protease production process employing a germination deficient derivative of B. licheniformis DSM13. Since a significant amount of genes in Bacillus strains are regulated posttranscriptionally, the generated data have been confirmed by 2D gel-based proteomics. Regulatory events affecting the coordinated activity of hundreds of genes have been analyzed. The data enabled the identification of genes involved in the adaptations to changing environmental conditions during the fermentation process. A special focus of the analyses was on genes contributing to central carbon metabolism, amino acid transport and metabolism, starvation and stress responses and protein secretion. Genes contributing to lantibiotics production and Tat-dependent protein secretion have been pointed out as potential optimization targets.ConclusionsThe presented data give unprecedented insights into the complex adaptations of bacterial production strains to the changing physiological demands during an industrial-oriented fermentation. These are, to our knowledge, the first publicly available data that document quantifiable transcriptional responses of the commonly employed production strain B. licheniformis to changing conditions over the course of a typical fermentation process in such extensive depth.
| S-EPMC3878961 | biostudies-literature
Project description: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.
| S-EPMC3695803 | biostudies-literature
Project description:Bacterial nitroreductase enzymes that can efficiently convert nitroaromatic prodrugs to a cytotoxic form have numerous applications in targeted cellular ablation. For example, the generation of cytotoxic metabolites that have low bystander potential (i.e., are largely confined to the activating cell) has been exploited for precise ablation of specific cell types in animal and cell-culture models; while enzyme-prodrug combinations that generate high levels of bystander cell killing are useful for anti-cancer strategies such as gene-directed enzyme-prodrug therapy (GDEPT). Despite receiving substantial attention for such applications, the canonical nitroreductase NfsB from Escherichia coli has flaws that limit its utility, in particular a low efficiency of conversion of most prodrugs. Here, we sought to engineer a superior broad-range nitroreductase, E. coli NfsA, for improved activity with three therapeutically-relevant prodrugs: the duocarmycin analogue nitro-CBI-DEI, the dinitrobenzamide aziridine CB1954 and the 5-nitroimidazole metronidazole. The former two prodrugs have applications in GDEPT, while the latter has been employed for targeted ablation experiments and as a precise 'off-switch' in GDEPT models to eliminate nitroreductase-expressing cells. Our lead engineered NfsA (variant 11_78, with the residue substitutions S41Y, L103M, K222E and R225A) generated reduced metabolites of CB1954 and nitro-CBI-DEI that exhibited high bystander efficiencies in both bacterial and 2D HEK-293 cell culture models, while no cell-to-cell transfer was evident for the reduced metronidazole metabolite. We showed that the high bystander efficiency for CB1954 could be attributed to near-exclusive generation of the 2-hydroxylamine reduction product, which has been shown in 3D cell culture to cause significantly greater bystander killing than the 4-hydroxylamine species that is also produced by NfsB. We similarly observed a high bystander effect for nitro-CBI-DEI in HCT-116 tumor spheroids in which only a small proportion of cells were expressing variant 11_78. Collectively, our data identify variant 11_78 as a broadly improved prodrug-activating nitroreductase that offers advantages for both targeted cellular ablation and suicide gene therapy applications.
| S-EPMC8215503 | biostudies-literature