RESUMO
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.
RESUMO
Directed enzyme prodrug therapy is a highly promising anti-cancer strategy. However, the current technology is limited by inefficient prodrug activation and the dose-limiting toxicity associated with the prodrugs being tested; to overcome these limitations, the dinitrobenzamide mustard prodrugs, PR-104A and SN27686, have been developed. The present study will assess both of these prodrugs for their potential uses in a novel magnetic-nanoparticle directed enzyme prodrug therapy strategy by determining their kinetic parameters, assessing the products formed during enzymatic reduction using HPLC and finally their ability to cause cell death in the ovarian cancer cell line, SK-OV-3. It was shown for the first time that the dinitrobenzamide mustard prodrugs are able to be reduced by the genetically modified nitroreductases, NfnB-cys and YfkO-cys, and that these enzyme/prodrug combinations can induce a significant cell death in the SK-OV-3 cell line, highlighting the potential for both enzyme/prodrug combinations for use in magnetic-nanoparticle directed enzyme prodrug therapy.
Assuntos
Antineoplásicos , Neoplasias , Compostos de Mostarda Nitrogenada , Pró-Fármacos , Humanos , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Compostos de Mostarda Nitrogenada/metabolismo , Antineoplásicos Alquilantes , Antineoplásicos/metabolismoRESUMO
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.
RESUMO
Hypoxia-activated prodrugs are bioactivated in oxygen-deficient tumour regions and represent a novel strategy to exploit this pharmacological sanctuary for therapeutic gain. The approach relies on the selective metabolism of the prodrug under pathological hypoxia to generate active metabolites with the potential to diffuse throughout the tumour microenvironment and potentiate cell killing by means of a "bystander effect". In the present study, we investigate the pharmacological properties of the nitrogen mustard prodrug CP-506 in tumour tissues using in silico spatially-resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) modelling. The approach employs a number of experimental model systems to define parameters for the cellular uptake, metabolism and diffusion of both the prodrug and its metabolites. The model predicts rapid uptake of CP-506 to high intracellular concentrations with its long plasma half-life driving tissue diffusion to a penetration depth of 190 µm, deep within hypoxic activating regions. While bioreductive metabolism is restricted to regions of severe pathological hypoxia (<1 µM O2), its active metabolites show substantial bystander potential with release from the cell of origin into the extracellular space. Model predictions of bystander efficiency were validated using spheroid co-cultures, where the clonogenic killing of metabolically defective "target" cells increased with the proportion of metabolically competent "activator" cells. Our simulations predict a striking bystander efficiency at tissue-like densities with the bis-chloro-mustard amine metabolite (CP-506M-Cl2) identified as a major diffusible metabolite. Overall, this study shows that CP-506 has favourable pharmacological properties in tumour tissue and supports its ongoing development for use in the treatment of patients with advanced solid malignancies.
RESUMO
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.
Assuntos
Aziridinas , Neoplasias , Terapia Viral Oncolítica , Pró-Fármacos , Adenoviridae , Aziridinas/farmacologia , Aziridinas/uso terapêutico , Humanos , Neoplasias/terapia , Compostos de Mostarda Nitrogenada , Nitrorredutases/genética , Nitrorredutases/metabolismo , Vírus Oncolíticos , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêuticoRESUMO
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.
Assuntos
Pró-Fármacos , Composição de Bases , Clostridium/genética , Clostridium/metabolismo , Humanos , Filogenia , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , RNA Ribossômico 16S , Análise de Sequência de DNARESUMO
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.
RESUMO
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.
Assuntos
Pró-Fármacos/uso terapêutico , Hipóxia Tumoral/efeitos dos fármacos , Animais , Humanos , Camundongos , Pró-Fármacos/farmacologiaRESUMO
OBJECTIVES: To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954. RESULTS: Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitize E. coli host cells to at least 2.4-fold lower concentrations of SN33623 than the native enzyme. Two of these variants were able to be purified in a functional form, and in vitro assays revealed these were twofold and fourfold improved in kcat/KM with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in kcat/KM versus wild type YfkO in converting CB1954 to a genotoxic drug. CONCLUSIONS: The enhanced activation of the PET imaging probe SN33623 and CB1954 prodrug exhibited by the lead evolved variant of YfkO offers prospects for improved enzyme-prodrug therapy.
Assuntos
Bacillus subtilis , Proteínas de Bactérias/genética , Evolução Molecular Direcionada/métodos , Nitroimidazóis/metabolismo , Nitrorredutases/genética , Antineoplásicos/metabolismo , Aziridinas/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Terapia Enzimática , Nitrorredutases/metabolismoRESUMO
The use of reporter genes to non-invasively image molecular processes inside cells has significant translational potential, particularly in the context of systemically administered gene therapy vectors and adoptively administered cells such as immune or stem cell based therapies. Bacterial nitroreductase enzymes possess ideal properties for reporter gene imaging applications, being of non-human origin and possessing the ability to metabolize a range of clinically relevant nitro(hetero)cyclic substrates. Methods: A library of eleven Escherichia coli nitroreductase candidates were screened for the ability to efficiently metabolize 2-nitroimidazole based positron emission tomography (PET) probes originally developed as radiotracers for hypoxic cell imaging. Several complementary methods were utilized to detect formation of cell-entrapped metabolites, including various in vitro and in vivo models to establish the capacity of the 2-nitroimidazole PET agent EF5 to quantify expression of a nitroreductase candidate. Proof-of-principle PET imaging studies were successfully conducted using 18F-HX4. Results: Recombinant enzyme kinetics, bacterial SOS reporter assays, anti-proliferative assays and flow cytometry approaches collectively identified the major oxygen-insensitive nitroreductase NfsA from E. coli (NfsA_Ec) as the most promising nitroreductase reporter gene. Cells expressing NfsA_Ec were demonstrably labelled with the imaging agent EF5 in a manner that was quantitatively superior to hypoxia, in monolayers (2D), multicellular layers (3D), and in human tumor xenograft models. EF5 retention correlated with NfsA_Ec positive cell density over a range of EF5 concentrations in 3D in vitro models and in xenografts in vivo and was predictive of in vivo anti-tumor activity of the cytotoxic prodrug PR-104. Following PET imaging with 18F-HX4, a significantly higher tumor-to-blood ratio was observed in two xenograft models for NfsA_Ec expressing tumors compared to the parental tumors thereof, providing verification of this reporter gene imaging approach. Conclusion: This study establishes that the bacterial nitroreductase NfsA_Ec can be utilized as an imaging capable reporter gene, with the ability to metabolize and trap 2-nitroimidazole PET imaging agents for non-invasive imaging of gene expression.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Proteínas de Escherichia coli/administração & dosagem , Genes Reporter , Neoplasias/diagnóstico por imagem , Nitrorredutases/administração & dosagem , Tomografia por Emissão de Pósitrons/métodos , Animais , Antineoplásicos Alquilantes/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Proteínas de Escherichia coli/genética , Etanidazol/administração & dosagem , Etanidazol/análogos & derivados , Etanidazol/farmacocinética , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/farmacocinética , Células HCT116 , Humanos , Hidrocarbonetos Fluorados/administração & dosagem , Hidrocarbonetos Fluorados/farmacocinética , Imidazóis/administração & dosagem , Indicadores e Reagentes/administração & dosagem , Indicadores e Reagentes/farmacocinética , Camundongos , Imagem Molecular/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Compostos de Mostarda Nitrogenada/farmacologia , Compostos de Mostarda Nitrogenada/uso terapêutico , Nitrorredutases/genética , Medicina de Precisão/métodos , Estudo de Prova de Conceito , Compostos Radiofarmacêuticos/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Triazóis/administração & dosagem , Hipóxia Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Monitoramento de Medicamentos/métodos , Proteínas de Escherichia coli/metabolismo , Etanidazol/análogos & derivados , Hidrocarbonetos Fluorados/metabolismo , Imagem Molecular/métodos , Nitroimidazóis/uso terapêutico , Nitrorredutases/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Pró-Fármacos/uso terapêutico , Antineoplásicos/uso terapêutico , Técnicas Biossensoriais/métodos , Hipóxia Celular/fisiologia , Ativação Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Etanidazol/química , Etanidazol/metabolismo , Terapia Genética/métodos , Células HCT116 , Humanos , Hidrocarbonetos Fluorados/química , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Nitroimidazóis/farmacologia , Nitrorredutases/genética , Pró-Fármacos/metabolismo , Engenharia de ProteínasRESUMO
A series of 2-oxo-3, 4-dihydropyrimido[4,5-d]-pyrimidinyl derivatives were designed and synthesized as new irreversible inhibitors of the FGFR family. One of the most promising compounds 2l potently inhibited FGFR1/2/3 with IC50 values of 1.06, 0.84 and 5.38 nM, respectively, whereas its potency against FGFR4 was diminished by an order of magnitude. Compound 2l strongly suppresses the proliferation of FGFR1-amplified H520 non-small cell lung cancer cells, FGFR2-amplified SUM52 breast cancer cells and FGFR3-amplified SW780 bladder cancer cells with low nanomolar IC50 values, but was significantly less potent against four FGFR-negative cancer cell lines, with low micromolar IC50 values. Biological investigation also confirmed the irreversible binding of the molecule with the FGFR1-3 target kinases. Compound 2l may serve as a promising new lead for further anticancer drug discovery.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Proteínas de Escherichia coli/metabolismo , Neoplasias/terapia , Compostos de Mostarda Nitrogenada/metabolismo , Nitrorredutases/metabolismo , Pró-Fármacos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Etanidazol/análogos & derivados , Etanidazol/química , Etanidazol/metabolismo , Células HCT116 , Humanos , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Concentração Inibidora 50 , Metronidazol/química , Metronidazol/metabolismo , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Neoplasias/diagnóstico , Neoplasias/patologia , Compostos de Mostarda Nitrogenada/química , Nitrorredutases/química , Nitrorredutases/genética , Tomografia por Emissão de Pósitrons , Pró-Fármacos/química , Estrutura Terciária de Proteína , Especificidade por Substrato , Triazóis/química , Triazóis/metabolismoRESUMO
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.
Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Antineoplásicos Alquilantes/uso terapêutico , Benzamidas/uso terapêutico , Carcinoma/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Desenho de Fármacos , Hidroxiprostaglandina Desidrogenases/metabolismo , Mesilatos/uso terapêutico , Modelos Moleculares , Organofosfonatos/uso terapêutico , Pró-Fármacos/uso terapêutico , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/genética , Ativação Metabólica/efeitos dos fármacos , Membro C3 da Família 1 de alfa-Ceto Redutase , Animais , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacologia , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Carcinoma/metabolismo , Carcinoma/patologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HCT116 , Humanos , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Hidroxiprostaglandina Desidrogenases/química , Hidroxiprostaglandina Desidrogenases/genética , Mesilatos/química , Mesilatos/metabolismo , Mesilatos/farmacologia , Camundongos Nus , Simulação de Acoplamento Molecular , Nitrorredutases/genética , Nitrorredutases/metabolismo , Organofosfonatos/química , Organofosfonatos/metabolismo , Organofosfonatos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Distribuição Aleatória , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Livres de Patógenos Específicos , Especificidade por Substrato , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Neoplasias , Pró-Fármacos/farmacologia , Antraquinonas/química , Antraquinonas/farmacologia , Antineoplásicos/química , Aziridinas/química , Aziridinas/farmacologia , Humanos , Indolquinonas/química , Indolquinonas/farmacologia , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/química , NAD(P)H Desidrogenase (Quinona)/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Compostos de Mostarda Nitrogenada/química , Compostos de Mostarda Nitrogenada/farmacologia , Nitroimidazóis/química , Nitroimidazóis/farmacologia , Mostardas de Fosforamida/química , Mostardas de Fosforamida/farmacologia , Pró-Fármacos/química , Tirapazamina , Triazinas/química , Triazinas/farmacologiaRESUMO
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.
Assuntos
Antineoplásicos/metabolismo , Aziridinas/metabolismo , Escherichia coli/enzimologia , Biblioteca Gênica , Terapia Genética , Compostos de Mostarda Nitrogenada/metabolismo , Nitrorredutases/genética , Pró-Fármacos/metabolismo , Células HCT116 , Humanos , Nitrorredutases/isolamento & purificação , Resposta SOS em GenéticaRESUMO
The synthesis of 19 (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate prodrugs containing sulfonate leaving groups and 7-substituted electron-withdrawing groups is reported. These were designed to undergo hypoxia-selective metabolism to form potent DNA minor groove-alkylating agents. Analogues 17 and 24, containing the benzyl sulfonate leaving group and a neutral DNA minor groove-binding side chain, displayed hypoxic cytotoxicity ratios (HCRs) of >1000 in HT29 human cancer cells in vitro in an antiproliferative assay. Four analogues maintained large HCRs across a panel of eight human cancer cell lines. In a clonogenic assay, 19 showed an HCR of 4090 in HT29 cells. Ten soluble phosphate preprodrugs were also prepared and evaluated in vivo, alone and in combination with radiation in SiHa human tumor xenografts at a nontoxic dose. Compounds 34 and 39 displayed hypoxic log(10) cell kills (LCKs) of 1.78 and 2.71, respectively, equivalent or superior activity to previously reported chloride or bromide analogues, thus showing outstanding promise as hypoxia-activated prodrugs.
Assuntos
Antineoplásicos Alquilantes/síntese química , Indóis/síntese química , Neoplasias/metabolismo , Nitrocompostos/síntese química , Pró-Fármacos/síntese química , Ácidos Sulfônicos/química , Animais , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Hipóxia Celular , Linhagem Celular Tumoral , Terapia Combinada , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis/química , Indóis/farmacologia , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/radioterapia , Nitrocompostos/química , Nitrocompostos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Nitro seco analogs (nitroCBIs) of the antitumor antibiotic duocarmycins are a new class of hypoxia activated prodrugs. These compounds undergo hypoxia-selective metabolism to form potent DNA alkylating agents. A series of four nitroCBI alcohol prodrugs containing a bromide rather than chloride or sulfonate leaving group was synthesized. In assays for in vitro hypoxia-selective cytotoxicity against human tumor cell lines the two bromides with DNA minor groove binding basic side chains displayed hypoxic cytotoxicity ratios (HCRs) of 52-286 in HT29 cells and 41-43 in SiHa cells. These values compare well with a related previously reported chloride analog. The corresponding more water soluble phosphate pre-prodrugs of the bromides were synthesized and evaluated for in vivo antitumor activity against SiHa human tumor xenografts. All four phosphates, with both neutral and basic side chains, demonstrated activity providing statistically significant hypoxic log(10) cell kills of 0.87-2.80 at non-toxic doses, matching or proving superior to those of their chloride analogs.
Assuntos
Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Brometos/química , Brometos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Hipóxia Celular/fisiologia , Feminino , Humanos , Indóis/química , Indóis/farmacologia , Masculino , Camundongos , Camundongos Nus , Fosfatos/química , Fosfatos/farmacologia , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-Atividade , Neoplasias do Colo do Útero/tratamento farmacológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of 3-substituted (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate (nitroCBI) prodrugs containing sulfonate leaving groups undergo hypoxia-selective metabolism to form potent DNA minor groove alkylating agents. They were evaluated (along with chloride leaving group analogs for comparison) for their cytotoxicity against cultures of SKOV3 and HT29 human tumor cell lines under both aerobic and hypoxic conditions. Sulfonates with neutral side chains (e.g., 5,6,7-trimethoxyindole; TMI) show consistently higher hypoxic cytotoxicity ratios (HCRs) (34-246) than the corresponding chloro analogs (2.8-3.1) in SKOV3 cells, but these trends do not hold for compounds with cationic or polar neutral side chains.