RESUMO
Hypoxia represents a remarkably exploitable target for cancer therapy, is encountered only in solid human tumors, and is highly associated with cancer resistance and recurrence. Here, a hypoxia-activated mitochondria-accumulated Ru(II) polypyridyl prodrug functionalized with conjugated azo (Az) and nitrogen mustard (NM) functionalities, RuAzNM, is reported. This prodrug has multimodal theranostic properties toward hypoxic cancer cells. Reduction of the azo group in hypoxic cell microenvironments gives rise to the generation of two primary amine products, a free aniline mustard, and the polypyridyl RuNH2 complex. Thus, the aniline mustard triggers generation of reactive oxygen species (ROS) and mtDNA crosslinking. Meanwhile, the resultant biologically benign phosphorescent RuNH2 gives rise to a diagnostic signal and signals activation of the phototherapy. This multimodal therapeutic effect eventually elevates ROS levels, depletes reduced nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP), and induces mitochondrial membrane damage, mtDNA damage, and ultimately cell apoptosis. This unique strategy allows controlled multimodal theranostics to be realized in hypoxic cells and multicellular spheroids, making RuAzNM a highly selective and effective cancer-cell-selective theranostic agent (IC50 = 2.3 µm for hypoxic HepG2 cancer cells vs 58.2 µm for normoxic THL-3 normal cells). This is the first report of a metal-based compound developed as a multimodal theranostic agent for hypoxia.
Assuntos
Mostarda de Anilina , Antineoplásicos , Neoplasias , Pró-Fármacos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Hipóxia/metabolismo , DNA Mitocondrial , Oxirredução , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológicoRESUMO
Aim: Steroidal prodrugs of nitrogen mustards such as estramustine and prednimustine have proven effective anticancer agents in clinical use since the 1970s. In this work, we aimed to develop steroidal prodrugs of the novel nitrogen mustard POPAM-NH2. POPAM-NH2 is a melphalan analogue that was coupled with three different steroidal lactams. Methodology: The new conjugates were preclinically tested for anticancer activity against nine human and one rodent cancer experimental models, in vitro and in vivo. Results & conclusion: All the steroidal alkylators showed high antitumor activity, in vitro and in vivo, in the experimental systems tested. Moreover, these hybrid compounds showed by far superior anticancer activity compared with the alkylating agents, melphalan and POPAM-NH2.
Assuntos
Mostarda de Anilina/análogos & derivados , Antineoplásicos/farmacologia , Descoberta de Drogas , Lactamas/farmacologia , Propionatos/farmacologia , Esteroides/farmacologia , Mostarda de Anilina/administração & dosagem , Mostarda de Anilina/química , Mostarda de Anilina/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HT29 , Humanos , Injeções Intraperitoneais , Lactamas/administração & dosagem , Lactamas/química , Masculino , Camundongos , Camundongos SCID , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Propionatos/administração & dosagem , Propionatos/química , Esteroides/administração & dosagem , Esteroides/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Directed Enzyme Prodrugs Therapy (DEPT) as an alternative method against conventional cancer treatments, in which the non-toxic prodrugs is converted to highly cytotoxic derivative, has attracted an ample attention in recent years for cancer therapy studies. OBJECTIVE: The metabolite profile, cell cytotoxicity and molecular modeling interactions of a series of nitro benzamides with Ssap-NtrB were investigated in this study. METHOD: A series of nitro-substituted benzamide prodrugs (1-4) were synthesized and firstly investigated their enzymatic reduction by Ssap-NtrB (S. saprophyticus Nitroreductase B) using HPLC analysis. Resulting metabolites were analyzed by LC-MS/MS. Molecular docking studies were performed with the aim of investigating the relationship between nitro benzamide structures (prodrugs 1-4) and Ssap-NtrB at the molecular level. Cell viability assay was conducted on two cancer cell lines, hepatoma (Hep3B) and colon (HT-29) cancer models and healthy cell model HUVEC. Upon reduction of benzamide prodrugs by Ssap-NtrB, the corresponding amine effectors were tested in a cell line panel comprising PC-3, Hep3B and HUVEC cells and were compared with the established NTR substrates, CB1954 (an aziridinyl dinitrobenzamide). RESULTS: Cell viability assay resulted in while prodrugs 1, 2 and 3 had no remarkable cytotoxic effects, prodrug 4 showed the differential effect, showing moderate cytotoxicity with Hep3B and HUVEC. The metabolites that obtained from the reduction of nitro benzamide prodrugs (1-4) by Ssap-NtrB, showed differential cytotoxic effects, with none toxic for HUVEC cells, moderate toxic for Hep3B cells, but highly toxic for PC3 cells. CONCLUSION: Amongst all metabolites of prodrugs after Ssap-NtrB reduction, N-(2,4- dinitrophenyl)-4-nitrobenzamide (3) was efficient and toxic in PC3 cells as comparable as CB1954. Kinetic parameters, molecular docking and HPLC results also confirm that prodrug 3 is better for Ssap-NtrB than 1, 2 and 4 or known cancer prodrugs of CB1954 and SN23862, demonstrating that prodrug 3 is an efficient candidate for NTR based cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Nitrobenzenos/farmacologia , Nitrorredutases/metabolismo , Pró-Fármacos/farmacologia , Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Aziridinas/farmacologia , Benzamidas/metabolismo , Benzamidas/toxicidade , Linhagem Celular Tumoral , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Simulação de Acoplamento Molecular , Nitrobenzenos/metabolismo , Nitrobenzenos/toxicidade , Nitrorredutases/química , Pró-Fármacos/metabolismo , Pró-Fármacos/toxicidade , Staphylococcus saprophyticus/enzimologiaRESUMO
AIM: Alkylating agents and antimetabolites are cytotoxic drugs commonly used in cancer treatment. These medications are often associated with serious side effects on normal tissues and organs. METHODOLOGY: To improve the pharmacological profile of the alkylating agent POPAM and the antimetabolite 5-fluorouracil, novel integrin-targeted delivery systems based on c(RGDyK) were successfully synthesized. The new conjugates were tested in vitro against different cancer cells such as PC3, SKOV3, A549, MCF7 and MBA-MB-321. RESULTS & CONCLUSION: The c(RGDyK) conjugates of POPAM demonstrated better inhibitory effects and selectivity compared with c(RGDyK) and POPAM. The c(RGDyK) conjugates of 5-FUA demonstrated diverse inhibitory effects compared with c(RGDyK) and 5-FUA related to the levels of integrin expression, the conjugate stability and sensitivity of cancer cells to 5-FUA.
Assuntos
Mostarda de Anilina/análogos & derivados , Antineoplásicos/química , Fluoruracila/química , Integrinas/metabolismo , Peptídeos Cíclicos/química , Propionatos/química , Células A549 , Sequência de Aminoácidos , Mostarda de Anilina/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fluoruracila/análise , Fluoruracila/farmacologia , Humanos , Integrinas/antagonistas & inibidores , Células MCF-7 , Espectroscopia de Ressonância Magnética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Oligopeptídeos/químicaRESUMO
Alkylating agents are still nowadays one of the most important classes of cytotoxic drugs, which display a wide range of therapeutic use for the treatment of various cancers. We have synthesized and tested four hybrid homo-azasteroidal alkylating esters for antileukemic activity against five sensitive to alkylating agents human leukemia cell lines in vitro and against P388 murine leukemia in vivo. Comparatively, melphalan and 3-(4-(bis(2-chloroethyl)amino)phenoxy)propanoic acid (POPAM) were also examined. All the homo-aza-steroidal alkylators showed relatively lower acute toxicity, very promising and antileukemic activity both in vitro and in vivo.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Lactamas/síntese química , Lactamas/uso terapêutico , Leucemia P388/tratamento farmacológico , Esteroides/síntese química , Esteroides/uso terapêutico , Mostarda de Anilina/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Células Jurkat , Células K562 , Lactamas/química , Lactamas/farmacologia , Masculino , Mecloretamina/química , Camundongos Endogâmicos BALB C , Esteroides/química , Esteroides/farmacologia , Relação Estrutura-AtividadeRESUMO
A new G-quadruplex (G-4)-directing alkylating agent BMVC-C3M was designed and synthesized to integrate 3,6-bis(1-methyl-4-vinylpyridinium iodide)carbazole (BMVC) with aniline mustard. Various telomeric G-4 structures (hybrid-2 type and antiparallel) and an oncogene promoter, c-MYC (parallel), were constructed to react with BMVC-C3M, yielding 35 % alkylation yield toward G-4 DNA over other DNA categories (<6 %) and high specificity under competition conditions. Analysis of the intact alkylation adducts by electrospray ionization mass spectroscopy (ESI-MS) revealed the stepwise DNA alkylation mechanism of aniline mustard for the first time. Furthermore, the monoalkylation sites and intrastrand cross-linking sites were determined and found to be dependent on G-4 topology based on the results of footprinting analysis in combination with mass spectroscopic techniques and in silico modeling. The results indicated that BMVC-C3M preferentially alkylated at A15 (H26), G12 (H24), and G2 (c-MYC), respectively, as monoalkylated adducts and formed A15-C3M-A21 (H26), G12-C3M-G4 (H24), and G2-C3M-G4/G17 (c-MYC), respectively, as cross-linked dialkylated adducts. Collectively, the stability and site-selective cross-linking capacity of BMVC-C3M provides a credible tool for the structural and functional characterization of G-4 DNAs in biological systems.
Assuntos
Mostarda de Anilina/química , Carbazóis/química , Quadruplex G , Hidrocarbonetos Iodados/química , Compostos de Piridínio/química , Alquilação , DNA/químicaRESUMO
A set of PCR primers based on the genome sequence were used to clone a gene encoding a hypothetical nitroreductases (named as Ssap-NtrB) from uropathogenic staphylococcus, Staphylococcus saprophyticus strain ATCC 15305, an oxygen insensitive flavoenzyme. Activity studies of the translation product revealed that the nitroreductase catalyses two electron reduction of a nitroaromatic drug of nitrofurazone (NFZ), cancer prodrugs of CB1954 and SN23862 at optimum temperature of 20 °C together with retaining its maximum activity considerably at 3 °C. The required electrons for such reduction could be supplied by either NADH or NADPH with a small preference for the latter. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a mostly soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined. The findings lead us to propose that Ssap-NtrB represents a novel nitro reductase with an unusual cold active property, which has not been described previously for prodrug activating enzymes of nitroreductases.
Assuntos
Nitrorredutases/metabolismo , Pró-Fármacos/metabolismo , Staphylococcus saprophyticus/enzimologia , Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/metabolismo , Aziridinas/metabolismo , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Escherichia coli/genética , Mononucleotídeo de Flavina/metabolismo , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Nitrofurazona/metabolismo , Nitrorredutases/genética , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus saprophyticus/genética , TemperaturaRESUMO
The antitumor agent 11ß (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11ß against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11ß; flow cytometry studies showed that 11ß exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11ß inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11ß blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11ß enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11ß, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11ß, which supplements conventional DNA adduct formation to promote cancer cell death.
Assuntos
Mostarda de Anilina/farmacologia , Antineoplásicos Alquilantes/farmacologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias Hepáticas/metabolismo , Acetilcisteína/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Adutos de DNA/metabolismo , Feminino , Sequestradores de Radicais Livres/farmacologia , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Nus , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Vitamina E/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
To improve the chemical stability and therapeutic efficacy of N-mustard, a series of phenyl N-mustard linked to DNA-affinic 9-anilinoacridines and acridine via a urea linker were synthesized and evaluated for antitumor studies. The new N-mustard derivatives were prepared by the reaction of 4-bis(2-chloroethyl)aminophenyl isocyanate with a variety of 9-anilinoacridines or 9-aminoacridine. The antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro without cross-resistance to taxol or vinblastine and showed potent antitumor therapeutic efficacy in nude mice against human tumor xenografts. It also showed that 24d was capable of inducing marked dose-dependent levels of DNA cross-linking by comet assay and has long half-life in rat plasma.
Assuntos
Amsacrina/análogos & derivados , Mostarda de Anilina/síntese química , Mostarda de Anilina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Ureia/química , Acridinas/química , Amsacrina/química , Mostarda de Anilina/análogos & derivados , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Nus , Estrutura Molecular , Ratos , Estereoisomerismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Antibody-directed enzyme prodrug therapy is a two-stage treatment whereby a tumor-targeted antibody-enzyme complex localizes in tumor for selective conversion of prodrug. The purpose of this study was to establish optimal variables for single administration of MFECP1, a recombinant antibody-enzyme fusion protein of an anti-carcinoembryonic antigen single-chain Fv antibody and the bacterial enzyme carboxypeptidase G2 followed by a bis-iodo phenol mustard prodrug. MFECP1 is manufactured in mannosylated form to facilitate normal tissue elimination. EXPERIMENTAL DESIGN: Pharmacokinetic, biodistribution, and tumor localization studies were used to test the hypothesis that MFECP1 localizes in tumor and clears from normal tissue via the liver. Firstly, safety of MFECP1 and a blood concentration of MFECP1 that would avoid systemic prodrug activation were tested. Secondly, dose escalation of prodrug was done. Thirdly, the dose of MFECP1 and timing of prodrug administration were optimized. RESULTS: MFECP1 was safe and well tolerated, cleared rapidly via the liver, and was less immunogenic than previously used products. Eighty-fold dose escalation from the starting dose of prodrug was carried out before dose-limiting toxicity occurred. Confirmation of the presence of enzyme in tumor and DNA interstrand cross-links indicating prodrug activation were obtained for the optimal dose and time point. A total of 28 of 31 patients was evaluable for response, the best response being a 10% reduction of tumor diameter, and 11 of 28 patients had stable disease. CONCLUSIONS: Optimal conditions for effective therapy were established. A study testing repeat treatment is currently being undertaken.
Assuntos
Mostarda de Anilina/análogos & derivados , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/tratamento farmacológico , Pró-Fármacos/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , gama-Glutamil Hidrolase/uso terapêutico , Idoso , Mostarda de Anilina/sangue , Mostarda de Anilina/farmacocinética , Mostarda de Anilina/uso terapêutico , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Relação Dose-Resposta a Droga , Feminino , História do Século XVI , História do Século XVII , Humanos , Imageamento Tridimensional , Imunoconjugados/sangue , Imunoconjugados/farmacocinética , Imunoconjugados/uso terapêutico , Masculino , Pró-Fármacos/efeitos adversos , Pró-Fármacos/farmacocinética , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/farmacocinética , gama-Glutamil Hidrolase/sangue , gama-Glutamil Hidrolase/farmacocinéticaRESUMO
A series of bifunctional compounds was prepared consisting of 17beta estradiol linked to a DNA damaging N,N-bis-(2-chloroethyl)aniline. The objective of our studies was to determine the characteristics of the linker that permitted both reaction with DNA and binding of the resultant covalent adducts to the estrogen receptor. Linker characteristics were pivotal determinants underlying the ability of the compounds to kill selectively breast cancer cells that express the estrogen receptor.
Assuntos
Compostos de Anilina/uso terapêutico , Antineoplásicos Alquilantes/síntese química , Neoplasias da Mama/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Estradiol/uso terapêutico , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Mostarda de Anilina , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Sítios de Ligação , Neoplasias da Mama/metabolismo , Adutos de DNA/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Estradiol/química , Estradiol/farmacologia , Estudos de Avaliação como Assunto , Feminino , Humanos , Receptores de Estrogênio/metabolismo , Células Tumorais CultivadasRESUMO
The potential antibody directed prodrug therapy half-mustard prodrug 4-[(2-chloroethyl)(2-ethyl)amino]-phenoxycarbonyl-L-glutamic acid was synthesised by reductive alkylation of 4-[(2-chloroethyl)amino]-phenoxycarbonyl-L-glutamic acid using acetaldehyde. 4-[(2-chloroethyl)[(11)C](2-ethyl)amino]phenoxycarbonyl-L-glutamic acid was synthesized with 18-22% decay corrected radiochemical yield in 45 min from EOB by reductive alkylation of 4-[(2-chloroethyl)amino]-phenoxycarbonyl-L-glutamic acid using [(11)C]acetaldehyde. [(11)C]Acetaldehyde was prepared in 60% decay corrected radiochemical yield by oxidation of [(11)C]ethanol over heated copper oxide. The radiosynthesis of [(11)C]ethanol was re-examined and optimized. 4-[(2-chloroethyl)(2-ethyl)amino]-phenoxycarbonyl-L-glutamic acid was found to have affinity for carboxypeptidase G2; the K(m) and V(max) were 99.4-115.9 microM (n=3) and 3.6-5.0 microM/min, respectively, at a carboxypeptidase G2 concentration of 0.0247 U/ml.
Assuntos
Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/síntese química , Acetaldeído , Mostarda de Anilina/farmacocinética , Indicadores e Reagentes , Marcação por Isótopo/métodos , Compostos Radiofarmacêuticos , Especificidade por Substrato , Tomografia Computadorizada de Emissão , gama-Glutamil HidrolaseRESUMO
Nine new nitrogen mustard compounds derived from 2,6-difluoro-4-hydroxy- (3a-e) and 2,6-difluoro-4-amino- (4a-d) aniline were synthesized as potential prodrugs. They were designed to be activated to their corresponding 3,5-difluorophenol and -aniline (4)-nitrogen mustards by the enzyme carboxypeptidase G2 (CPG2) in gene-directed enzyme prodrug therapy (GDEPT) models. The compounds were tested for cytotoxicity in the MDA MB-361 breast adenocarcinoma. The cell line was engineered to express stably either CPG2 tethered to the cell surface stCPG2-(Q)3 or beta-galactosidase (beta-Gal) as control. The cytotoxicity differentials were calculated between CPG 2-expressing and -nonexpressing cells and yielded different results for the two series of prodrugs despite their structural similarities. While the phenol compounds are ineffective as prodrugs, their aniline counterparts exhibit outstanding activity in the tumor cell lines expressing CPG2. [3,5-Difluoro-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl-l-glutamic acid gave a differential of >227 in MDA MB361 cells as compared with 19 exhibited by 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-l-glutamic acid, 1a, which has been in clinical trials.
Assuntos
Antineoplásicos/metabolismo , Ácido Glutâmico/metabolismo , Compostos de Mostarda Nitrogenada/metabolismo , Pró-Fármacos/metabolismo , gama-Glutamil Hidrolase/metabolismo , Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/síntese química , Mostarda de Anilina/metabolismo , Mostarda de Anilina/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Derivados de Benzeno/síntese química , Derivados de Benzeno/metabolismo , Derivados de Benzeno/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Terapia Genética , Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/síntese química , Ácido Glutâmico/farmacologia , Meia-Vida , Humanos , Camundongos , Transplante de Neoplasias , Compostos de Mostarda Nitrogenada/síntese química , Compostos de Mostarda Nitrogenada/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade , Transplante Heterólogo , gama-Glutamil Hidrolase/química , gama-Glutamil Hidrolase/genéticaRESUMO
Artificial recombinant receptors may be useful for selectively targeting imaging and therapeutic agents to sites of gene expression. To evaluate this approach, we developed transgenes to express highly on cells a single-chain antibody (scFv) against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx). A phOx enzyme conjugate was created by covalently attaching phOx molecules to polyethylene glycol (PEG)-modified beta-glucuronidase. Cells expressing phOx scFv but not control scFv receptors were selectively killed after exposure to ss-glucuronidase derivatized with phOx and PEG (phOx-beta G-PEG) and a glucuronide prodrug (p-hydroxy aniline mustard beta-D-glucuronide, HAMG) of p-hydroxyaniline mustard. Targeted activation of HAMG produced bystander killing of receptor-negative cells in mixed populations containing as few as 10% phOx-receptor-positive cells. Functional phOx scFv receptors were stably expressed on B16-F1 melanoma tumors in vivo. Treatment of mice bearing established phOx-receptor-positive tumors with phOx-beta G-PEG and HAMG significantly (P< or =.0005) suppressed tumor growth as compared with treatment with beta G-PEG and HAMG or prodrug alone. phOx was unstable in the serum, suggesting alternative haptens may be more suitable for in vivo applications. Our results show that therapeutic agents can be targeted to artificial hapten receptors in vitro and in vivo. The expression of artificial receptors on target cells may allow preferential delivery of therapeutic or imaging molecules to sites of transgene expression.
Assuntos
Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/administração & dosagem , Anticorpos Monoclonais/imunologia , Sistemas de Liberação de Medicamentos , Melanoma/tratamento farmacológico , Neoplasias Experimentais/imunologia , Oxazóis/imunologia , Polietilenoglicóis/administração & dosagem , Pró-Fármacos/administração & dosagem , Mostarda de Anilina/síntese química , Mostarda de Anilina/imunologia , Animais , Anticorpos Monoclonais/genética , Linhagem Celular Tumoral , Expressão Gênica , Haptenos , Imunoterapia , Injeções Intravenosas , Melanoma/genética , Melanoma/imunologia , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/terapia , Oxazóis/química , Polietilenoglicóis/síntese química , Pró-Fármacos/síntese química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours. Activation of the aziridinyl dinitrobenzamide CB 1954 by E. coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite. We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2- and 4-hydroxylamines and their corresponding amines). The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines. Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line. These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours. The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites. These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.
Assuntos
Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/metabolismo , Antineoplásicos/metabolismo , Aziridinas/metabolismo , Efeito Espectador , Terapia Genética , Neoplasias/terapia , Nitrorredutases/genética , Mostarda de Anilina/farmacologia , Antineoplásicos/farmacologia , Aziridinas/farmacologia , Cromatografia Líquida de Alta Pressão , Escherichia coli/enzimologia , Vetores Genéticos , Humanos , Espectrometria de Massas , Neoplasias/enzimologia , Nitrorredutases/metabolismo , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The E. coli nitroreductase enzyme (NTR) has been widely used in suicide gene therapy (GDEPT and ADEPT) applications as a activating enzyme for nitroaromatic prodrugs of the dinitrobenzamide class. NTR has been previously shown to be a homodimeric enzyme with two active sites. We present here the crystal structures of the reduced form of NTR and its complexes with the inhibitor dicoumarol and three dinitrobenzamide prodrugs. Comparison of the structures of the oxidized and reduced forms of the native enzyme shows that the principal structural changes occur in the FMN cofactor and indicate that the enzyme itself is a relatively rigid structure that primarily provides a rigid structural framework on which hydride transfer occurs. The aziridinyldinitrobenzamide prodrug CB 1954 binds in nonidentical ways in both of the two active sites of the homodimeric enzyme, employing both hydrophobic and (in active site B) a direct H-bond contact to the side chain of Lys14. In active site A the 2-nitro group stacks above the FMN, and in active site B the 4-nitro group does, explaining why reduction of either nitro group is observed. In contrast, the larger mustard group of the dinitrobenzamide mustard compound SN 23862 forces the prodrug to bind at both active sites with only the 2-nitro group able to participate in hydride transfer from the FMN, explaining why only the 2-hydroxylamine reduction product is observed. In each site, the nitro groups of the prodrug make direct H-bond contacts with the enzyme; in active Site A the 2-nitro to Ser40 and the 4-nitro to Asn71, while in active Site B the 2-nitro contacts the main chain nitrogen of Thr41 and the 4-nitro group the Lys14 side chain. The related amide-substituted mustard SN 27217 binds in a broadly similar fashion, but with the larger amide group substituent able to reach and contact the side chain of Arg107, further restricting the prodrug conformations in the binding site. The inhibitor dicoumarol appears to bind primarily by pi-stacking interactions and hydrophobic contacts, with no conformational changes in the enzyme. One of the hydroxycoumarin subunits stacks above the plane of the FMN via pi-overlap with the isoalloxazine ring, penetrating deep into the groove, with the other less well-defined. These studies suggest guidelines for further prodrug design. Steric bulk (e.g., mustard rather than aziridine) on the ring can limit the possible binding orientations, and the reducible nitro group must be located para to the mustard. Substitution on the carboxamide side chain still allows the prodrugs to bind, but also limits their orientation in the binding site. Finally, modulating substrate specificity by alteration of the structure of the enzyme rather than the prodrug might usefully focus on modifying the Phe124 residue and those surrounding it.
Assuntos
Mostarda de Anilina/análogos & derivados , Benzamidas/química , Dicumarol/química , Nitrorredutases/química , Nitrorredutases/metabolismo , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Mostarda de Anilina/química , Mostarda de Anilina/metabolismo , Aziridinas/química , Aziridinas/metabolismo , Benzamidas/farmacocinética , Benzamidas/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Dicumarol/farmacocinética , Dicumarol/farmacologia , Desenho de Fármacos , Ativação Enzimática , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Modelos Moleculares , Nitrorredutases/antagonistas & inibidores , Oxirredução , Ligação ProteicaRESUMO
Nineteen novel potential self-immolative prodrugs and their corresponding drugs have been synthesized for gene-directed enzyme prodrug therapy (GDEPT) with carboxypeptidase G2 (CPG2) as the activating enzyme. The compounds are derived from o- and p-amino and p-methylamino aniline nitrogen mustards. Their aqueous stability, kinetics of drug release by CPG2, and cytotoxicity in the colon carcinoma cell line WiDr, expressing either surface-tethered CPG2 (stCPG2(Q)3) or control beta-galactosidase, are assessed. The effect of various structural features on stability, kinetics of activation, and biological activity is discussed. The p-methylamino prodrugs are the most stable compounds from this series, with the largest cytotoxicity differentials between CPG2-expressing and nonexpressing cells. The most potent compounds in all series are prodrugs of bis-iodo nitrogen mustards. 4-[N-[4'-Bis(2' '-iodoethyl)aminophenyl]-N'-methylcarbamoyloxymethyl]phenylcarbamoyl-l-glutamic acid, compound 39b, is 124-fold more cytotoxic to WiDr cells expressing CPG2 than to cells expressing beta-galactosidase. An additional six compounds show better cytotoxicity differential than the published N-[4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl]-l-glutamic acid (CMDA) prodrug.
Assuntos
Mostarda de Anilina/síntese química , Antineoplásicos Alquilantes/síntese química , Carbamatos/síntese química , Ácido Glutâmico/síntese química , Pró-Fármacos/síntese química , gama-Glutamil Hidrolase/metabolismo , Mostarda de Anilina/química , Mostarda de Anilina/farmacocinética , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacocinética , Carbamatos/química , Carbamatos/farmacocinética , Técnicas de Transferência de Genes , Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/química , Ácido Glutâmico/farmacocinética , Meia-Vida , Humanos , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Relação Quantitativa Estrutura-Atividade , Células Tumorais Cultivadas , gama-Glutamil Hidrolase/genéticaRESUMO
The dinitrobenzamide aziridine CB 1954 (1) and its nitrogen mustard analogue SN 23862 (6) are prodrugs that are activated by enzymatic nitroreduction in tumors. Bioactivation of 1 is considered to be due to reduction of its 4-nitro group to the hydroxylamine and subsequent formation of the N-acetoxy derivative; this acts as a reactive center, in concert with the aziridine moiety, to provide a bifunctional DNA cross-linking agent (Knox model). It is currently unclear whether bioactivation of 6 occurs by the same mechanism or results from the electronic effects of nitroreduction on reactivity of the nitrogen mustard moiety. To discriminate between these mechanisms, we have synthesized the hydroxylamine and amine derivatives of 1 and 6, plus related compounds, and determined their alkylating reactivities in aqueous solution, using LC/MS to identify reaction pathways. The relationships between substituent electronic effects, reactivity, and cytotoxicity were determined using the UV4 cell line, which is defective in nucleotide excision repair (thus avoiding differences in repair kinetics). Alkylating reactivity correlated with the electron-donating character of the ortho or para substituent in the case of the mustards, with a less marked electronic effect for the aziridines. Importantly, there was a highly significant linear relationship between cytotoxic potency and alkylating reactivity in both the aziridine and the mustard series, with the notable exception of 4, the 4-hydroxylamine of 1, which was 300-fold more toxic than predicted by this relationship. This demonstrates that the high potency of 4 does not result from activation of the aziridine ring, supporting the Knox model. The single-step bioactivation of 6, to amino or hydroxylamine metabolites with similar potency to 4, is a potential advantage in the use of dinitrobenzamide mustards as prodrugs for activation by nitroreductases.
Assuntos
Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/metabolismo , Antineoplásicos Alquilantes/metabolismo , Aziridinas/metabolismo , Nitrorredutases/metabolismo , Pró-Fármacos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Cricetinae , Eletroquímica , Hidrólise , Cinética , Espectrometria de Massas , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The efficacy of cancer gene therapy depends critically on "bystander effects" by which genetic modification of tumor cells results in killing of unmodified cells in the local microenvironment. In gene-dependent enzyme-prodrug therapy, expression of a prodrug-activating suicide gene is used to generate a cytotoxic metabolite that diffuses to nontransduced cells. The objective of this study was to develop a physiologically relevant tissue culture model for quantifying bystander effects and to validate the model using as an example the activation of dinitrobenzamide prodrugs (e.g., CB 1954) by Escherichia coli aerobic nitroreductase (NTR). Bystander effects were measured in three-dimensional multilayer cocultures of NTR+ and NTR- cells by determining clonogenic survival curves for both cell types using V79, Skov3, or WiDr as parental cell lines. Bystander killing by CB 1954 was much more efficient in multilayers than monolayers at equivalent cell:medium ratios, whereas the chloromustard analogue of CB 1954 showed even greater efficiency. For a series of dinitrobenzamides, bystander killing in multilayers showed a positive correlation with prodrug lipophilicity and also correlated with the bystander effect in mixed tumor xenografts grown from the same NTR+ and NTR- WiDr cell lines (r(2) = 0.84; P < 0.001). The multilayer model identified a bromomustard prodrug (SN 24927) with superior therapeutic activity to CB 1954 that provided curative activity against WiDr tumors comprising 1:1 mixtures of NTR+ and NTR- cells. This study demonstrates the utility of the multilayer tissue culture model for quantifying and optimizing bystander effects in tumors and identifies a new lead prodrug for NTR gene-dependent enzyme-prodrug therapy.
Assuntos
Efeito Espectador , Técnicas de Cultura de Células/métodos , Terapia Genética/métodos , Neoplasias/terapia , Nitrorredutases/genética , Pró-Fármacos/metabolismo , Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/farmacologia , Animais , Aziridinas/farmacologia , Cricetinae , Humanos , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
We describe a novel strategy to increase the selective toxicity of genotoxic compounds. The strategy involves the synthesis of bifunctional molecules capable of forming DNA adducts that have high affinity for specific proteins in target cells. It is proposed that the association of such proteins with damaged sites in DNA can compromise protein function and/or DNA repair resulting in increased toxicity. We describe the synthesis of a bifunctional compound consisting of an aniline mustard linked to the 7alpha position of estradiol. This novel compound can form covalent DNA adducts that have high affinity for the estrogen receptor. Breast cancer cells that express high levels of the estrogen receptor showed increased sensitivity to the cytotoxic effects of the new compound.