RESUMO
Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex with the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P(R) enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P(S) isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P(S) isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.
Assuntos
Hidrolases de Éster Carboxílico/química , Substâncias para a Guerra Química/química , Inibidores Enzimáticos/química , Compostos Organofosforados/química , Sarina/química , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Cristalização , Humanos , Hidrólise , Modelos Moleculares , Compostos Organofosforados/farmacologia , Oximas/farmacologia , Sarina/farmacologia , EstereoisomerismoRESUMO
Neural stem cells and progenitor cells migrate selectively to tumor loci in vivo. We exploited the tumor-tropic properties of HB1.F3.C1 cells, an immortalized cell line derived from human fetal telencephalon, to deliver the cDNA encoding a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors in mice. This enzyme activates the prodrug CPT-11 more efficiently than do human enzymes. Mice bearing multiple tumors were treated with rCE-expressing HB1.F3.C1 cells and schedules of administration of CPT-11 that produced levels of active drug (SN-38) tolerated by patients. Both HB1.F3.C1 cells and CPT-11 were given i.v. None of the untreated mice and 30% of mice that received only CPT-11 survived long term. In contrast, 90% of mice treated with rCE-expressing HB1.F3.C1 cells and 15 mg/kg CPT-11 survived for 1 year without detectable tumors. Plasma carboxylesterase activity and SN-38 levels in mice receiving both rCE-expressing HB1.F3.C1 cells (HB1.F3.C1/AdCMVrCE) and CPT-11 were comparable with those in mice receiving CPT-11 only. These data support the hypothesis that the antitumor effect of the described neural stem/progenitor cell-directed enzyme prodrug therapy (NDEPT) is mediated by production of high concentrations of active drug selectively at tumor sites, thereby maximizing the antitumor effect of CPT-11. NDEPT approaches merit further investigation as effective, targeted therapy for metastatic tumors. We propose that the described approach may have greatest use for eradicating minimum residual disease.
Assuntos
Camptotecina/análogos & derivados , Carboxilesterase/metabolismo , Terapia Genética/métodos , Neuroblastoma/terapia , Pró-Fármacos/farmacologia , Telencéfalo/fisiologia , Adenoviridae/genética , Animais , Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacocinética , Camptotecina/farmacologia , Carboxilesterase/biossíntese , Carboxilesterase/genética , Linhagem Celular Tumoral , Terapia Combinada , DNA Complementar/administração & dosagem , DNA Complementar/genética , Intervalo Livre de Doença , Humanos , Irinotecano , Camundongos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/enzimologia , Neuroblastoma/genética , Pró-Fármacos/farmacocinética , Telencéfalo/citologia , Telencéfalo/enzimologia , Transdução Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Carboxylesterases (CEs) are ubiquitous enzymes responsible for the hydrolysis of numerous clinically useful drugs. As ester moieties are frequently included in molecules to improve their water solubility and bioavailability, de facto they become substrates for CEs. OBJECTIVE: In this review, we describe the properties of human CEs with regard to their ability to activate anticancer prodrugs and demonstrate how structure-based design can be used to modulate substrate specificity and to increase efficiency of hydrolysis. METHODS: A specific example using CPT-11 and a human liver CE is discussed. However, these techniques can be applied to other enzymes and their associated prodrugs. RESULTS: Structure-guided mutagenesis of CEs can be employed to alter substrate specificity and generate novel enzymes that are efficacious at anticancer prodrug activation.
Assuntos
Antineoplásicos/farmacocinética , Hidrolases de Éster Carboxílico/metabolismo , Pró-Fármacos/farmacocinética , Animais , Disponibilidade Biológica , Camptotecina/análogos & derivados , Camptotecina/farmacocinética , Hidrolases de Éster Carboxílico/química , Desenho de Fármacos , Humanos , Hidrólise , Irinotecano , Fígado/enzimologia , Mutagênese , SolubilidadeRESUMO
PURPOSE: The anaplastic lymphoma kinase (ALK) has been demonstrated to be a valid clinical target in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. Recent studies have indicated that ALK is overexpressed in pediatric rhabdomyosarcoma (RMS) and hence we hypothesized that this kinase may be a suitable candidate for therapeutic intervention in this tumor. METHODS: We evaluated the expression of ALK in a panel of pediatric RMS cell lines and patient-derived xenografts (PDX), and sensitivity to ALK inhibitors was assessed both in vitro and in vivo. RESULTS: Essentially, all RMS lines were sensitive to crizotinib, NVP-TAE684 or LDK-378 in vitro, and molecular analyses demonstrated inhibition of RMS cell proliferation following siRNA-mediated reduction of ALK expression. However, in vivo PDX studies using ALK kinase inhibitors demonstrated no antitumor activity when used as single agents or when combined with standard of care therapy (vincristine, actinomycin D and cyclophosphamide). More alarmingly, however, crizotinib actually accelerated the growth of these tumors in vivo. CONCLUSIONS: While ALK appears to be a relevant target in RMS in vitro, targeting this kinase in vivo yields no therapeutic efficacy, warranting extreme caution when considering the use of these agents in pediatric RMS patients.
Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/biossíntese , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/enzimologia , Quinase do Linfoma Anaplásico/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Crizotinibe/administração & dosagem , Crizotinibe/farmacologia , Ciclofosfamida/administração & dosagem , Dactinomicina/administração & dosagem , Interações Medicamentosas , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-met/biossíntese , Proteínas Proto-Oncogênicas c-met/genética , Pirimidinas/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Rabdomiossarcoma/genética , Transfecção , Vincristina/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Carboxylesterases (CE) are ubiquitous enzymes thought to be responsible for the metabolism and detoxification of xenobiotics. Numerous clinically used drugs including Demerol, lidocaine, capecitabine, and CPT-11 are hydrolyzed by these enzymes. Hence, the identification and application of selective CE inhibitors may prove useful in modulating the metabolism of esterified drugs in vivo. Having recently identified benzil (diphenylethane-1,2-dione) as a potent selective inhibitor of CEs, we sought to evaluate the inhibitory activity of related 1,2-diones toward these enzymes. Biochemical assays and kinetic studies demonstrated that isatins (indole-2,3-diones), containing hydrophobic groups attached at a variety of positions within these molecules, could act as potent, specific CE inhibitors. Interestingly, the inhibitory potency of the isatin compounds was related to their hydrophobicity, such that compounds with clogP values of <1.25 were ineffective at enzyme inhibition. Conversely, analogs demonstrating clogP values>5 routinely yielded Ki values in the nM range. Furthermore, excellent 3D QSAR correlates were obtained for two human CEs, hCE1 and hiCE. While the isatin analogues were generally less effective at CE inhibition than the benzils, the former may represent valid lead compounds for the development of inhibitors for use in modulating drug metabolism in vivo.
Assuntos
Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/química , Isatina/análogos & derivados , Isatina/síntese química , Relação Quantitativa Estrutura-Atividade , Acetilcolinesterase/química , Butirilcolinesterase/química , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isatina/química , Modelos MolecularesRESUMO
Carboxylesterases are ubiquitous proteins responsible for the detoxification of xenobiotics. However, these enzymes also activate prodrugs, such as the anticancer agents capecitabine and CPT-11. As a consequence, overexpression of carboxylesterases within tumor cells sensitizes these cells to CPT-11. We have recently identified two classes of carboxylesterase inhibitors based on either a benzil (diphenylethane-1,2-dione) or a benzene sulfonamide scaffold and showed that these compounds inhibit carboxylesterases with Kis in the low nanomolar range. Because both classes of inhibitors show reversible enzyme inhibition, conventional in vitro biochemical assays would not accurately reflect the in situ levels of carboxylesterase activity or inhibition. Therefore, we have developed a novel assay for the determination of intracellular carboxylesterase activity using 4-methylumbelliferone as a substrate. These studies show that benzil and a dimethylbenzil analogue efficiently enter cells and inhibit human intestinal carboxylesterase and rabbit liver carboxylesterase intracellularly. This inhibition results in reduced cytotoxicity to CPT-11 due to the lack of carboxylesterase-mediated conversion of the prodrug to SN-38. These results suggest that intracellular modulation of carboxylesterase activity with benzil or its analogues may be applied to minimize the toxicity of normal cells to CPT-11.
Assuntos
Camptotecina/análogos & derivados , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Fenilglioxal/análogos & derivados , Animais , Camptotecina/antagonistas & inibidores , Camptotecina/farmacocinética , Camptotecina/farmacologia , Linhagem Celular Tumoral , Interações Medicamentosas , Resistencia a Medicamentos Antineoplásicos , Humanos , Intestinos/enzimologia , Irinotecano , Fígado/enzimologia , Fenilglioxal/farmacologia , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , CoelhosRESUMO
Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of ester-containing xenobiotics. This hydrolysis reaction results in the formation of the corresponding carboxylic acid and alcohol. Due to their highly plastic active site, CEs can hydrolyze structurally very distinct and complex molecules. Because ester groups significantly increase the water solubility of compounds, they are frequently used in the pharmaceutical industry to make relatively insoluble compounds more bioavailable. By default, this results in CEs playing a major role in the distribution and metabolism of these esterified drugs. However, this can be exploited to selectively improve compound hydrolysis, and using specific in vivo targeting techniques can be employed to generate enhanced drug activity. Here, we seek to detail the human CEs involved in esterified molecule hydrolysis, compare and contrast these with CEs present in small mammals and describe novel methods to improve drug therapy by specific delivery of CEs to cells in vivo. Finally, we will discuss the development of such approaches for their potential application towards malignant disease.
Assuntos
Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Metástase Neoplásica/tratamento farmacológico , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Animais , Hidrolases de Éster Carboxílico/metabolismo , Inibidores Enzimáticos/química , Humanos , Hidrólise , Metástase Neoplásica/patologia , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents).
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Animais , Hidrolases de Éster Carboxílico/química , Humanos , Inativação Metabólica , Modelos Moleculares , Organofosfatos/metabolismo , Especificidade por SubstratoRESUMO
Benzil has been identified as a potent selective inhibitor of carboxylesterases (CEs). Essential components of the molecule required for inhibitory activity include the dione moiety and the benzene rings, and substitution within the rings affords increased selectivity toward CEs from different species. Replacement of the benzene rings with heterocyclic substituents increased the K(i) values for the compounds toward three mammalian CEs when using o-nitrophenyl acetate as a substrate. Logarithmic plots of the K(i) values versus the empirical resonance energy, the heat of union of formation energy, or the aromatic stabilization energy determined from molecular orbital calculations for the ring structures yielded linear relationships that allowed prediction of the efficacy of the diones toward CE inhibition. Using these data, we predicted that 2,2'-naphthil would be an excellent inhibitor of mammalian CEs. This was demonstrated to be correct with a K(i) value of 1 nM being observed for a rabbit liver CE. In addition, molecular simulations of the movement of the ring structures around the dione dihedral indicated that the ability of the compounds to inhibit CEs was due, in part, to rotational constraints enforced by the dione moiety. Overall, these studies identify subdomains within the aromatic ethane-1,2-diones, that are responsible for CE inhibition.
Assuntos
Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/química , Furanos/química , Fenilglioxal/análogos & derivados , Piridinas/química , Tiofenos/química , Animais , Benzoína/química , Bromo/química , Cristalografia por Raios X , Naftalenos/síntese química , Naftalenos/química , Fenilglioxal/síntese química , Fenilglioxal/química , Teoria Quântica , Coelhos , Relação Estrutura-Atividade , Termodinâmica , Tiofenos/síntese químicaRESUMO
Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2) > 0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.
Assuntos
Carboxilesterase/antagonistas & inibidores , Fenilglioxal/análogos & derivados , Fenilglioxal/química , Acetilcolinesterase/química , Animais , Butirilcolinesterase/química , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/química , Inibidores da Colinesterase/química , Bases de Dados Factuais , Humanos , Intestinos/enzimologia , Modelos Moleculares , Fenilglioxal/síntese química , Relação Quantitativa Estrutura-Atividade , Ratos , Relação Estrutura-Atividade , Umbeliferonas/químicaRESUMO
The goal of cancer gene therapy is the selective and efficient eradication of tumor cells without significant systemic toxicity. Although several different gene therapy approaches have been developed and tested both in preclinical and clinical trials, none of these methods are suitable for the safe and efficient treatment of cancer. Recent advances in tumor cell biology have accelerated the identification of novel proteins as targets for gene transfer strategies. However, the development of vectors and delivery systems for specific and efficient gene therapy has not kept pace with these discoveries. Below, we describe the most widely used gene therapy approaches and discuss the caveats of using these techniques in the clinic.
Assuntos
Terapia Genética , Neoplasias/terapia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos de Neoplasias/imunologia , Técnicas de Transferência de Genes/efeitos adversos , Técnicas de Transferência de Genes/tendências , Genes Transgênicos Suicidas , Terapia Genética/efeitos adversos , Humanos , Mutação/genética , Terapia Viral Oncolítica/métodosRESUMO
Rhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.
Assuntos
Estresse Oxidativo , Rabdomiossarcoma Embrionário/genética , Animais , Evolução Clonal , Dosagem de Genes , Homeostase , Humanos , Perda de Heterozigosidade , Camundongos , Mutação , Rabdomiossarcoma Embrionário/tratamento farmacológico , Rabdomiossarcoma Embrionário/metabolismoRESUMO
Carboxylesterases (CEs) are ubiquitous enzymes responsible for the detoxification of xenobiotics. In humans, substrates for these enzymes are far-ranging, and include the street drug heroin and the anticancer agent irinotecan. Hence, their ability to bind and metabolize substrates is of broad interest to biomedical science. In this study, we focused our attention on dynamic motions of a CE from B. subtilis (pnbCE), with emphasis on the question of what individual domains of the enzyme might contribute to its catalytic activity. We used a 10 ns all-atom molecular dynamics simulation, normal mode calculations, and enzyme kinetics to understand catalytic consequences of structural changes within this enzyme. Our results shed light on how molecular motions are coupled with catalysis. During molecular dynamics, we observed a distinct C-C bond rotation between two conformations of Glu310. Such a bond rotation would alternately facilitate and impede protonation of the active site His399 and act as a mechanism by which the enzyme alternates between its active and inactive conformation. Our normal mode results demonstrate that the distinct low-frequency motions of two loops in pnbCE, coil_5 and coil_21, are important in substrate conversion and seal the active site. Mutant CEs lacking these external loops show significantly reduced rates of substrate conversion, suggesting this sealing motion prevents escape of substrate. Overall, the results of our studies give new insight into the structure-function relationship of CEs and have implications for the entire family of α/ß fold family of hydrolases, of which this CE is a member.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Carboxilesterase/química , Simulação de Dinâmica Molecular , Algoritmos , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Biocatálise , Carboxilesterase/genética , Domínio Catalítico , Ligação de Hidrogênio , Hidrólise , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Substâncias para a Guerra Química/farmacocinética , Compostos Organofosforados/farmacocinética , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Domínio Catalítico , Substâncias para a Guerra Química/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Mutação , Compostos Organofosforados/metabolismo , Conformação ProteicaRESUMO
Abstract Structures of mammalian carboxylesterases (CEs) reveal the presence of a 'side door' that is proposed to act as an alternative pore for the trafficking of substrates and products. p-Nitrobenzyl esterase (pnb CE) from Bacillus subtilis exhibits close structural homology and a similar side-door domain as mammalian CEs. We investigated the role of a specific 'gate' residue at the side door (i.e., Leu 362) during pnb CE-catalyzed hydrolysis of model esters, pesticides, and lipids. Recombinant pnb CE proteins containing mutations at position 362 demonstrated markedly lower kcat and kcat/Km values. The mutation with the most significant impact on catalysis was the L362R mutant (kcat/Km was 22-fold lower). Moreover, the ability of the L362R mutant to be inhibited by organophosphates (OP) was also lower. Investigation into the altered catalytic proficiency using pH-activity studies indicated that the catalytic triad of the mutant enzyme was preserved. Furthermore, viscosity variation and carbamate inhibition experiments indicated that rates of substrate association and acylation/deacylation were lower. Finally, recombinant CEs were found to possess lipolytic activity toward cholesteryl oleate and 2-arachidonylglycerol. In summary, the L362R mutant CE markedly slowed the rate of ester hydrolysis and was less sensitive to OP inhibition. The apparent causes of the diminished catalysis are discussed.
Assuntos
Bacillus subtilis/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Sítios de Ligação , Catálise , Ésteres/metabolismo , Cinética , Metabolismo dos Lipídeos , Mutação de Sentido Incorreto , Praguicidas/metabolismo , Estrutura Terciária de ProteínaRESUMO
We have synthesized and assessed the ability of symmetrical fluorobenzoins and fluorobenzils to inhibit mammalian carboxylesterases (CE). The majority of the latter were excellent inhibitors of CEs however unexpectedly, the fluorobenzoins were very good enzyme inhibitors. Positive correlations were seen with the charge on the hydroxyl carbon atom, the carbonyl oxygen, and the Hammett constants for the derived K(i) values with the fluorobenzoins.
Assuntos
Benzoína/análogos & derivados , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/farmacologia , Fenilglioxal/análogos & derivados , Humanos , Fenilglioxal/química , Relação Quantitativa Estrutura-AtividadeRESUMO
CPT-11 is a prodrug that is converted in vivo to the topoisomerase I poison SN-38 by carboxylesterases (CEs). Among the CEs studied thus far, a rabbit liver CE (rCE) converts CPT-11 to SN-38 most efficiently. Despite extensive sequence homology, however, the human homologues of this protein, hCE1 and hiCE, metabolize CPT-11 with significantly lower efficiencies. To understand these differences in drug metabolism, we wanted to generate mutations at individual amino acid residues to assess the effects of these mutations on CPT-11 conversion. We identified a Bacillus subtilis protein (pnb CE) that could be used as a model for the mammalian CEs. We demonstrated that pnb CE, when expressed in Escherichia coli, metabolizes both the small esterase substrate o-NPA and the bulky prodrug CPT-11. Furthermore, we found that the pnb CE and rCE crystal structures show an only 2.4 A rmsd variation over 400 residues of the alpha-carbon trace. Using the pnb CE model, we demonstrated that the "side-door" residues, S218 and L362, and the corresponding residues in rCE, L252 and L424, were important in CPT-11 metabolism. Furthermore, we found that at position 218 or 252 the size of the residue, and at position 362 or 424 the hydrophobicity and charge of the residue, were the predominant factors in influencing drug activation. The most significant change in CPT-11 metabolism was observed with the L424R variant rCE that converted 10-fold less CPT-11 than the wild-type protein. As a result, COS-7 cells expressing this mutant were 3-fold less sensitive to CPT-11 than COS-7 cells expressing the wild-type protein.
Assuntos
Camptotecina/análogos & derivados , Camptotecina/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Modelos Moleculares , Animais , Arginina/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células COS , Camptotecina/química , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Catálise , Simulação por Computador , Análise Mutacional de DNA , Humanos , Irinotecano , Cinética , Leucina/genética , Leucina/metabolismo , Mutagênese Sítio-Dirigida , Coelhos , Serina/metabolismo , ViscosidadeRESUMO
The recently introduced camptothecin-derived chemotherapeutic agents have demonstrated remarkable promise in cancer therapy and as such have been approved for use in humans for the treatment of ovarian, lung, and colon cancer. CPT-11 is a prodrug that is activated by esterases to yield the potent topoisomerase I inhibitor, SN-38. Considerable success has been achieved in the treatment of both naïve and drug-resistant colon cancer with CPT-11. However, mechanisms of resistance to this agent have not been explored in detail. The role of the ATP-dependent drug transporter ABCG2 in CPT-11 cytotoxicity is unclear because some ABCG2 mutants confer camptothecin resistance, whereas others do not. Because CPT-11 is activated by carboxylesterases (CEs), we assessed the relative contribution of each protein in mediating CPT-11 toxicity by both drug accumulation and cell growth-inhibition assays. Our results indicate that the expression of ABCG2 protects cells from CPT-11 toxicity, even in the presence of high levels of a rabbit liver carboxylesterase (rCE), which can efficiently activate the drug. However, this can be partially overcome by the ABCG2 inhibitor reserpine. These studies indicate that overexpression of ABCG2 in vivo would probably overcome any increased drug activation that might be achieved by gene delivery or antibody-directed enzyme prodrug therapy methods using rCE.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Hidrolases de Éster Carboxílico/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas de Neoplasias , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Benzimidazóis/farmacologia , Transporte Biológico/efeitos dos fármacos , Camptotecina/farmacocinética , Divisão Celular/efeitos dos fármacos , Humanos , Irinotecano , Coelhos , Células Tumorais CultivadasRESUMO
We have exploited the ability of wild-type (wt) p53 to repress gene expression and produce tumor-selective cytotoxicity using viral-directed enzyme prodrug therapy. Vectors containing either the cytomegalovirus or Rous sarcoma virus promoter regulating transcription of a rabbit liver carboxylesterase (CE) have been constructed. Upon transfection of these plasmids into cells expressing either wt or mutant p53, differential expression of the CE has been observed, resulting in sensitization of the cells expressing the latter protein to the anticancer prodrug irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carb- onyloxycamptothecin (CPT-11). Transduction of isogenic cell lines with adenovirus containing CE under control of the Rous sarcoma virus promoter confirmed the decreased sensitization of cells expressing wtp53 to CPT-11. These studies indicate that the inactivation of wtp53 by mutant p53 in human tumor cells may be sufficient enough to generate a therapeutic window for enhanced cytotoxicity with CPT-11.