RESUMEN
[structure: see text] 2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.
Asunto(s)
Acetamidas/química , Hidrocarburos Fluorados/química , Hidrocarburos Fluorados/síntesis química , Proteína 1A de Unión a Tacrolimus/química , Tacrolimus/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Hidrocarburos Fluorados/metabolismo , Inmunosupresores/química , Inmunosupresores/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Unión Proteica , Tacrolimus/metabolismo , Proteína 1A de Unión a Tacrolimus/metabolismo , Proteínas de Unión a Tacrolimus/antagonistas & inhibidoresRESUMEN
Conjugates of the L49 monoclonal antibody (binds to the p97 antigen on melanomas and carcinomas) were formed by attaching Enterobacter cloacae beta-lactamase (bL) to the L49-Fab' fragment using a heterobifunctional cross-linking reagent or by linking the enzyme to L49-sFv using DNA recombinant technology. The conjugates thus formed, L49-Fab'-bL and L49-sFv-bL, were designed to activate cephalosporin containing anticancer prodrugs at the surfaces of antigen positive tumor cells. Results from in vitro experiments using two lung carcinoma cell lines demonstrated that the conjugates were equally active in effecting the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug CCM. While treatment with either of the conjugates combined with the maximum tolerated doses of CCM led to cures of established SN12P renal cell carcinoma tumors in nude mice, only the L49-sFv-bL conjugate maintained its ability to do so at 1/4 the maximum tolerated dose of CCM. L49-sFv-bL was also superior to L49-Fab'-bL in the 1934J renal cell carcinoma tumor model and was shown to be quite active in two in vivo models of human lung carcinoma. These results demonstrate that the recombinant fusion protein leads to more pronounced therapeutic windows than the chemical conjugate and is active in an array of human tumor models.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Antineoplásicos/metabolismo , Profármacos/metabolismo , beta-Lactamasas/química , beta-Lactamasas/genética , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/inmunología , Cefalosporinas/metabolismo , Cefalosporinas/uso terapéutico , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Melanoma/inmunología , Antígenos Específicos del Melanoma , Ratones , Ratones Desnudos , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Compuestos de Mostaza Nitrogenada/metabolismo , Compuestos de Mostaza Nitrogenada/uso terapéutico , Proteínas Recombinantes , Células Tumorales CultivadasRESUMEN
The synthesis of C-Mel, a cephalosporin carbamate derivative of the clinically used alkylating agent melphalan, is described. C-Mel was designed as an anticancer nitrogen mustard prodrug that releases melphalan upon tumor-specific activation by targeted beta-lactamase (bL). The Km and kcat values for bL hydrolysis of C-Mel were 218 microM and 980 s(-1), respectively. In vitro cytotoxicity assays with 3677 human melanoma cells demonstrated that C-Mel was 40-fold less toxic than melphalan and was activated in an immunologically specific manner by L49-sFv-bL, a recombinant fusion protein that binds to the melanotransferrin antigen on melanomas and on some carcinomas. L49-sFv-bL in combination with C-Mel led to regressions and cures of established subcutaneous 3677 tumors in nude mice. The effects were significantly greater than those of melphalan, which did not result in any long-term regressions in this tumor model. The therapeutic effects were comparable to those obtained in mice treated with the previously described L49-sFv-bL/7-(4-carboxybutanamido)-cephalosporin mustard (CCM) combination. However, C-Mel may be more attractive than CCM for clinical development since the released drug is clinically approved.
Asunto(s)
Antineoplásicos Alquilantes , Cefalosporinas/síntesis química , Melfalán/análogos & derivados , Melfalán/química , Profármacos/síntesis química , Animales , Anticuerpos Monoclonales/genética , Cefalosporinas/metabolismo , Cefalosporinas/uso terapéutico , Humanos , Inmunotoxinas/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Melfalán/síntesis química , Melfalán/metabolismo , Melfalán/uso terapéutico , Ratones , Ratones Desnudos , Proteínas Recombinantes de Fusión , Células Tumorales Cultivadas , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
The syntheses of two cephalosporin derivatives 2 and 3 of mitomycin C (1) containing 7-phenylacetamido and 7-delta-carboxybutanamido side chains, respectively, are described. These compounds were prepared for evaluation as cephalosporin prodrugs capable of being activated by mAb-beta-lactamase conjugates. In vitro cytotoxicity assays performed on H2987 lung adenocarcinoma and clone 62 melanoma cell lines indicated that compound 2 was comparable in cytotoxicity to the parent drug. In an effort to improve upon the cytotoxic differential of 2, an alternative prodrug 3 containing a polar carboxyl group in the side chain of the cephalosporin moiety was prepared. Compound 3 consistently behaved as a prodrug and was approximately 40- and 10-fold less toxic than 1 toward H2987 and clone 62, respectively. Determination of kinetic constants for hydrolysis by beta-lactamase from Enterobacter cloacae P99 indicated kcat values of 476 +/- 170 and 248 +/- 15.1 s-1 for 2 and 3, respectively. The kcat/Km ratios for 2 and 3 were found to be approximately 9.7 and 2.1 microM/s, respectively. Comparison of these kcat/Km values with those obtained for similar cephalosporin derivatives of other antitumor agents demonstrated that compounds with delta-carboxybutanamido side chains generally have slightly diminished efficiency of enzymatic hydrolysis compared to the corresponding 7-phenylacetamido analog. It was also demonstrated that the less toxic prodrug 3 was activated in an immunologically specific manner by L6-F(ab')-beta-lactamase and 96.5-F(ab')-beta-lactamase conjugates, selective for H2987 and clone 62 cells, respectively.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Cefalosporinas/química , Mitomicinas , beta-Lactamasas/inmunología , Cefalosporinas/inmunología , Humanos , Sustancias Macromoleculares , Mitomicina/inmunología , Mitomicina/metabolismo , Modelos Químicos , Profármacos/síntesis química , Células Tumorales CultivadasRESUMEN
The L49 (IgG1) monoclonal antibody binds to p97 (melanotransferrin), a tumor-selective antigen that is expressed on human melanomas and carcinomas. A recombinant fusion protein, L49-sFv-bL, that contains the antibody binding regions of L49 fused to the Enterobacter cloacae r2-1 beta-lactamase (bL) was constructed, expressed, and purified to homogeneity in an Escherichia coli soluble expression system. The variable regions of L49 were cloned by reverse transcription-polymerase chain reaction from L49 hybridoma mRNA using signal sequence and constant region primers. Construction of the gene encoding L49-sFv-bL was accomplished by hybridization insertion of VH, VL, and sFv linker sequences onto a pET phagemid template containing the bL gene fused to the pelB leader sequence. Optimal soluble expression of L49-sFv-bL in E. coli was found to take place at 23 degrees C with 50 microM isopropyl beta-D-thiogalactopyranoside induction and the use of the nonionic detergent Nonidet P-40 for isolation from the bacteria. Construction and expression of a soluble form of the p97 antigen in Chinese hamster ovary cells allowed affinity-based methods for analysis and purification of the fusion protein. Surface plasmon resonance, fluorescent activated cell sorting, and Michaelis-Menten kinetic analyses showed that L49-sFv-bL retained the antigen binding capability of monovalent L49 as well as the enzymatic activity of bL. In vitro experiments demonstrated that L49-sFv-bL bound to 3677 melanoma cells expressing the p97 antigen and effected the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin nitrogen mustard prodrug. On the basis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, and localization was determined by measuring bL activity. Tumor to blood conjugate ratios of 13 and 150 were obtained 4 and 48 h post conjugate administration, respectively, and the tumor to liver, spleen, and kidney ratios were even higher. A chemically produced L49-Fab'-bL conjugate yielded a much lower tumor to blood ratio (5.6 at 72 h post administration) than L49-sFv-bL. Therapy experiments established that well-tolerated doses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumors in nude mice. The favorable pharmacokinetic properties of L49-sFv-bL allowed prodrug treatment to be initiated 12 h after the conjugate was administered. Thus, L49-sFv-bL appears to have promising characteristics for site-selective anticancer prodrug activation.
Asunto(s)
Antineoplásicos/farmacocinética , Inmunoglobulina G/genética , Profármacos/farmacocinética , beta-Lactamasas/genética , Animales , Biotransformación , Células CHO , Clonación Molecular , Cricetinae , Enterobacter cloacae/genética , Escherichia coli/genética , Humanos , Inmunoglobulina G/farmacología , Inmunoglobulina G/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/terapia , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Células Tumorales Cultivadas , beta-Lactamasas/farmacología , beta-Lactamasas/uso terapéuticoRESUMEN
Cephalosporin doxorubicin (C-Dox) and 7-(4-carboxybutanamido)-cephalosporin mustard (CCM) are prodrugs that are catalytically converted by Enterobacter cloacae beta-lactamase (bL) to the active anticancer agents doxorubicin and phenylenediamine mustard, respectively. Both prodrugs were less cytotoxic to the 3677 human melanoma line than their respective drugs and were activated in an immunologically specific manner by 96.5-bL, a mAb-bL conjugate that binds to 3677 cell surface antigens. Similar results were obtained using the CCM prodrug on SK-MEL 28 human melanoma cells. Experiments in mice with established s.c. 3677 tumors demonstrated that although no tumors were cured in mice receiving the 96.5-bL/C-Dox combination, the activities were greater than those obtained from systemic doxorubicin treatment or from administration of the nonbinding conjugate P1.17-bL in combination with C-Dox. In contrast, when CCM was used as a prodrug, cures of established 3677 tumors were obtained in 80% of the 96.5-bL treated animals. This combination was also able to induce regressions of large 3677 tumor masses (800 mm3) without any apparent toxic side effects. We conclude that 96.5-bL in combination with C-Dox or CCM has greater antitumor activity than systemic treatment with the corresponding drugs and that CCM is a more effective prodrug than C-Dox for treating human 3677 melanoma xenografts.
Asunto(s)
Doxorrubicina/administración & dosificación , Melanoma/tratamiento farmacológico , Compuestos de Mostaza Nitrogenada/administración & dosificación , Profármacos/administración & dosificación , beta-Lactamasas/administración & dosificación , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Neoplasias , Cefalosporinas , Femenino , Humanos , Inmunoconjugados , Antígenos Específicos del Melanoma , Ratones , Ratones Desnudos , Proteínas de Neoplasias/inmunología , Trasplante de NeoplasiasRESUMEN
7-Aminocephalosporin doxorubicin (AC-Dox) was condensed with monomethoxypoly(ethylene glycol)-propionic acid N-hydroxysuccinimide ester (5 kDa) or with a branched form of poly(ethylene glycol)-propionic acid N-hydroxysuccinimide ester (10 kDa), forming M-PEG-AC-Dox and B-PEG-AC-Dox, respectively. These polymer drug derivatives were designed such that doxorubicin would be released upon Enterobacter cloacae beta-lactamase (bL)-catalyzed hydrolysis. Both M-PEG-AC-Dox (IC50 = 80 microM) and B-PEG-AC-Dox (IC50 = 8 microM) were less toxic to H2981 human lung adenocarcinoma cells than doxorubicin (IC50 = 0.1-0.2 microM) and could be activated in an immunologically specific manner by L6-bL, a monoclonal antibody-bL conjugate that bound to H2981 cell surface antigens. In addition, the polymers were relatively stable in mouse plasma (< 26% hydrolysis after 24 h at 37 degrees C) and were less toxic to mice (maximum tolerated dose > 52 mumol/kg) than doxorubicin (maximum tolerated dose = 13.8 mumol/kg). Pharmacokientic studies were performed in mice bearing subcutaneous 3677 melanoma tumors. B-PEG-AC-Dox cleared from the blood more slowly than M-PEG-AC-Dox and was retained to a 2.1-fold greater extent in human 3677 melanoma tumor xenografts over a 4 h period. The intratumoral concentrations of both polymers far exceeded that of doxorubicin. Thus, the PEG-AC-Dox polymers offer the possibility of generating large intratumoral doxorubicin concentrations owing to their reduced toxicities, the amounts that accumulate in tumors, and the fact that doxorubicin is released upon beta-lactam ring hydrolysis.
Asunto(s)
Cefalosporinas/síntesis química , Cefalosporinas/toxicidad , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Inmunotoxinas/farmacocinética , Inmunotoxinas/toxicidad , Melanoma/tratamiento farmacológico , Polietilenglicoles , Polietilenglicoles/síntesis química , Polietilenglicoles/toxicidad , beta-Lactamasas/metabolismo , Adenocarcinoma , Animales , Anticuerpos Monoclonales , Supervivencia Celular/efectos de los fármacos , Cefalosporinas/farmacocinética , Doxorrubicina/síntesis química , Doxorrubicina/toxicidad , Enterobacter cloacae/enzimología , Femenino , Humanos , Inmunotoxinas/uso terapéutico , Neoplasias Pulmonares , Ratones , Ratones Desnudos , Estructura Molecular , Polietilenglicoles/farmacocinética , Relación Estructura-Actividad , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
A cephalosporin derivative of doxorubicin (C-Dox) was evaluated as a prodrug for activation by mAb conjugates of the beta-lactamase from Enterobacter cloacae P99 (beta L; EC 3.5.2.6). The conjugates consisted of beta L and the F(ab') fragments of either of the mAbs L6, P1.17, or 96.5. L6 binds to antigens on a variety of carcinomas, including the two lung adenocarcinoma cell lines H2981 and H2987 used in this study. 96.5 binds to the melanoma-associated antigen p97, and P1.17 was used for the control conjugate. C-Dox was found to be less cytotoxic to three different tumor cell lines in vitro compared to the parent drug doxorubicin (Dox). Immunospecific activation took place when the cells were pretreated with beta L conjugates that could bind to antigens on the tumor cells. In vivo toxicity and pharmacokinetics studies in athymic female nu/nu mice revealed that C-Dox was at least 7-fold less toxic than Dox (on a molar basis), despite the fact that a > or = 320-fold greater area-under-the-curve (blood concentration versus time) of C-Dox compared to Dox was obtained 0-2 h after administration of the two agents. Pharmacokinetic studies at maximum tolerated doses in mice bearing xenografts of either H2981 or H2987 revealed that the intratumoral levels of Dox after treatment with L6-beta L in combination with C-Dox were higher than were obtained by either systemic treatment with Dox or a combination of P1.17-beta L and C-Dox. This finding suggested that the conversion of C-Dox to Dox was tumor specific and dependent on the presence of the targeted antigen. Furthermore, the best antitumor activity against both H2981 and H2987 tumors was obtained by treatment with L6-beta L and C-Dox compared to P1.17-beta L and C-Dox or Dox alone. Thus, higher levels of Dox corresponded to greater therapeutic effects in both of the tumor models studied.
Asunto(s)
Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Inmunotoxinas/farmacocinética , Inmunotoxinas/toxicidad , Profármacos/administración & dosificación , Profármacos/farmacocinética , beta-Lactamasas/administración & dosificación , beta-Lactamasas/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/toxicidad , Biotransformación , Doxorrubicina/toxicidad , Femenino , Humanos , Hidrólisis , Fragmentos de Inmunoglobulinas/metabolismo , Fragmentos de Inmunoglobulinas/toxicidad , Cinética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Profármacos/toxicidad , Sensibilidad y Especificidad , Distribución Tisular , Trasplante Heterólogo , beta-Lactamasas/farmacocinéticaRESUMEN
The synthesis of a series of cephalosporin doxorubicin derivatives that differ with respect to the substituent at position 7 of the cephem nucleus is described. These compounds are designed as prodrugs of doxorubicin for activation by monoclonal antibody-beta-lactamase conjugates. The key step in the synthesis of this series of compounds involves the use of the phenylacetamido group as an enzymatically removable protecting group for the 7-amino group on the cephem. In vitro cytotoxicity assays with H2981 lung adenocarcinoma cells revealed that cephalosporin doxorubicin derivatives were all less toxic than the released drug. Prodrugs containing negatively charged groups in the side chain, such as the delta-carboxybutanamido derivative 4 and the alpha-sulfophenylacetyl derivative 5, displayed the least cytotoxic activity and were 46- and 26-fold less toxic than doxorubicin, respectively. The efficiency of activation of all the prodrugs was evaluated in cytotoxicity assays on H2981 cells with the beta-lactamases from Enterobacter cloacae P99, Escherichia coli TEM-1, and Bacillus cereus (type II). In general, the E. cloacae enzyme was found to most rapidly activate the majority of these prodrugs. Phenylacetamido prodrug 2 and delta-carboxybutanamido prodrug 4 were both activated in an immunospecific manner by L6-E. cloacae beta-lactamase, a monoclonal antibody conjugate that binds to receptors on H2981 lung adenocarcinoma cells.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/síntesis química , Cefalosporinas/síntesis química , Doxorrubicina/química , Profármacos/síntesis química , beta-Lactamasas/farmacología , Anticuerpos Monoclonales/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Secuencia de Carbohidratos , Línea Celular , Cefalosporinas/farmacocinética , Cefalosporinas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Datos de Secuencia Molecular , Profármacos/farmacocinética , Profármacos/farmacología , beta-Lactamasas/químicaRESUMEN
7-(Phenylacetamido)cephalosporin mustard (CM) and 7-(4-carboxybutanamido)cephalosporin mustard (CCM) were developed as anticancer prodrugs that could be activated site selectively by monoclonal antibody-beta-lactamase conjugates targeted to antigens present on tumor cell surfaces. Both CM and CCM were hydrolyzed by purified beta-lactamases from Escherichia coli (EC beta L), Bacillus cereus (BC beta L), and Enterobacter cloacae (ECl beta L). This resulted in the release of phenylenediamine mustard (PDM), a potent cytotoxic drug. The Km and kcat values of the reactions were determined, and it was found that ECl beta L effected the hydrolysis of CM and CCM more rapidly than the other enzymes. Conjugates of ECl beta L were prepared by reacting maleimide-substituted F(ab')2 fragments of the monoclonal antibodies L6 and P1.17 to ECl beta L that had been modified with sulfhydryl groups. In vitro experiments indicated that CCM (IC50 = 25-45 microM) was less toxic than PDM (IC50 = 1.5 microM) to H2981 lung adenocarcinoma cells (L6 antigen positive, P1.17 antigen negative) and that immunologically specific prodrug activation took place when the cells were treated with L6-ECl beta L. In vivo experiments in nude mice demonstrated that CCM was less toxic than CM, and that both prodrugs were much less toxic than PDM. Neither CCM nor PDM exerted antitumor activity on subcutaneous H2981 tumors in vivo. However, a significant antitumor effect was obtained in mice that received L6-ECl beta L 96 h prior to the administration of CCM. The effect was immunologically specific (P < 0.05), since a smaller degree of antitumor activity was obtained in mice that received the nonbinding control conjugate P1.17-ECl beta L prior to CCM.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antineoplásicos/farmacología , Cefalosporinas/síntesis química , Cefalosporinas/farmacología , Compuestos de Mostaza Nitrogenada/síntesis química , Profármacos/farmacología , beta-Lactamasas/inmunología , Animales , Cromatografía Líquida de Alta Presión , Portadores de Fármacos , Femenino , Hidrólisis , Ratones , Ratones Desnudos , Compuestos de Mostaza Nitrogenada/farmacología , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
The syntheses and cytotoxic activities of substituted N-phenylacetamido derivatives of doxorubicin and melphalan are described. The derivatives were designed as prodrugs which could be activated in a site-specific manner by monoclonal antibody-penicillin-G amidase (mAb-PGA) conjugates. N-(Phenylacetamido)doxorubicin (2) and N-(phenylacetyl)melphalan (6) were found to be 10- and 20-fold less cytotoxic against H2981 lung adenocarcinoma cells than doxorubicin and melphalan, respectively. When incubated with PGA, the cytotoxicity of 2 and 6 increased and became equivalent to that of the corresponding drugs from which they were made. The poor solubility characteristics of 2 in aqueous solutions provided the basis for the development of the more soluble doxorubicin derivatives, N-(4-aminophenylacetyl)doxorubicin (3) and N-(4-phosphonooxy)phenylacetyl)-doxorubicin (4). In vitro cytotoxicity assays indicated that 3 and 4 were at least 1000-fold less toxic than doxorubicin against H2981 cells. PGA and the mAb conjugate L6-PGA were able to effect the activation of 3 and 6 on H2981 cells (L6-antigen positive). Hydrolysis of the phosphate group of 4 was required prior to activation with PGA or L6-PGA. This was achieved using alkaline phosphatase, or by exposing 4 to phosphatases present in cell culture medium. The activation of 3, 4, and 6 on H2981 cells by L6-PGA occurred in an immunologically specific manner, since activation could be blocked by saturating cell surface antigens with L6 prior to treatment with L6-PGA. These results demonstrate that 3, 4, and 6 are prodrugs that can be specifically activated to release clinically approved anticancer agents by a mAb-PGA conjugate.
Asunto(s)
Antibióticos Antineoplásicos/síntesis química , Doxorrubicina/análogos & derivados , Melfalán/análogos & derivados , Fenilacetatos/síntesis química , Profármacos/síntesis química , Adenocarcinoma/tratamiento farmacológico , Antibióticos Antineoplásicos/farmacología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Penicilina Amidasa/metabolismo , Fenilacetatos/farmacología , Profármacos/farmacología , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacosAsunto(s)
Antineoplásicos/metabolismo , Enzimas/metabolismo , Profármacos/metabolismo , Fosfatasa Alcalina/administración & dosificación , Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/uso terapéutico , Anticuerpos Monoclonales , Antineoplásicos/uso terapéutico , Portadores de Fármacos , Terapia Enzimática , Enzimas/administración & dosificación , Humanos , Profármacos/uso terapéutico , gamma-Glutamil Hidrolasa/administración & dosificación , gamma-Glutamil Hidrolasa/metabolismo , gamma-Glutamil Hidrolasa/uso terapéuticoRESUMEN
Cephalosporin mustard (CM) was designed as an anticancer prodrug that could be activated in a site-specific manner by monoclonal antibody-beta-lactamase conjugates targeted to antigens present on tumor cell surfaces. Purified beta-lactamases from Bacillus cereus (BC beta L) and Escherichia coli (EC beta L) catalyzed the release of phenylenediamine mustard (PDM) from CM through a fragmentation reaction which occurs after the beta-lactam ring of CM is hydrolyzed. The Km and Vmax values were 5.7 microM and 201 mumol/min per mg for BC beta L and 43 microM and 29 mumol/min per mg for EC beta L, respectively. Conjugates of BC beta L were prepared by combining the F(ab')2 fragments of the maleimide-substituted monoclonal antibodies L6 and 1F5 with thiolated BC beta L. The conjugates showed little loss in enzymatic activity and bound nearly as well as the unmodified F(ab')2 monoclonal antibodies to antigens expressed on the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). PDM was approximately 50-fold more cytotoxic than CM to H2981 cells. Treatment of the cells with L6-BC beta L followed by CM resulted in a level of cytotoxic activity that was comparable to that of PDM. This was most likely due to activation of CM by conjugate that bound to cell-surface antigens, since pretreatment of H2981 cells with BC beta L or 1F5-BC beta L enhanced the activity of CM to a lesser extent. Thus, we have shown that CM is a prodrug, and that it can be activated with immunological specificity by a monoclonal antibody-beta-lactamase conjugate.
Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos/química , Cefalosporinas/química , Compuestos de Mostaza Nitrogenada/química , Profármacos/química , beta-Lactamasas/química , Anticuerpos Monoclonales/inmunología , Antineoplásicos/metabolismo , Bacillus cereus/enzimología , Biotransformación , Supervivencia Celular/efectos de los fármacos , Cefalosporinas/metabolismo , Cromatografía Líquida de Alta Presión , Escherichia coli/enzimología , Humanos , Compuestos de Mostaza Nitrogenada/metabolismo , Compuestos de Mostaza Nitrogenada/farmacología , Profármacos/metabolismo , Células Tumorales Cultivadas , beta-Lactamasas/metabolismoRESUMEN
N6,N6-Dibenzoyl-2',3'-O-isopropylideneadenosine, which is readily synthesized by one-pot 5'-O-trimethylsilylation, N6-benzoylation, and desilylation, was converted to the corresponding 5'-aldehyde. This was treated with CH2 = CHMgBr to afford, after debenzoylation, a 1:3 mixture of the 5'S and 5'R epimers, respectively, of 5'-C-vinyl-2',3'-O-isopropylideneadenosine. The configurations were established by single-crystal X-ray diffraction analysis of the 5'R epimer. Hydroboration of the 5'-O-tetrahydropyranyl derivative of the mixed epimeric 5'-C-vinyl nucleosides readily furnished 5'(S,R)-C-(2-hydroxyethyl)-2',3'-O-isopropylideneadenosine. Treatment of the 5'(S,R)-C-(2-O-tosyl) derivative of this with disodium L-homocysteinate permitted facile introduction of the L-ethionine system. By means of methods developed earlier in the synthesis of homologous methionine-ATP adducts, the alpha-amino acid group was protected, a beta,gamma-imidotriphosphoryl group was introduced at O5', and blocking groups were removed to give the title adduct as a 2:3 mixture of its two 5' epimers. It was a powerful inhibitor [KM(ATP)/Ki = 520 and 340] of the M-2 (normal tissue) and M-T (hepatoma tissue) forms, respectively, of the title enzyme and displayed predominantly competitive kinetics with the two substrates L-methionine and MgATP. It inhibited M-2 and M-T slightly less effectively than its homologue possessing one less CH2 between sulfur and C5' and gave kinetic evidence of an increased tendency to form L-methionine-enzyme-adduct and MgATP-enzyme-adduct complexes.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Adenilil Imidodifosfato/farmacología , Etionina/análogos & derivados , Isoenzimas/antagonistas & inhibidores , Metionina Adenosiltransferasa/antagonistas & inhibidores , Transferasas/antagonistas & inhibidores , Adenosina Trifosfato/farmacología , Adenilil Imidodifosfato/síntesis química , Animales , Unión Competitiva , Fenómenos Químicos , Química , Cromatografía Líquida de Alta Presión , Etionina/síntesis química , Etionina/farmacología , Cinética , Espectroscopía de Resonancia Magnética , Metionina/metabolismo , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
With 2',3'-O-isopropylideneadenosine or its N6-benzoyl derivative as starting material, synthetic routes to two novel adducts of L-methionine and beta,gamma-imido-ATP have been devised. One adduct, 14 (2:3 mixture of 6' epimers), had a P alpha OCH(R1)CH2 system [R1 = CH2-L-SCH2CH2CH2CH(NH2)CO2H] in place of the P alpha OC(5')H2 system of ATP, while the other, 16 (2:3 mixture of 5' epimers), had a P alpha OCH2CH2CH(R2) system [R2 = L-SCH2CH2CH(NH2)CO2H]. The ribose-P alpha bridge in 14 and 16 contained one more methylene group than in two homologous methionine-ATP adducts studied previously. Adduct 14 was a potent inhibitor of the rat M-2 (normal tissue) and M-T (Novikoff ascitic hepatoma) variants of methionine adenosyltransferase and gave competitive kinetics vs MgATP (Ki = 0.39 and 0.63 microM, respectively) or vs L-methionine (Ki = 2.2 and 2.7 microM). Adduct 16 was likewise a potent inhibitor competitive vs MgATP (Ki = 0.44 and 0.81 microM, respectively) or L-methionine (Ki = 2.1 and 1.5 microM). The kinetic data indicate that 14 and 16 inhibit by binding simultaneously to the MgATP and L-methionine substrate sites and that the extra methylene group facilitates the interaction of their methionine residues with these methionine sites.
Asunto(s)
Adenosina Trifosfato/metabolismo , Isoenzimas/antagonistas & inhibidores , Metionina Adenosiltransferasa/antagonistas & inhibidores , Metionina/metabolismo , Transferasas/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/síntesis química , RatasRESUMEN
The title compound is a covalent adduct of L-methionine (Met) and beta,gamma-imido-ATP. In its synthesis the N-Boc derivative of 5'(R)-C-(aminomethyl)-N6-benzoyl-5'-O-tosyl-2',3'-O- isopropylidenadenosine was converted by the successive actions of CF3CO2H and HNO2 into the corresponding 5'(R)-C-hydroxymethyl derivative. Treatment of this with disodium L-homocysteinate led to attack of sulfur at C6', apparently via a 5',6'-epoxide, and to total stereoselective inversion at C5' to furnish, after debenzoylation, 5'(R)-C-(L-homocystein-S-ylmethyl)-2',3'-O-isopropylidene ade nosine. The 5' configuration was established by conversion of this into the known 5'(S)-C-methyl-2',3'-O-isopropylidene adenosine with Raney nickel. The alpha-amino acid residue was protected as an N-Boc methyl ester, after which the 5'-hydroxyl was phosphorylated with benzyl phosphate and dicyclohexylcarbodiimide. The phosphoanhydride bond with inorganic imidodiphosphate was then created by established methods. Finally, blocking groups were removed under conditions that gave the desired adduct with no racemization of its L-methionine residue. It was a potent inhibitor [KM(ATP)/Ki = 1080; KM(Met)/Ki = 7.7] of the M-2 (normal tissue) form of rat methionine adenosyltransferase and of the M-T (hepatoma tissue) form [KM(ATP)/Ki = 670; KM(Met)/Ki = 22]. Inhibitions were competitive with respect to ATP or to L-methionine, indicating a dual substrate site mode of binding to the enzyme forms.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Isoenzimas/antagonistas & inhibidores , Metionina Adenosiltransferasa/antagonistas & inhibidores , Transferasas/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Fenómenos Químicos , Química Física , Metionina/metabolismo , RatasRESUMEN
A synthesis is described of the title compound and its 5'S epimer, which are two-substrate adducts of adenosine 5'-triphosphate (ATP) and L-methionine (Met) in which the C(5')H2OP system in ATP is replaced by CH(R)CH2NHP [R = L-S(CH2)2CH(NH2)CO2H]. The 5'R epimer was a potent nonselective competitive inhibitor [averaged Ki = 0.32 microM; KM(ATP)/Ki = 440] vs. ATP of the rat M-2 (normal tissue) and M-T (Novikoff ascitic hepatoma) variants of methionine adenosyltransferase. It produced simple noncompetitive inhibition (averaged Ki = 2.7 microM) vs. Met with both variants. The 5'S epimer inhibited M-T competitively vs. ATP, but was 74-fold less effective than the 5'R epimer. Replacement of the homocysteine moiety in the 5'R epimer by hydrogen markedly reduced inhibitory potency, as indicated by Ki values of 14 microM for competitive inhibition vs. ATP and 580 microM for noncompetitive inhibition vs. Met with M-2. The data suggest that the 5'R epimer can interact simultaneously with two enzymic sites. Information on the kinetic mechanism of a human counterpart of M-2 and inhibitor properties of a previously studied Met-ATP adduct are consistent with the view that the two sites might resemble those that interact with the initial products of the reaction, S-adenosylmethionine and triphosphate.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Homocisteína/análogos & derivados , Isoenzimas/antagonistas & inhibidores , Metionina Adenosiltransferasa/antagonistas & inhibidores , Transferasas/antagonistas & inhibidores , Adenosina Trifosfato/síntesis química , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Sitios de Unión , Unión Competitiva , Fenómenos Químicos , Química , Homocisteína/síntesis química , Homocisteína/metabolismo , Homocisteína/farmacología , Cinética , Metionina/metabolismo , Polifosfatos/metabolismo , Ratas , S-Adenosilmetionina/metabolismoRESUMEN
Verrucarol was obtained from a simple procedure that involved the hydrolysis of a crude extract of a culture of Myrothecium verrucaria ATCC 24571.