RESUMEN

A correction to this paper has been published and can be accessed via a link at the top of the paper.

RESUMEN

Chronic myelogenous leukaemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukaemia (ALL) are caused by the BCR-ABL oncogene. Imatinib inhibits the tyrosine kinase activity of the BCR-ABL protein and is an effective, frontline therapy for chronic-phase CML. However, accelerated or blast-crisis phase CML patients and Ph+ ALL patients often relapse due to drug resistance resulting from the emergence of imatinib-resistant point mutations within the BCR-ABL tyrosine kinase domain. This has stimulated the development of new kinase inhibitors that are able to over-ride resistance to imatinib. The novel, selective BCR-ABL inhibitor, AMN107, was designed to fit into the ATP-binding site of the BCR-ABL protein with higher affinity than imatinib. In addition to being more potent than imatinib (IC50< 30 nM) against wild-type BCR-ABL, AMN107 is also significantly active against 32/33 imatinib-resistant BCR-ABL mutants. In preclinical studies, AMN107 demonstrated activity in vitro and in vivo against wild-type and imatinib-resistant BCR-ABL-expressing cells. In phase I/II clinical trials, AMN107 has produced haematological and cytogenetic responses in CML patients, who either did not initially respond to imatinib or developed imatinib resistance. Dasatinib (BMS-354825), which inhibits Abl and Src family kinases, is another promising new clinical candidate for CML that has shown good efficacy in CML patients. In this review, the early characterisation and development of AMN107 is discussed, as is the current status of AMN107 in clinical trials for imatinib-resistant CML and Ph+ ALL. Future trends investigating prediction of mechanisms of resistance to AMN107, and how and where AMN107 is expected to fit into the overall picture for treatment of early-phase CML and imatinib-refractory and late-stage disease are discussed.

Asunto(s)

Antineoplásicos , Genes abl/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Pirimidinas , Animales , Ensayos Clínicos como Asunto , Humanos

RESUMEN

Exponential tumor growth is angiogenesis-dependent. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are potent angiogenic inducers that act synergistically in vivo and in vitro. We assessed the effect of specific inhibitors of VEGF receptor (VEGFR)-2 tyrosine kinase activity in in vivo and in vitro models of VEGF- and bFGF-induced angiogenesis. In an implant mouse model of angiogenesis, VEGFR-2 inhibitors completely blocked angiogenesis induced by VEGF, and, surprisingly, also inhibited the effect of bFGF to various extents. In vitro, VEGF- and bFGF-induced bovine microvascular and aortic endothelial (BME and BAE) cell collagen gel invasion could be blocked by the VEGFR-2 inhibitors by 100 and approximately 90%, respectively. Similar results were obtained with VEGFR-1-IgG and VEGFR-3-IgG fusion proteins and with VEGFR-2 blocking antibodies. Both BME and BAE cells produce VEGF and VEGF-C, which is not modulated by bFGF. Thus, the unexpected inhibition of bFGF-induced angiogenesis by VEGFR-2 antagonists reveals a requirement for endogenous VEGF and VEGF-C in this process. These findings broaden the spectrum of mediators of angiogenesis that can be inhibited by VEGFR-2 antagonists and highlight the importance of these compounds as agents for inhibiting tumor growth sustained by both VEGF and bFGF.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/efectos de los fármacos , Linfocinas/fisiología , Neovascularización Fisiológica/efectos de los fármacos , Piridinas , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Animales , Células CHO , Células Cultivadas , Cricetinae , Factores de Crecimiento Endotelial/biosíntesis , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/inmunología , Endotelio Vascular/fisiología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Humanos , Inmunoglobulina G/farmacología , Linfocinas/genética , Linfocinas/inmunología , Ftalazinas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular , Factor C de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin-protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G(1) progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-Ras(G12V) to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Proteínas Oncogénicas/metabolismo , Proteínas Supresoras de Tumor , Agar , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/genética , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Progresión de la Enfermedad , Células Epiteliales/citología , Células Epiteliales/patología , Epitelio/metabolismo , Epitelio/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias de la Boca/metabolismo , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ratas , Proteínas Quinasas Asociadas a Fase-S , Transfección

RESUMEN

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Dominios Homologos src/genética , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Caenorhabditis elegans/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Citoplasma/química , Dimerización , Proteína Adaptadora GRB2 , Células HeLa , Humanos , Datos de Secuencia Molecular , Familia de Multigenes/genética , Unión Proteica , Proteínas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/farmacología , Seudogenes , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas , Técnicas del Sistema de Dos Híbridos , Proteína X Asociada a bcl-2

RESUMEN

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC(50) values < 0.1 microM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED(50) = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

Asunto(s)

Inhibidores de la Angiogénesis/síntesis química , Compuestos de Anilina/síntesis química , Inhibidores Enzimáticos/síntesis química , Ftalazinas/síntesis química , Piridinas , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Administración Oral , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacocinética , Inhibidores de la Angiogénesis/farmacología , Compuestos de Anilina/química , Compuestos de Anilina/farmacocinética , Compuestos de Anilina/farmacología , Animales , Disponibilidad Biológica , Células CHO , Línea Celular , Cricetinae , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Modelos Moleculares , Neoplasias/irrigación sanguínea , Neovascularización Patológica , Fosforilación , Ftalazinas/química , Ftalazinas/farmacocinética , Ftalazinas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Relación Estructura-Actividad , Transfección , Receptor 1 de Factores de Crecimiento Endotelial Vascular

RESUMEN

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Linfocinas/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Ftalazinas , Piridinas , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Inhibidores de la Angiogénesis/efectos adversos , Inhibidores de la Angiogénesis/sangre , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Carcinoma/irrigación sanguínea , División Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Humanos , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Leucocitos/citología , Leucocitos/efectos de los fármacos , Linfocinas/farmacología , Ratones , Ratones Desnudos , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Trasplante de Neoplasias , Neovascularización Patológica/patología , Fosforilación/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

CGP 41251 was originally identified as an inhibitor of protein kinase C (PKC), inhibiting mainly the conventional PKC subtypes, and subsequently shown to inhibit the vascular endothelial growth factor (VEGF) receptor kinase insert domain-containing receptor, which is involved in angiogenesis. CGP 41251 inhibits reversibly intracellular PKC activity, induction of c-fos and the corresponding activation of the mitogen-activated protein kinase induced by either tumor promoting phorbol esters, platelet-derived growth factor, or basic fibroblast growth factor, but not by the epidermal growth factor. CGP 41251 inhibited the ligand-induced autophosphorylation of the receptors for platelet-derived growth factor, stem cell factor, and VEGF (kinase insert domain-containing receptor) that correlated with the inhibition of the mitogen-activated protein kinase activation, but did not affect the ligand-induced autophosphorylation of the receptors for insulin, insulin-like growth factor-I, or epidermal growth factor. CGP 41251 showed broad antiproliferative activity against various tumor and normal cell lines in vitro, and is able to reverse the p-glycoprotein-mediated multidrug resistance of tumor cells in vitro. CGP 41251 showed in vivo antitumor activity as single agent and inhibited angiogenesis in vivo. Thus, CGP 41251 may suppress tumor growth by inhibiting tumor angiogenesis (via its effects on the VEGF receptor tyrosine kinases) in addition to directly inhibiting tumor cell proliferation (via its effects on PKCs).

Asunto(s)

Antineoplásicos/uso terapéutico , Inhibidores de Proteínas Quinasas , Receptores de Factores de Crecimiento/efectos de los fármacos , Receptores del Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Estaurosporina/análogos & derivados , Factores de Crecimiento Endotelial/fisiología , Inhibidores Enzimáticos/uso terapéutico , Predicción , Humanos , Técnicas In Vitro , Unión Proteica , Proteínas Quinasas/clasificación , Estaurosporina/farmacología , Estaurosporina/uso terapéutico

RESUMEN

On the basis of previously described X-ray studies of an enzyme/aza-dipeptide complex,8 aza-dipeptide analogues carrying N-(bis-aryl-methyl) substituents on the (hydroxethyl)hydrazine moiety have been designed and synthesized as HIV-1 protease inhibitors. By using either equally (12) or orthogonally (13) protected dipeptide isosteres, symmetrically and asymmetrically acylated aza-dipeptides can be synthesized. This approach led to the discovery of very potent inhibitors with antiviral activities (ED50) in the subnanomolar range. Acylation of the (hydroxethyl)hydrazine dipeptide isostere with the L-tert-leucine derivative 29 increased the oral bioavailability significantly when compared to the corresponding L-valine or L-isoleucine derivatives. The bis(L-tert-leucine) derivatives CGP 75355, CGP 73547, CGP 75136, and CGP 75176 combine excellent antiviral activity with high blood concentration after oral administration. Furthermore, they show no cross-resistance with saquinavir-resistant strains and maintain activity against indinavir-resistant ones. Consequently they qualify for further profiling as potential clinical candidates.

Asunto(s)

Fármacos Anti-VIH , Compuestos Aza , Dipéptidos , Inhibidores de la Proteasa del VIH , Proteasa del VIH/metabolismo , Administración Oral , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Compuestos Aza/administración & dosificación , Compuestos Aza/síntesis química , Compuestos Aza/farmacología , Disponibilidad Biológica , Dipéptidos/administración & dosificación , Dipéptidos/síntesis química , Dipéptidos/farmacología , Evaluación Preclínica de Medicamentos , Farmacorresistencia Microbiana , Femenino , Inhibidores de la Proteasa del VIH/administración & dosificación , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/enzimología , VIH-1/fisiología , Indinavir/farmacología , Ratones , Ratones Endogámicos BALB C , Saquinavir/farmacología , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos

RESUMEN

Polyclonal antibodies to native chicken pectoral fast-twitch myosin are directed to all subfragments of the molecule (S1, S2 and LMM), as seen in the ELISA and Western blotting techniques. The antibodies inhibit the Ca(2+)-activated myosin ATPase. Absorption of the antibodies with native myosin abolishes these reactions. Heat treatment of myosin for 2h at 40 degrees C will inactivate myosin ATPase and alter its antibody binding pattern: the binding of antibodies to the rod fractions is reduced, that to the globular head (S1) completely abolished. Thus, these antibodies are useful as sensitive probes for the structural integrity of the myosin head.

Asunto(s)

Anticuerpos/metabolismo , Calor , Miosinas/antagonistas & inhibidores , Miosinas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Reacciones Antígeno-Anticuerpo , Western Blotting , Pollos , Ensayo de Inmunoadsorción Enzimática , Técnicas de Inmunoadsorción , Fibras Musculares de Contracción Rápida/química , Miosinas/química , Miosinas/inmunología , Fragmentos de Péptidos/inmunología , Conformación Proteica , Desnaturalización Proteica

RESUMEN

Protein binding can impair the potency of human immunodeficiency virus (HIV) protease inhibitors. Therefore, the activity of a novel compound, CGP 61755, was studied in the absence or presence of alpha1-acid glycoprotein (alpha1AGP). In MT-2 cells, the activity loss was 4-fold (EC90 without alpha1AGP, 29 nM vs. 122 nM with alpha1AGP). In primary lymphocytes, the loss was 8-fold (EC90, 45 nM vs. 364 nM). In identical experiments, the activity loss in MT-2 cells and lymphocytes was 2- and 3-fold, respectively, for indinavir, 11- and 10-fold for saquinavir, and 11- and 48-fold for ritonavir. For SC-52151, a 17-fold loss was seen in MT-2 cells, whereas no EC90 with alpha1AGP was reached in lymphocytes. This study demonstrates that the impact of alpha1AGP on in vitro activity varies greatly among different HIV protease inhibitors. The magnitude of such differences is greater in human lymphocytes than in a standard cell line.

Asunto(s)

Fármacos Anti-VIH/farmacología , Etilenos/farmacología , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/fisiología , Orosomucoide/farmacología , Replicación Viral/efectos de los fármacos , Carbamatos , Línea Celular , Células Cultivadas , Furanos , Seronegatividad para VIH/inmunología , VIH-1/efectos de los fármacos , Humanos , Indinavir/farmacología , Cinética , Linfocitos/virología , Ritonavir/farmacología , Saquinavir/farmacología , Sulfonamidas/farmacología , Urea/análogos & derivados , Urea/farmacología

RESUMEN

CGP 61755 is a novel hydroxyethylene derivative produced by a high yield 10 step chemical synthesis. It is highly specific for HIV-1 protease with an IC50 of 1 nM. The ED90 in MT-2, PBLs and macrophages is infected with laboratory strains of HIV-1 or clinical isolates is 30-100 nM. In chronically infected macrophages the ED90 is 1000 nM (1000 nM for saquinavir and 10 microM for indinavir). When the antiviral activity of CGP 61755 on HIV-1 infected lymphocytes was examined using serum free medium an ED99 of 60 nM was determined, while in the presence of 10% human serum the same activity was achieved with 120 nM. When examined in combination with RT inhibitors or protease inhibitors, either in a co-culture of CEM-SS and chronically infected H9IIIB cells or in a free virus lymphocyte infection, cooperativity of the antiviral activities was observed. Dog pharmacokinetic studies comparing p.o. and i.v. data indicate that CGP 61755 has a bioavailability between 50 and 80%. Following oral administration the area under the concentration curve (AUC) values increased in a dose proportional manner. The plasma levels of the drug at 6 hours after oral administration were above the ED90. Based on these properties we believe that CGP 61755 has an attractive profile that justifies further preclinical evaluation of the drug.

Asunto(s)

Fármacos Anti-VIH/síntesis química , Etilenos/síntesis química , Inhibidores de la Proteasa del VIH/síntesis química , Replicación Viral/efectos de los fármacos , Animales , Fármacos Anti-VIH/farmacocinética , Proteínas Sanguíneas/metabolismo , Perros , Etilenos/farmacocinética , Inhibidores de la Proteasa del VIH/farmacocinética , VIH-1/efectos de los fármacos , VIH-1/enzimología , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Unión Proteica

RESUMEN

A series of aza-peptide analogs with a (hydroxyethyl)hydrazine isostere has been synthesized as HIV-1 protease inhibitors using a simple synthetic scheme. Structure-activity studies based on the X-ray of a previously described inhibitor-enzyme complex led to potent inhibitors with antiviral activity in the low-nanomolar range. The S-configuration of the transition-state hydroxyl group was preferred in this series. Small modifications of the P2P3 and P2'P3' substituents had little effect on enzyme inhibition but greatly influenced the pharmacokinetic profile. As a result of these studies, the symmetrically acylated compound 8a and its close analog 24a bearing a methyl carbamate in P3 and an ethyl carbamate in P3' position were identified as potent inhibitors with plasma concentrations exceeding antiviral ED50 values 150-fold following oral application in mice.

Asunto(s)

Aminoácidos/síntesis química , Antivirales/síntesis química , Inhibidores de la Proteasa del VIH/síntesis química , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , Hidrazinas/síntesis química , Administración Oral , Secuencia de Aminoácidos , Aminoácidos/administración & dosificación , Aminoácidos/farmacocinética , Aminoácidos/farmacología , Animales , Antivirales/administración & dosificación , Antivirales/farmacocinética , Antivirales/farmacología , Disponibilidad Biológica , Células Cultivadas , Femenino , Proteasa del VIH/metabolismo , Inhibidores de la Proteasa del VIH/administración & dosificación , Inhibidores de la Proteasa del VIH/farmacocinética , VIH-1/enzimología , Hidrazinas/administración & dosificación , Hidrazinas/farmacocinética , Hidrazinas/farmacología , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Estructura Molecular , Relación Estructura-Actividad

RESUMEN

A series of potent HIV-protease inhibitors has been prepared. Several of the newly synthesized compounds showed high plasma even after oral administration to animals. Based on the overall biological profile, CGP 61755 was chosen for further preclinical evaluation. For this compound, a 10 step synthesis potentially suitable for large scale production was developed.

Asunto(s)

Inhibidores de la Proteasa del VIH/síntesis química , Administración Oral , Antivirales/síntesis química , Antivirales/química , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Inhibidores de la Proteasa del VIH/farmacología , Estructura Molecular , Renina/antagonistas & inhibidores , Relación Estructura-Actividad

RESUMEN

Similar to interferon alpha, pentostatin is highly effective in hairy cell leukemia and moderately active in other chronic lymphoid malignancies. In ten patients with hairy cell leukemia (HCL) and seven patients with other B-cell chronic leukemias (BCL), we have studied the intracellular 2',5'-oligoadenylate synthetase (2,5OAS) activity of the mononuclear cells before, 4 h, 24 h, and 48 h after pentostatin administration. In patients with HCL the median level of intracellular 2,5OAS increased 4.6-fold at 4 h and 11.5-fold at 24 h compared to the pretreatment value. Among the other seven patients, the median intracellular 2,5OAS remained unchanged in three patients and rose slightly by 2 to 14 times in four patients. Eleven patients (eight with HCL and two with BCL) responded to pentostatin. The median increase in 2,5OAS among the responders was 13.0-fold (range 4.8-30.0) whereas that among non-responders was 2.2-fold (range 0.2-6.3). The difference was highly significant (p less than 0.0001). In five of the total seventeen patients, the plasma levels of 2,5OAS activity were also determined and changes in plasma levels paralleled those measured intracellularly. To determine if the elevation of 2,5OAS is mediated by induction of interferon alpha, the expressions of mRNA for interferon alpha and beta were investigated by means of reverse transcription and polymerase chain reaction using the corresponding sense primers. In none of the five patients thus studied could we find an induction of mRNA for interferon alpha or beta in the leukemic cells during treatment with pentostatin. Thus, response to pentostatin correlates with induction of 2,5OAS directly and the 2',5'-oligoadenylate system seems to be involved in cytotoxicity.

Asunto(s)

2',5'-Oligoadenilato Sintetasa/biosíntesis , Leucocitos Mononucleares/efectos de los fármacos , Pentostatina/uso terapéutico , 2',5'-Oligoadenilato Sintetasa/sangre , Adulto , Anciano , Anciano de 80 o más Años , Inducción Enzimática/efectos de los fármacos , Femenino , Humanos , Interferón-alfa/metabolismo , Interferón beta/metabolismo , Leucemia de Células Pilosas/tratamiento farmacológico , Leucemia de Células Pilosas/enzimología , Leucemia de Células Pilosas/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucocitos Mononucleares/enzimología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Inducción de Remisión

RESUMEN

Tumor necrosis factor (TNF) induces an antiviral state in various cell lines. This antiviral state is quite similar to that established by interferon (IFN), e.g., TNF treatment of HEp-2 cells induces 2',5'-oligoadenylate synthetase activity. Both antiviral activity and synthetase induction are greatly reduced when TNF treatment occurs in the presence of a beta interferon subtype 1 (IFN-beta 1)-neutralizing antiserum. However, no one has yet directly demonstrated IFN-beta 1 induction, either as an antiviral activity in supernatants from TNF-treated cells or as IFN-specific mRNA by Northern (RNA) blot analysis. We have adopted a recently described in vitro DNA amplification protocol for the detection of specific RNAs. By applying this method to RNA from HEp-2 cells, we could demonstrate increased levels of IFN-beta 1-specific transcripts after TNF treatment. Dose response and kinetics of IFN-beta 1 induction coincided with the TNF-induced antiviral state. Nuclear run-on analysis showed enhanced transcriptional activity of the IFN-beta 1 gene in TNF-treated cells. Our data substantiate a role of IFN-beta 1 as mediator of the biological activity of TNF in HEp-2 cells.

Asunto(s)

Regulación de la Expresión Génica , Interferón Tipo I/biosíntesis , Factor de Necrosis Tumoral alfa/fisiología , Northern Blotting , Células Cultivadas , ADN/biosíntesis , ADN/genética , ADN Polimerasa Dirigida por ADN , Amplificación de Genes , Humanos , Interferón Tipo I/genética , Cinética , Pruebas de Neutralización , Oligonucleótidos , ARN Mensajero/genética , Virus de la Estomatitis Vesicular Indiana/inmunología

RESUMEN

Tumour necrosis factor (TNF) induces antiviral activity in HEp-2 cells. Virus yield reduction assays with vesicular stomatitis virus as challenging virus demonstrated that the antiviral state was more pronounced in confluent cultures under low serum conditions. A significant enhancement of the antiviral state was obtained by combining TNF with low concentrations of either interferon (IFN)-beta 1 or IFN-gamma. The reduction in virus yield was significantly higher than that expected from summation of the independent antiviral activities of either substance alone, i.e. TNF and IFN acted synergistically as antiviral agents. Synergism of TNF with IFN-beta or IFN-gamma appeared to be mediated by different pathways, since different requirements for pretreatment and different effects on oligo-2',5'-adenylate synthetase (2-5AS) induction were observed. Induction of 2-5AS by TNF could be shown to be an indirect event that was sensitive to an antiserum against natural IFN-beta 1.

Asunto(s)

2',5'-Oligoadenilato Sintetasa/biosíntesis , Antivirales , Interferones/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Células Cultivadas , Medios de Cultivo/farmacología , Sinergismo Farmacológico , Inducción Enzimática , Humanos , Interferón Tipo I/farmacología , Interferón gamma/farmacología , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Virus de la Estomatitis Vesicular Indiana/crecimiento & desarrollo

RESUMEN

This paper shows that peritoneal murine macrophages become preactivated in vivo during the course of a Schistosoma mansoni infection. Thus, less macrophage-activating factor (MAF) was required to induce in vitro tumoricidal and schistosomulicidal activity in macrophages from S. mansoni-infected mice than in macrophages from uninfected control animals. Moreover, the respiratory burst activity, as measured by chemiluminescence, was enhanced in macrophages from S. mansoni-infected mice as compared to controls, whether or not lymphokine (LK) was present in the macrophage cultures. This response appeared at 3 weeks and persisted at least until 12 weeks after infection. Interferon-gamma (IFN-gamma) is most likely involved in the mechanisms leading to such an increased cytolytic and oxidative activity, since in vitro experiments showed: 1) that less IFN-gamma was required to induce tumoricidal activity in macrophages from infected as compared to macrophages from uninfected animals, 2) that the activity of (2'-5')-adenylate synthetase (2'-5' A-synthetase), an enzyme strongly induced by IFN, was elevated in cells from livers of S. mansoni-infected mice.

Asunto(s)

Activación de Macrófagos , Esquistosomiasis mansoni/inmunología , Animales , Citotoxicidad Inmunológica , Técnicas In Vitro , Interferón gamma/farmacología , Linfocinas/farmacología , Factores Activadores de Macrófagos , Masculino , Ratones , Ratones Endogámicos , Oxígeno/metabolismo , Cavidad Peritoneal/citología

RESUMEN

Tumour necrosis factor (TNF) was first described as a factor in the serum of mice injected with tubercle bacilli (BCG) and several days later with lipopolysaccharide (LPS). The gene encoding TNF has recently been cloned and pure recombinant human TNF is now available. TNF is known for its in vivo antitumour effect and in vitro cytotoxicity on certain transformed cell lines. Similarities in amino acid sequence and biological activity to lymphotoxin and cachectin have been reported, and very recently a growth-factor-like activity on diploid fibroblasts was observed. There is no similarity between these proteins and interferons (IFNs), which are also induced during in vivo induction of TNF. Here we describe the antiviral activity of pure recombinant human TNF in a typical in vitro antiviral assay which we discovered while investigating the possible role of TNF as an inducer of IFN.

Asunto(s)

Antivirales/uso terapéutico , Glicoproteínas/uso terapéutico , Proteínas Recombinantes/uso terapéutico , 2',5'-Oligoadenilato Sintetasa/metabolismo , Animales , Línea Celular , Virus de la Encefalomiocarditis/efectos de los fármacos , Activación Enzimática , Humanos , Técnicas In Vitro , Interferón Tipo I/farmacología , Ratones , Ratas , Simplexvirus/efectos de los fármacos , Factores de Tiempo , Factor de Necrosis Tumoral alfa , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos