RESUMEN
An NMR fragment screen for binders to the bromodomains of BRD4 identified 2-methyl-3-ketopyrroles 1 and 2. Elaboration of these fragments guided by structure-based design provided lead molecules with significant activity in a mouse tumor model. Further modifications to the methylpyrrole core provided compounds with improved properties and enhanced activity in a mouse model of multiple myeloma.
Asunto(s)
Antineoplásicos/química , Proteínas Nucleares/antagonistas & inhibidores , Pirroles/química , Factores de Transcripción/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Semivida , Humanos , Ratones , Simulación de Dinámica Molecular , Mieloma Múltiple/tratamiento farmacológico , Proteínas Nucleares/metabolismo , Pirroles/síntesis química , Pirroles/farmacocinética , Pirroles/uso terapéutico , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Trasplante HeterólogoRESUMEN
Delivering small interfering RNA (siRNA) to tumors is the major technical hurdle that prevents the advancement of siRNA-based cancer therapy. One of the difficulties associated with the development of clinically relevant delivery systems is the lack of reliable tools for monitoring siRNA delivery to tumors in vivo. We describe here a novel, positive-readout system where siRNA-mediated target knockdown elicits a rapid and robust increase of reporter activity. Using the positive-readout system, we created (1) ß-galactosidase-based tumor models that allow the detection of target knockdown in 1%-2% of tumor cells and can distinguish between tumor areas where effective target knockdown occurs versus tumor areas that are not accessible to delivery, and (2) luciferase-based tumor models that allow the quantitative assessment of a large number of delivery systems. Using these positive-readout models, we screened a number of literature-described siRNA delivery systems and identified lipid nanoparticles as a promising delivery platform for siRNA-based cancer therapy.
Asunto(s)
Técnicas de Silenciamiento del Gen , Monitoreo Fisiológico/métodos , Neoplasias/terapia , ARN Interferente Pequeño/administración & dosificación , Animales , Secuencia de Bases , Línea Celular Tumoral , Femenino , Genes Reporteros , Vectores Genéticos , Liposomas , Ratones , Ratones SCID , Datos de Secuencia Molecular , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , beta-Galactosidasa/genéticaRESUMEN
This study describes the synthesis and characterization of five conjugates of poly(ethylene glycol) modified polyethylenimine (PEG-PEIs) coupled in two different synthesis routes to a nonpeptidic pentacyclic RDG-mimetic for integrin receptor-targeted gene delivery. Synthesis of this panel of different conjugates allowed for systematic analysis of structure-activity relationships. Conjugates were therefore characterized regarding molecular composition, DNA condensation, size, and zeta potential of self-assembled polyplexes. In vitro characterization included investigation of blood compatibility, binding affinity to receptor-positive and receptor-negative cells measured by flow cytometry, cellular uptake quantified by scintillation counting, and efficiency and specificity of transfection assayed by reporter gene expression. In a first synthetic approach, low molecular weight PEI (LMW-PEI) was PEGylated using a heterobifunctional PEG linker and coupling of the RGD-mimetic was achieved at the distal end of PEG chains. In a second synthesis route, the RGD-mimetic was directly coupled to AB-block-copolymers of PEI (25 kDa) and PEG (30 kDa). Interactions of RGD-PEG-LMW-PEI conjugates with DNA were strongly impaired, whereas PEG-PEI-RGD conjugates were more promising candidates due to their physicochemical properties and higher receptor specificity. The binding, uptake, and transfection efficiency in receptor-positive cells was strongly increased upon conjugation of the RGD-mimetic to AB-block-copolymers of PEG-PEI and depended on the degree of peptide substitution. The conjugates of PEG-PEI AB-block-copolymers with low ligand density of the RGD-mimetic appear to be promising candidates for in vivo cancer gene therapy.
Asunto(s)
Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Técnicas de Transferencia de Gen , Integrina alfaVbeta3/metabolismo , Oligopéptidos/química , Polietilenglicoles/química , Polietileneimina/química , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Materiales Biomiméticos/síntesis química , Línea Celular Tumoral , ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Ligandos , Maleimidas/química , Propionatos/química , Compuestos de Sulfhidrilo/química , TransfecciónRESUMEN
Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 µM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.
Asunto(s)
Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Piridonas/química , Piridonas/farmacología , Animales , Cristalografía por Rayos X , Descubrimiento de Drogas , Compuestos Macrocíclicos/farmacocinética , Estructura Molecular , Piridonas/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
Matrix metalloproteinases (MMPs) have been implicated in several pathologies. At Abbott Laboratories, the matrix metalloproteinases inhibitor drug discovery program has focused on the discovery of a potent, selective, orally bioavailable MMP inhibitor for the treatment of cancer. The program evolved from early succinate-based inhibitors to utilizing in-house technology such as SAR by NMR to develop a novel class of biaryl hydroxamate MMP inhibitors. The metabolic instability of the biaryl hydroxamates led to the discovery of a new class of N-formylhydroxylamine (retrohydroxamate) biaryl ethers, exemplified by ABT-770 (16). Toxicity issues with this pre-clinical candidate led to the discovery of another novel class of retrohydroxamate MMP inhibitors, the phenoxyphenyl sulfones such as ABT-518 (19j). ABT-518 is a potent, orally bioavailable, selective inhibitor of MMP-2 and 9 over MMP-1 that has been evaluated in Phase I clinical trials in cancer patients.
Asunto(s)
Industria Farmacéutica , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Animales , Diseño de Fármacos , Humanos , Espectroscopía de Resonancia Magnética , Relación Estructura-ActividadRESUMEN
A novel series of sulfone N-formylhydroxylamines (retrohydroxamates) have been investigated as matrix metalloproteinases (MMP) inhibitors. The substitution of the ether linkage of ABT-770 (5) with a sulfone group 13a led to a substantial increase in activity against MMP-9 but was accompanied by a loss of selectivity for inhibition of MMP-2 and -9 over MMP-1 and diminished oral exposure. Replacement of the biphenyl P1' substituent with a phenoxyphenyl group provided compounds that are highly selective for inhibition of MMP-2 and -9 over MMP-1. Optimization of the substituent adjacent to the retrohydroxamate center in this series led to the clinical candidate ABT-518 (6), a highly potent, selective, orally bioavailable MMP inhibitor that has been shown to significantly inhibit tumor growth in animal cancer models.
Asunto(s)
Antineoplásicos/síntesis química , Formamidas/síntesis química , Hidroxilaminas/síntesis química , Metaloendopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Disponibilidad Biológica , Línea Celular , Formamidas/química , Formamidas/farmacocinética , Formamidas/farmacología , Hidroxilaminas/química , Hidroxilaminas/farmacocinética , Hidroxilaminas/farmacología , Macaca fascicularis , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Successful siRNA therapeutics requires the optimal integration of multiple components, including an efficient delivery system, a disease indication that is appropriate for siRNA-based therapy, and a potent and nontoxic siRNA against a robust therapeutic target. Although all currently available delivery systems have limitations, it is important to recognize that a careful selection of the disease indication, therapeutic target, and siRNA molecule could partially compensate for deficiencies associated with the delivery system and makes it possible to advance a therapeutic siRNA regimen. In this study, we present the development of siRNA therapeutics for hepatocellular carcinoma using an integrated approach, including the development of an efficient lipid nanoparticle delivery system, the identification of a robust therapeutic target that does not trigger liver toxicity upon target knockdown, and the selection of potent and nonimmunogenic siRNA molecules against the target. The resulting siRNA-containing lipid nanoparticles produced significant antitumor efficacy in orthotopic hepatocellular carcinoma models, and, thus, represent a promising starting point for the development of siRNA therapeutics for hepatocellular carcinoma.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Terapia Genética/métodos , Neoplasias Hepáticas/tratamiento farmacológico , Nanopartículas , ARN Interferente Pequeño/administración & dosificación , Animales , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Femenino , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas Experimentales , Ratones , Ratones SCID , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/toxicidadRESUMEN
Trifluoromethyl ketones were found to be inhibitors of histone deacetylases (HDACs). Optimization of this series led to the identification of submicromolar inhibitors such as 20 that demonstrated antiproliferative effects against the HT1080 and MDA 435 cell lines.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Cetonas/química , Cetonas/farmacología , Acetilación , Western Blotting , Neoplasias de la Mama/metabolismo , Ciclo Celular/efectos de los fármacos , Fibrosarcoma/metabolismo , Histonas/metabolismo , Humanos , Hidrocarburos Fluorados/química , Hidrocarburos Fluorados/farmacología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos/farmacología , Inhibidores de Histona Desacetilasas , Cetonas/farmacología , Inhibidores Enzimáticos/química , Cetonas/química , Cinética , Relación Estructura-ActividadRESUMEN
Alpha-keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The alpha-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.